BACKGROUND
background is first given on biophysics information flows that result in stationary statistics observations.
then background is provided on the use of stationary statistics signal processing in device enhancement and communication.
ponderable media flow phenomenology and related information phenomenology
an important part of the ntd methodology is to have a stable ion-channel sized such that it can be modulated by a single, non-translocating, molecule, where the channel is modulated such that it is significantly blockaded, and not at a fixed level -- e.g., where at least one of the transducer blockade states has multi-level structure .
using a properly-sized channel, it is possible to establish a modulated ion-flow, through the alpha-hemolysin ion-channel for example, with a single ntd transducer molecule, where the ntd molecule is electrophoretically drawn into the channel.
biology provides highly stable, nanometer-scale , protein-based ion-channels appropriately sized for the ntd methodology with single-molecule blockaders.
a single molecule’s blockading interaction upon capture in an ion channel can be self-modulating upon capture , and this has been found in a number of experiments  <cit> .
the mechanism of interaction involves transient chemical bond formation between transducer and protein, where each bound state between transducer and channel imprints on the surrounding ionic current flow to provide a fixed level which then transitions to a different fixed level upon the bond dissociation or transitions to a different bound state.
self-modulatory blockaders each have unique blockade signatures that can be resolved to very high confidence .
given the engineering freedom to design the self-modulatory molecules, and the generalizations in the standard periodic carrier based signal processing to stationary statistics carrier based signal processing, we arrive at a means to leverage ponderable media flow phenomena, and blockader interaction kinetics, into a stochastic carrier wave signal processing problem that can be solved using efficient dynamic programming table computational methods as described here.
a single molecule that is captured in an ion channel is not in its natural state, especially if made to modulate the channel , e.g., to provide a multi-level  modulation of the channel current.
if the captured molecule is engineered  for use in transducing events, then the captured molecule will have at least two ion-channel blockade states.
given the freedom to adjust applied potential, ph, salt, and other factor so as to increase the number or distinctiveness of blockade states, highly sensitive biosensing arrangements can be established.
detector operation typically involves capturing and establishing flow modulators to produce a biosensing arrangement that responds to specifically designed, or selected, target stimuli .
the captured molecules are altered from their natural form, via stretching and conformational change, in the high electrophoretic force environment at the channel’s internal limiting aperture .
as mentioned, the most unnatural aspect of a captured molecule, if an ntd modulator, is the unique channel blocking dance that is established upon capture.
multi-state capture configurations are possible because the captured molecules are brought into contact with the channel walls such that transient chemical interactions take place between portions of the captured molecule and portions of the channel wall, where chemical interactions are taken to include the following binding interactions, among others: electrostatic, pi-bond, ionic, polar covalent, dipole-dipole, hydrogen-bond, van der waals bond, hydrophobic effect bond, and water-of-hydration effect bond.
in a general sense, the nanopore transduction detection method could be broadened to include a detection situation involving functionalization of any flow with stationary statistics, by use of flow modulators that have more than one stationary statistics phase of channel blockade, or flow modulation, where those phases are associated with states of the blockader.
anything that can be bathed in a stationary statistics flow can, thus, be observed by how it modulates that flow.
this takes any widget and makes it a smart widget by allowing us to be better informed about its state, and state transitions.
all that is required is establishing a stationary statistics flow that is modulated according to state of blockader in some desirable way.
for flow modulation at the nanometer-scale, it is important to note that we are at the scale of single molecules, for which the inherent coherence, and the time-frames for stationary statistics and clearly discernable states , can be established for greater periods, allowing for greater statistical stability on the modulations produced by a single-molecule interaction .
the lipid bilayer is the main weakness in the protein-channel based approach.
device hardening may eventually be done using solid-state channels, or via hybrid protein channel & ‘soft’-state devices .
thus, a single molecule’s blockading interaction upon capture in an ion channel can be self-modulating upon capture , and this has been found in a number of experiments .
self-modulatory blockaders each have unique blockade signatures that can be resolved to very high confidence over time.
given the engineering freedom to design the self-modulatory molecules, and the generalizations in the standard periodic carrier based signal processing to stationary statistics carrier based signal processing  <cit> , we arrive at a means to leverage ponderable media flow phenomena, and interaction kinetics, into a stochastic carrier wave signal processing problem that can be solved by efficient dynamic programming table computational methods  <cit> .
nanopore ion-flow based biosensing methods
the standard nanopore detector  detection paradigm, that is predominantly translocation  based, is shown in fig.
<dig> side-by-side with the nanopore transduction detector paradigm.
figure  <dig> elaborates on the possible nd detection platform topologies possible with translocation-based approaches, where the difference in translocation times is often the critical information that is used.
the difference in dwell times can depend on the off-binding time of the target binding entity , where binding failure allows polymer  translocation to complete .
by this mechanism, and its variants, bound probes can be distinguished from unbound.
there are specificity limits on the melting-based detection, however, that are not a problem in the ntd approach.
fig.
<dig> translocation information and transduction information.
open channel.
a channel blockade event with feature extraction that is typically dwell-time based.
a single-molecule coulter counter.
single-molecule transduction detection is shown with a transduction molecule modulating current flow .
fig.
<dig> nanopore detector detection topologies involving polymer translocation or threading.
the detection event is given by polymer  translocations that are delayed if bound .
if bound entity is on the trans side , and bound entity is a processive dna enzyme, then sequencing may be possible as described in  <cit> .
methods and
RESULTS
ntd-based applications and implementations are described for:  biosensing;  assaying; and  ntd nanoscope deployments for both device and unassembled kit platforms.
ntd biosensing
ntd biosensing methods can involve a dna modulator with linkages to an aptamer, antibody, or some other binding moiety, including simply a ssdna overhang.
the linkages needed to connect a dna-based channel-modulator to a dna-based aptamer involves a trivial join of the underlying ssdna sequences involved.
the linkage needed to connect a dna-based channel-modulator to an antibody could involve use of linker technology, and this has been used in the past with dsdna hairpins  <cit> , but another, more commoditized route, easily accessible with use of the nadir refined y-shaped dna channel modulators  <cit> , is that the antibody need merely be tagged with the appropriate ssdna strand, e.g., where the dna sequence is complement to part of the ‘y’ shaped dna channel modulator, and antibody tagging with dna is a standard service for use in immuno-pcr.
proof-of-concept experiments are described next for the streptavidin-biotin and dna annealing model systems, a pathogen/snp detection prototype, and for aptamer and antibody based detection.
model system based on streptavidin and biotin
a biotinylated dna-hairpin has been engineered to generate two signals depending on whether or not a streptavidin molecule is bound to the biotin.
in  <cit>  we show transduction of bound/unbound signals at three different transducer concentrations and a range of binding target  concentrations.
rescaling is done on counts, with the count of events at the  <dig>  µm bt-8gc scaled up by a factor of  <dig>  for example, for comparing event rate observations at different concentrations with the  <dig> µm bt-8gc behavior .
thousands of individual blockade observations are used to obtain the observation counts, and from this the apparent concentration at the various streptavidin concentrations.
model system based on dna annealing
a unique, y-shaped, ntd-aptamer is described in fig.
<dig>  in this experiment a stable modulator is established using a y-shaped molecule, where one arm is loop terminated such that it can’t be captured in the channel, leaving one arm with a ssdna extension for annealing to complement target.
fig.
<dig> the y-anneal transducer, and its annealing target.
a test of dna annealing has been performed with the y-shaped dna transduction molecule indicated, where the molecule is engineered to have an eight-base overhang for annealing studies.
a dna hairpin with complementary  <dig> base overhang is used as the binding partner.
in  <cit>  we show the binding results at the population-level , where the effects of binding are discernible as are potential isoforms.
the introduction of urea at  <dig>  m concentration is easily tolerated  and actually helps in discerning collective binding interactions such as with the dna annealing.
snp detection efforts
a possible test of dna snp annealing is with the y-shaped dna transduction molecule shown in fig.
<dig>  that is minimally altered, and such that the snp variant occurs in the y-nexus region.
for the case where digestion can’t conveniently provide extension only to one-side, a y-shaped annealed dsdna molecule can still be obtained, but such that the ssdna extensions outside the annealed region are now free to extend on both arms of the y-molecule.
snp variant detection is reduced to resolving the signals of two y-shaped duplex dna molecules, one with mismatch at snp, one with watson-crick base-pairing match at snp.
in studies of y-shaped dna molecules, numerous y-shaped dna molecules were considered.
three variants that successfully demonstrated the easily discernible, modulatory, channel blockade signals are shown in fig.
<dig> <cit> .
in those variants we considered the y-nexus with and without an extra base .
if an extra base is inserted we explore the three positions at the y .
the blunt ended arm has to be carefully designed such that when it is captured by the nanopore it produces a toggling blockade.
one of the arms of the y-shaped aptamer  has a tata sequence, and is meant to be a binding target for tbp.
in general, any transcription factor binding site could be studied  in this manner.
similarly, transcription factor could be verified by such constructions, or the efficacy of a synthetic transcription factor could be examined.
in using the nadir refinement process to arrive at the y-transducer used in the dna annealing test  <cit> , we have demonstrated how single-base insertions or modifications at the nexus of the y-shaped molecule can have clearly discernible changes in channel-blockade signal.
y-molecules as dna probes with single point mutations discernible at the y-nexus are explored in  <cit>  .
what is described in  <cit>  is a linkage to a nanopore-detector directed  search for aptamers that is based on bound-state lifetime measurements .
nadir complements and augments selex in usage.
further discussion of ntd biosensing experiments along these lines is in the discussion, for snp, aptamer, and antibody based detection systems.
extensive further results in these areas are described in  <cit> , as well.
ntd assaying
using a ntd nanoscope, a single bound/unbound system can be held, observed, and its history of bound/unbound states can be tracked.
the single molecule state-tracking with lengthy time averages allows measurement of molecular characteristics that are obscured in ensemble-based measurements.
for complex biomolecules there is likely to be a tremendous diversity in molecular behavior, and in many cases this diversity may be the basis for their function.
dna termini are of critical importance for certain retroviral integrases and other biological processes – being able to study them, even comparatively, offers new avenues for understanding and drug selection .
information on the dna molecules’ variation in structure and flexibility is important to understanding the dynamically enhanced  dna complex formations that are found with strong affinities to other, specific, dna and protein molecules.
an important example of this is the hiv attack on cells.
the dna terminus properties of retroviral dna molecules are found to exhibit greater flexibility than similar sequences, often marked by an increase in the number of blockade states, such as in the upper-level fine structure for the molecule terminating with gacg-3’  <cit> .
one of the most critical stages in hiv’s attack is the binding between viral and human dna.
the dna molecule studied in this instance consists of the hiv consensus terminus at the end of the y-aptamer arm – where it is exposed for binding to integrase.
since this molecule presents another blunt-ended dsdna for capture, it is no surprise that such events occur.
the signal analysis must separate between two classes of signal associated with these two dominant forms of capture -- associated with capture of the two blunt-ended dna regions .
with appropriate capture of the molecule at the base of the y, this permits direct examination of protein binding to the terminal dna region.
the ntd approach may provide the best means for examining other enzymes, and other complex biomolecules, particularly their activity in the presence of different co-factors.
there are two ways that these studies can be performed:  the enzyme is linked to the channel transducer, such that the enzyme’s binding and conformational change activity may be directly observed and tracked or,  the enzyme’s substrate may be linked to the channel transducer and observation of enzyme activity on that substrate may then be examined.
case  provides a means to perform dna sequencing if the enzyme is a nuclease, such as lambda exonuclease.
case  provides a means to do screening, for example, against hiv integrase activity .
further details can be found in  <cit> .
ntd nanoscope deployment: device & kit
the ntd single-molecule nanoscope device
a ntd nanoscope for single molecule experiments is being developed by meta logos inc. the starting point for the device deployment is a fully assembled bilayer workstation interfaced with a preconfigured computer for data acquisition and signal processing.
further details on the fully assembled deployment will not be discussed further in this paper.
the ntd nanoscope kit device
a kit version of the ntd nanoscope is possible where the components can be bought separately  for as little as $ <dig>  in total.
two elements are missing, however, in a practical deployment of this very new technology:  model test systems – in response to this meta logos is developing test buffers and test molecules with its kit along the lines of the successful experiments described in  <cit> ; and  software with both local acquisition tools  and off-site server or web-interfaced tool-sets , the latter with more elaborate data analysis methods and services to be made available via meta logos server-linked signal processing via their kit or consulting arrangements.
the ntd kit deployment involves the following:
the ntd device machined components and parts , and a shopping list of recommended commercially available components , and chemicals.
the ntd set-up and operation manual .
a configured ntd kit computer with local ssa software and meta-logos ssa server-link .
a set of specialized buffer controls used for calibration .
selected model systems/controls
data analysis and data repository services .
setup, calibration, and troubleshooting services .
the ntd nanoscope is designed to be as self-calibrating as possible to provide enhanced, autonomous reliability: signals are normalized computationally with respect to physical parameters , eliminating the need for complex  physical feedback systems to stabilize the device.
in what follows, further results are specifically provided involving  the kit components;  the ntd model systems; and  the ssa toolset & server.
kit components
in fig.
<dig> is shown the ntd kit core assembly with peltier-based temperature controller model 350b manufactured by newport temperature.
the a-m systems patch clamp amplifier model  <dig> is used to impose a constant voltage across electrodes while monitoring picoampere fluctuations.
a micro manipulator is used to deliver profusion tubing to cis chamber during channel acquisition.
a tmc bench top vibration isolation table is used to support the core assembly and reduce vibration shock.
with the ntd kit we, thus, achieve a nanoscale single-molecule lab construction.
this is obviously a low-cost method to perform nanoscale experiments.
there is also a remarkable versatility in the incorporation of transducer, as will become more apparent in what follows.
fig.
<dig> the ntd kit components.
one inch diameter teflon core machined to contain two chambers that are connected by a segment of teflon tubing.
this tubing has one end open while the other is capped to restrict it’s opening to a tiny pin hole  approximately  <dig> microns in diameter .
aluminum square block is machined to support the core and provide access for illumination and electrode ports, and have easy assembly and mounting on the peltier device.
the teflon and aluminum components are shown seated on the peltier device for temperature control of the wells.
silver chloride  pellet electrodes are shown inserted into the cis and trans chambers port wells.
a ntd nanoscope kit arrangement that uses a table-top vibration isolation table, has perfusion apparatus, faraday cage, and linkage to a computer .
model systems
specially engineered calibration probes have been designed to enable real-time self-calibration by generating a standard “carrier signal.” these probes can also be added to samples being analyzed to provide a run-by-run self-calibration.
these redundant self-calibration capabilities result in a stable, user-friendly device that can be operated by an entry level lab technician.
similarly, model systems based on experiments described in  <cit>  have been established to allow quick entre into various snp-based and biosensing scenarios, including the following model systems:
the streptavidin biosensor model system  <cit> .
the dna annealing model system  <cit> .
the targeted pathogen model system  <cit> .
the snp minority/majority population detection system  <cit> .
the aptamer-transduced detection of thrombin system  <cit>
the antibody-transduced detection system using immuno-pcr style tagging with dna, where complement is engineered to form a y-transducer, or related, detection system  <cit> .
ssa toolset & server
although the nanopore transduction detector can be a self-contained device in a lab, external information can be used, for example, to update and broaden the operational information on control molecules .
for the general ntd kit user, carrier reference signals and other systemically-engineered constructs can be used, for example, for a wide range of thin-client arrangements .
the paradigm for both nanopore device and ntd kit implementations involve system-oriented interactions, where the kit implementation may operate on more of a data service/data repository level and thus need real-time  system processing of data-service requests or data-analysis requests.
although not as system-dependent on database-server linkages, the more self-contained device implementation will still typically have, for example, local networked  data-warehousing, and fast-access, for distributed processing speedup on real-time experimental operations.
for the general user and the ntd-kit user, the server interfaces for the ssa server facilities are being designed to provide services ranging from web-interface based services to an active, streaming, experimental calibration/troubleshooting effort.
nanopore device and nanopore kit users will have the signal acquisition module, the tfsa, bundled with their hardware .
this allows for bare-bones functionality with the bundling as a stand-alone device.
when there is need for more refined signal processing, such as when using the weakness recovery protocol to acquire signal when the fsa fails, the ssa server will have the full panoply of methods available to get a lock on the signal, and examine it further.
the transfer of data at this juncture will also be efficiently pre-processed  and will thereby allow for much lower bandwidth and latency in the networked signal processing.
biology has invented such structure repeatedly, such as with the retinal pre-processing in the human eye.
*specialized enhancement packages to be made available from meta logos in future refinements to their nanoscope effort.
discussion
in what follows we describe efforts underway with ntd biosensing using y-aptamers; ntd-based assaying; and for molecular capture for preprocessing and biofouling prevention.
ntd y-aptamer detection systems
snp detection
a test of dna snp annealing can be done with the y-shaped dna transduction molecule shown in fig.
<dig>  which is minimally altered  from the y-annealing transducer studied in fig.
<dig>
fig.
<dig> the y-snp with test complex is shown at the base-level specification and at the diagrammatic level, where a snp base is as indicated.
if the snp is its variant form , then a base-pairing will not occur at the nexus of the y-snp shown .
this allows discrimination between the annealed forms with high accuracy, while also discerning from the signals produced by the non-annealed y-snp, where there is no target-bound, or only non-specific molecular interactions imparting much less conformational structure as occurs with the matching  annealing interaction.
once the y-snp transducer has been tested on a single-species of short overhang length test molecules the next experimental challenge will be to detect snp variants using the y-snp transducer probe in the presence of a heterogeneous length mixture , with overhang as shown in figure  <dig>
fig.
<dig> the y-snp test complex with  <dig> dt length overhang is shown at the base-level specification, where a snp base is as shown.
if the snp is its variant form , then a base-pairing will not occur at the nexus of the y-snp shown.
this allows discrimination between the annealed forms with high accuracy, while also discerning from the signals produced by the non-annealed y-snp, where there is no target-bound, or only non-specific molecular interactions imparting much less conformational structure as occurs with the matching  annealing interaction.
the value of  <dig> ‘t’s on the extension is to also match the approximate extension, with the same ‘y’-sequence  as the previously blunt-ended annealed conformation.
snp variant detection is reduced to resolving the signals of two y-shaped duplex dna molecules, one with mismatch at snp, one with watson-crick base-pairing match at snp.
from the above, it is clear that the ntd method provides a viable prospect for snp variant detection to very high accuracy .
snp detection via translocation-based methods, on the other hand, must discern between two snp variants according to the different dwell times of the complement-template annealed snps, until dissociation from the template allows translocation of the blockading dsdna annealed conformation.
aptamer-based detection
aptamers are especially appropriate for study by nanopore detection due to the fact they can be designed with an end to be captured by and modulate a nanopore , while other parts of the aptamer are designed to bind a specific target.
this directly provides a ntd transducer if one or both of the bound/unbound states  provides distinctive channel modulations.
the binding statistics derived from the study of aptamers in a nanopore detector can also be used in the design of the aptamer itself, e.g., nadir selection instead of further selex-based selection  <cit> .
in fig.
<dig> we see the first aptamer test case to be considered, where we seek to detect thrombin  <cit>  in one case, and igg  <cit>  in another.
in one effort, we use the thrombin aptamer found by ikebukuro et al  <cit> , which is selected via selex and ema and is a 31-mer,that we link by a  <dig> dt spacer to the y-transducer .
fig.
<dig> the thrombin aptamer from  <cit>  is 5’-cactggtaggttggtgtggttggggccagtg-3’.
antibody-based detection
linkage of ssdna to antibody is commonly done in immuno-pcr preparations, so another path with rapid deployment is to make use of a linkage technology that is already commoditized, e.g., the molecules required for the antibody-based biosensing with this approach are simple  molecular components.
the core issue to be resolved here is whether a good ntd signal can be produced with immuno-pcr tagged antibodies that are designed to anneal to another dna molecule to form an ntd ‘y-transducer’ .
from previous efforts  <cit> , with more complicated edc linkages between a modified thymine and an antibody, it is clear that there are strong prospects for success with this method.
what is sought is not just further validation of the method, however, but a less expensive, accessible, platform from which to refine and develop ntd-based systems.
ntd based assaying systems
part of the complexity of glycoforms, and other modifications, of proteins such as hemoglobin and tsh, is that these glycoforms are present as a heterogeneous mixture, and it is the relative populations of the different glycoforms that may relate to clinical diagnosis or identification of disease .
to this end, a protein’s heterogeneous mixture of glycations and other modified forms might be directly observable with a ntd nanoscope, and this constitutes the clinically relevant data of interest in many situations, not simply the concentration of some particular glycoform.
furthermore, it is the transient, dynamic, changes of the glycoform profile that is often the data of interest, such that a real-time profile of glycoform populations may be of clinical relevance, and obtaining such real-time profiling of modified forms  would be another area of natural advantage for the ntd approach.
ntd assaying provides a new technology for characterization of transient glycoprotein complexes, antibody assays, and multicomponent enzyme assays.
for further discussion see  <cit> .
molecular capture & terisa
it is possible to couple ntd methods with antibody capture systems, or any specific-binding capture system .
this allows for a pre-processing step that can concentrate the analyte of interest in a capture matrix, as well as provide a means to eliminate biofouling components from entering the nanopore detector and possibly damaging the bilayer  <cit>  .
antibody capture in the terisa approach  <cit>  allows an ntd experiment to report on the presence of the target molecules via indirect observation of transduction molecule signal corresponding to uv cleaved ntd substrate molecules .
commercially produced systems are available with matrices pre-loaded with immobilized fc-binding antibodies; the secondary antibody can then be introduced and bound by the fc-binding ab’s to establish the desired, immobilized, specific-binding matrix .
if solution with target molecule is now repeatedly washed across the immunosorbant surface, an immobilized concentration of that target molecule can be obtained.
we can now introduce our primary antibody that targets the immobilized antigen .
the further novel aspect of this setup is to now have the primary antibody linked to an enzyme that acts on a ntd transducer substrate .
by taking some of the methodology from the elisa approach , and merging it with unique aspects of the nanopore detection approach, we have the ‘transducer enzyme-release with immunoabsorbent assay’  approach,  <cit> , where “sandwich terisa” is assumed to typically be the case, since specific immobilization is desired.
analogous to real-time pcr, where a qualitative pcr result is self-calibrated according to real-time values to obtain quantitative pcr results, we can do the same with the terisa and tarisa biosensing methods outlined here.
in other words, for all three methods with real-time observation , we can shift to a more quantitative footing .
in our case this is trivially achieved, since the data-acquisition and signal processing is already in use and operating in real-time.
this real-time tracking information helps to stabilize the method and complements the biosensing capability with a self-calibrating quantitative assaying capability .
see  <cit>  for other applications of note, such as: ntd operation as a gel assay; ntd use as a
nanopore processing unit ; ntd use in pathogen detection; and ntd-based stationary statistics based dna sequencing approach.
CONCLUSIONS
ntd methods offer prospects for improved snp detection and improved detection of other localized dna regions in general, with a profound impact on how snp detections are done in diagnostics.
ntd methods are shown to offer significant advantage in the aptamer and antibody based biosensing systems as well.
currently there are a huge variety of tests for various forms of cancer or genetic disorders, for example, and the ntd approach offers a highly-accurate, inexpensive, and fast test result on a platform functionalized for all tests – where the tests may be done simultaneously on the same sample using a mixture of the appropriate ntd probes.
nanopore transduction detection provides a highly discriminative method for biosensing, assaying and performing medical diagnostics using highly specific binding to some biomarker .
kidney damage, for example, is often undiagnosed until significant damage has already occurred and preventive action is limited  <cit> .
it is hypothesized that ntd probes can also be developed for early stage kidney disease detection and other disease detection, via biomarker biosensing with high sensitivity, without loss of necessary specificity in the standard clinical tests of the future.
the overall market impacted by this technology is very broad and includes the diagnostics, pharmaceutical, and biotechnology industries.
we describe ntd nanoscope device deployments that provide an inexpensive, quick, accurate and versatile method for biosensing, assaying and performing medical diagnostics.
we also describe the model systems examined on the ntd nanoscope.
the ntd approach provides a significant new tool for biology, biotechnology, public health and biodefense.
general methods
experimental setup
each experiment described in what follows was conducted using one alpha-hemolysin channel inserted into a diphytanoyl-phosphatidylcholine/hexadecane bilayer, where the bilayer was formed across a 20-micron diameter horizontal teflon aperture  <cit> .
the bilayer separates two seventy-microliter chambers containing  <dig>  m kcl buffered at ph  <dig>  .
a completed bilayer between the chambers was indicated by the lack of ionic current flow when a voltage was applied across the bilayer .
once the bilayer was in place, a dilute solution of α–hemolysin  was added to the cis chamber.
self-assembly of the α–hemolysin heptamer and insertion into the bilayer results in a stable, highly reproducible, nanometer-scale channel with a steady current of  <dig> pa under an applied potential of  <dig> mv at 23°c .
once one channel formed, further pores were prevented from forming by thoroughly perfusing the cis chamber with buffer.
molecular blockade signals were then observed by mixing analytes into the cis chamber.
the experiment is performed on a vibration isolation table to keep the bilayer from rupturing .
as mentioned earlier, the bilayer is the main weakness in the device operability, and it is primarily due to the problem of bilayer rupture, although permeability is another complicating factor in some circumstances as well.
these are ongoing areas of research and refinements to the methods along these lines will not be described in this paper.
channel current cheminformatics architecture
a protocol has been developed for the discovery, characterization, and classification of localizable, approximately-stationary, statistical signal structures in stochastic sequential data, such as channel current data  <cit> .
the channel current cheminformatics  pattern recognition informed signal processing architecture is described in  <cit> .
the processing is designed to rapidly extract useful information from noisy blockade signals using feature extraction protocols, wavelet analysis, hidden markov models  and support vector machines .
for blockade signal acquisition and simple, time-domain, feature-extraction, a finite state automaton  approach is used  <cit>  that is based on tuning a variety of threshold parameters.
a generic hmm is then used to characterize current blockades by identifying a sequence of sub-blockades as a sequence of state emissions  <cit> .
the parameters of the generic-hmm can then be estimated using a method called expectation/maximization, or em  <cit> , to effect de-noising.
the hmm method with em is part of the standard implementation used in what follows.
classification of feature vectors obtained by the hmm for each individual blockade event is then done using svms.
for the nanopore detector augmented with auxiliary molecules, much more data is usually needed to properly train the machine learning algorithms.
the distributed training of these algorithms  is a critical component in real-time signal processing  <cit> .
the ccc software helps in the discovery, characterization, and classification of localizable, approximately-stationary, statistical signal structures in channel current data, and changes between such structures.
along the lines of previous work in channel current cheminformatics  <cit> , the ccc real-time implementation is used for analysis of the data to be measured, and refined as needed.
the ssa server
the ssa protocol entails methods for signal acquisition, feature extraction, classification and clustering .
for nanoscope device or nanoscope kit users, a further refinement in the standard web-interfaces to ssa web-server methods  <cit>  is to provide a streaming-data ssa-server access.
the latter setting offers nanoscope calibration and troubleshooting capabilities in operational settings, as well as access to distributed real-time speedup on core computational methods.
thus, the entire pipeline of signal processing needed to go from raw streaming data to concise results can be passed to the ssa-server.
usually it is more efficient, however, to separate the acquisition module  and bundle that with the device  to enable a bare-bones operational capability locally for signal acquisition, then enable feature extraction and classification/clustering enhancements via centralized maintenance of an ssa-server algorithm toolset and data library.
many of the latter analyses can be done in real-time, for the experiments shown in the methods for example, where a networked device is already set up as described.
this same deployment is, thus, also sought in the nanoscope device or nanoscope kit development and is thereby already validated with the results on the existing system over the past several years.
what this means for the signal processing needs is that the latency of a large raw data transfer is largely eliminated by the local pre-processing to perform rudimentary signal acquisition, which only passes acquired signal onto the network.
for gene finding in mammals, for example, the gene regions amount to about 3% of the genome, so crude recognition of such would reduce acquisition and transmission of data by a factor of  <dig>  the drop in bandwidth needs in ntd channel current signal processing can be even more pronounced.
competing interests
the authors declare that they have no competing interests.

 background is first given on biophysics information flows that result in stationary statistics observations. then background is provided on the use of stationary statistics signal processing in device enhancement and communication. ponderable media flow phenomenology and related information phenomenology an important part of the ntd methodology is to have a stable ion-channel sized such that it can be modulated by a single, non-translocating, molecule, where the channel is modulated such that it is significantly blockaded, and not at a fixed level -- e.g., where at least one of the transducer blockade states has multi-level structure. using a properly-sized channel, it is possible to establish a modulated ion-flow, through the alpha-hemolysin ion-channel for example, with a single ntd transducer molecule, where the ntd molecule is electrophoretically drawn into the channel. biology provides highly stable, nanometer-scale, protein-based ion-channels appropriately sized for the ntd methodology with single-molecule blockaders. a single molecule’s blockading interaction upon capture in an ion channel can be self-modulating upon capture, and this has been found in a number of experiments. the mechanism of interaction involves transient chemical bond formation between transducer and protein, where each bound state between transducer and channel imprints on the surrounding ionic current flow to provide a fixed level which then transitions to a different fixed level upon the bond dissociation or transitions to a different bound state. self-modulatory blockaders each have unique blockade signatures that can be resolved to very high confidence. given the engineering freedom to design the self-modulatory molecules, and the generalizations in the standard periodic carrier based signal processing to stationary statistics carrier based signal processing, we arrive at a means to leverage ponderable media flow phenomena, and blockader interaction kinetics, into a stochastic carrier wave signal processing problem that can be solved using efficient dynamic programming table computational methods as described here. a single molecule that is captured in an ion channel is not in its natural state, especially if made to modulate the channel, e.g., to provide a multi-level modulation of the channel current. if the captured molecule is engineered for use in transducing events, then the captured molecule will have at least two ion-channel blockade states. given the freedom to adjust applied potential, ph, salt, and other factor so as to increase the number or distinctiveness of blockade states, highly sensitive biosensing arrangements can be established. detector operation typically involves capturing and establishing flow modulators to produce a biosensing arrangement that responds to specifically designed, or selected, target stimuli. the captured molecules are altered from their natural form, via stretching and conformational change, in the high electrophoretic force environment at the channel’s internal limiting aperture. as mentioned, the most unnatural aspect of a captured molecule, if an ntd modulator, is the unique channel blocking dance that is established upon capture. multi-state capture configurations are possible because the captured molecules are brought into contact with the channel walls such that transient chemical interactions take place between portions of the captured molecule and portions of the channel wall, where chemical interactions are taken to include the following binding interactions, among others: electrostatic, pi-bond, ionic, polar covalent, dipole-dipole, hydrogen-bond, van der waals bond, hydrophobic effect bond, and water-of-hydration effect bond. in a general sense, the nanopore transduction detection method could be broadened to include a detection situation involving functionalization of any flow with stationary statistics, by use of flow modulators that have more than one stationary statistics phase of channel blockade, or flow modulation, where those phases are associated with states of the blockader. anything that can be bathed in a stationary statistics flow can, thus, be observed by how it modulates that flow. this takes any widget and makes it a smart widget by allowing us to be better informed about its state, and state transitions. all that is required is establishing a stationary statistics flow that is modulated according to state of blockader in some desirable way. for flow modulation at the nanometer-scale, it is important to note that we are at the scale of single molecules, for which the inherent coherence, and the time-frames for stationary statistics and clearly discernable states, can be established for greater periods, allowing for greater statistical stability on the modulations produced by a single-molecule interaction. the lipid bilayer is the main weakness in the protein-channel based approach. device hardening may eventually be done using solid-state channels, or via hybrid protein channel & ‘soft’-state devices. thus, a single molecule’s blockading interaction upon capture in an ion channel can be self-modulating upon capture, and this has been found in a number of experiments. self-modulatory blockaders each have unique blockade signatures that can be resolved to very high confidence over time. given the engineering freedom to design the self-modulatory molecules, and the generalizations in the standard periodic carrier based signal processing to stationary statistics carrier based signal processing, we arrive at a means to leverage ponderable media flow phenomena, and interaction kinetics, into a stochastic carrier wave signal processing problem that can be solved by efficient dynamic programming table computational methods. nanopore ion-flow based biosensing methods the standard nanopore detector detection paradigm, that is predominantly translocation based, is shown in fig. side-by-side with the nanopore transduction detector paradigm. figure elaborates on the possible nd detection platform topologies possible with translocation-based approaches, where the difference in translocation times is often the critical information that is used. the difference in dwell times can depend on the off-binding time of the target binding entity, where binding failure allows polymer translocation to complete. by this mechanism, and its variants, bound probes can be distinguished from unbound. there are specificity limits on the melting-based detection, however, that are not a problem in the ntd approach. fig. translocation information and transduction information. open channel. a channel blockade event with feature extraction that is typically dwell-time based. a single-molecule coulter counter. single-molecule transduction detection is shown with a transduction molecule modulating current flow. fig. nanopore detector detection topologies involving polymer translocation or threading. the detection event is given by polymer translocations that are delayed if bound. if bound entity is on the trans side, and bound entity is a processive dna enzyme, then sequencing may be possible as described in. methods and  ntd-based applications and implementations are described for: biosensing; assaying; and ntd nanoscope deployments for both device and unassembled kit platforms. ntd biosensing ntd biosensing methods can involve a dna modulator with linkages to an aptamer, antibody, or some other binding moiety, including simply a ssdna overhang. the linkages needed to connect a dna-based channel-modulator to a dna-based aptamer involves a trivial join of the underlying ssdna sequences involved. the linkage needed to connect a dna-based channel-modulator to an antibody could involve use of linker technology, and this has been used in the past with dsdna hairpins, but another, more commoditized route, easily accessible with use of the nadir refined y-shaped dna channel modulators, is that the antibody need merely be tagged with the appropriate ssdna strand, e.g., where the dna sequence is complement to part of the ‘y’ shaped dna channel modulator, and antibody tagging with dna is a standard service for use in immuno-pcr. proof-of-concept experiments are described next for the streptavidin-biotin and dna annealing model systems, a pathogen/snp detection prototype, and for aptamer and antibody based detection. model system based on streptavidin and biotin a biotinylated dna-hairpin has been engineered to generate two signals depending on whether or not a streptavidin molecule is bound to the biotin. in we show transduction of bound/unbound signals at three different transducer concentrations and a range of binding target concentrations. rescaling is done on counts, with the count of events at the µm bt-8gc scaled up by a factor of for example, for comparing event rate observations at different concentrations with the µm bt-8gc behavior. thousands of individual blockade observations are used to obtain the observation counts, and from this the apparent concentration at the various streptavidin concentrations. model system based on dna annealing a unique, y-shaped, ntd-aptamer is described in fig. in this experiment a stable modulator is established using a y-shaped molecule, where one arm is loop terminated such that it can’t be captured in the channel, leaving one arm with a ssdna extension for annealing to complement target. fig. the y-anneal transducer, and its annealing target. a test of dna annealing has been performed with the y-shaped dna transduction molecule indicated, where the molecule is engineered to have an eight-base overhang for annealing studies. a dna hairpin with complementary base overhang is used as the binding partner. in we show the binding results at the population-level, where the effects of binding are discernible as are potential isoforms. the introduction of urea at m concentration is easily tolerated and actually helps in discerning collective binding interactions such as with the dna annealing. snp detection efforts a possible test of dna snp annealing is with the y-shaped dna transduction molecule shown in fig. that is minimally altered, and such that the snp variant occurs in the y-nexus region. for the case where digestion can’t conveniently provide extension only to one-side, a y-shaped annealed dsdna molecule can still be obtained, but such that the ssdna extensions outside the annealed region are now free to extend on both arms of the y-molecule. snp variant detection is reduced to resolving the signals of two y-shaped duplex dna molecules, one with mismatch at snp, one with watson-crick base-pairing match at snp. in studies of y-shaped dna molecules, numerous y-shaped dna molecules were considered. three variants that successfully demonstrated the easily discernible, modulatory, channel blockade signals are shown in fig.. in those variants we considered the y-nexus with and without an extra base. if an extra base is inserted we explore the three positions at the y. the blunt ended arm has to be carefully designed such that when it is captured by the nanopore it produces a toggling blockade. one of the arms of the y-shaped aptamer has a tata sequence, and is meant to be a binding target for tbp. in general, any transcription factor binding site could be studied in this manner. similarly, transcription factor could be verified by such constructions, or the efficacy of a synthetic transcription factor could be examined. in using the nadir refinement process to arrive at the y-transducer used in the dna annealing test, we have demonstrated how single-base insertions or modifications at the nexus of the y-shaped molecule can have clearly discernible changes in channel-blockade signal. y-molecules as dna probes with single point mutations discernible at the y-nexus are explored in. what is described in is a linkage to a nanopore-detector directed search for aptamers that is based on bound-state lifetime measurements. nadir complements and augments selex in usage. further discussion of ntd biosensing experiments along these lines is in the discussion, for snp, aptamer, and antibody based detection systems. extensive further results in these areas are described in, as well. ntd assaying using a ntd nanoscope, a single bound/unbound system can be held, observed, and its history of bound/unbound states can be tracked. the single molecule state-tracking with lengthy time averages allows measurement of molecular characteristics that are obscured in ensemble-based measurements. for complex biomolecules there is likely to be a tremendous diversity in molecular behavior, and in many cases this diversity may be the basis for their function. dna termini are of critical importance for certain retroviral integrases and other biological processes – being able to study them, even comparatively, offers new avenues for understanding and drug selection. information on the dna molecules’ variation in structure and flexibility is important to understanding the dynamically enhanced dna complex formations that are found with strong affinities to other, specific, dna and protein molecules. an important example of this is the hiv attack on cells. the dna terminus properties of retroviral dna molecules are found to exhibit greater flexibility than similar sequences, often marked by an increase in the number of blockade states, such as in the upper-level fine structure for the molecule terminating with gacg-3’. one of the most critical stages in hiv’s attack is the binding between viral and human dna. the dna molecule studied in this instance consists of the hiv consensus terminus at the end of the y-aptamer arm – where it is exposed for binding to integrase. since this molecule presents another blunt-ended dsdna for capture, it is no surprise that such events occur. the signal analysis must separate between two classes of signal associated with these two dominant forms of capture -- associated with capture of the two blunt-ended dna regions. with appropriate capture of the molecule at the base of the y, this permits direct examination of protein binding to the terminal dna region. the ntd approach may provide the best means for examining other enzymes, and other complex biomolecules, particularly their activity in the presence of different co-factors. there are two ways that these studies can be performed: the enzyme is linked to the channel transducer, such that the enzyme’s binding and conformational change activity may be directly observed and tracked or, the enzyme’s substrate may be linked to the channel transducer and observation of enzyme activity on that substrate may then be examined. case provides a means to perform dna sequencing if the enzyme is a nuclease, such as lambda exonuclease. case provides a means to do screening, for example, against hiv integrase activity. further details can be found in. ntd nanoscope deployment: device & kit the ntd single-molecule nanoscope device a ntd nanoscope for single molecule experiments is being developed by meta logos inc. the starting point for the device deployment is a fully assembled bilayer workstation interfaced with a preconfigured computer for data acquisition and signal processing. further details on the fully assembled deployment will not be discussed further in this paper. the ntd nanoscope kit device a kit version of the ntd nanoscope is possible where the components can be bought separately for as little as $ in total. two elements are missing, however, in a practical deployment of this very new technology: model test systems – in response to this meta logos is developing test buffers and test molecules with its kit along the lines of the successful experiments described in; and software with both local acquisition tools and off-site server or web-interfaced tool-sets, the latter with more elaborate data analysis methods and services to be made available via meta logos server-linked signal processing via their kit or consulting arrangements. the ntd kit deployment involves the following: the ntd device machined components and parts, and a shopping list of recommended commercially available components, and chemicals. the ntd set-up and operation manual. a configured ntd kit computer with local ssa software and meta-logos ssa server-link. a set of specialized buffer controls used for calibration. selected model systems/controls data analysis and data repository services. setup, calibration, and troubleshooting services. the ntd nanoscope is designed to be as self-calibrating as possible to provide enhanced, autonomous reliability: signals are normalized computationally with respect to physical parameters, eliminating the need for complex physical feedback systems to stabilize the device. in what follows, further results are specifically provided involving the kit components; the ntd model systems; and the ssa toolset & server. kit components in fig. is shown the ntd kit core assembly with peltier-based temperature controller model 350b manufactured by newport temperature. the a-m systems patch clamp amplifier model is used to impose a constant voltage across electrodes while monitoring picoampere fluctuations. a micro manipulator is used to deliver profusion tubing to cis chamber during channel acquisition. a tmc bench top vibration isolation table is used to support the core assembly and reduce vibration shock. with the ntd kit we, thus, achieve a nanoscale single-molecule lab construction. this is obviously a low-cost method to perform nanoscale experiments. there is also a remarkable versatility in the incorporation of transducer, as will become more apparent in what follows. fig. the ntd kit components. one inch diameter teflon core machined to contain two chambers that are connected by a segment of teflon tubing. this tubing has one end open while the other is capped to restrict it’s opening to a tiny pin hole approximately microns in diameter. aluminum square block is machined to support the core and provide access for illumination and electrode ports, and have easy assembly and mounting on the peltier device. the teflon and aluminum components are shown seated on the peltier device for temperature control of the wells. silver chloride pellet electrodes are shown inserted into the cis and trans chambers port wells. a ntd nanoscope kit arrangement that uses a table-top vibration isolation table, has perfusion apparatus, faraday cage, and linkage to a computer. model systems specially engineered calibration probes have been designed to enable real-time self-calibration by generating a standard “carrier signal.” these probes can also be added to samples being analyzed to provide a run-by-run self-calibration. these redundant self-calibration capabilities result in a stable, user-friendly device that can be operated by an entry level lab technician. similarly, model systems based on experiments described in have been established to allow quick entre into various snp-based and biosensing scenarios, including the following model systems: the streptavidin biosensor model system. the dna annealing model system. the targeted pathogen model system. the snp minority/majority population detection system. the aptamer-transduced detection of thrombin system the antibody-transduced detection system using immuno-pcr style tagging with dna, where complement is engineered to form a y-transducer, or related, detection system. ssa toolset & server although the nanopore transduction detector can be a self-contained device in a lab, external information can be used, for example, to update and broaden the operational information on control molecules. for the general ntd kit user, carrier reference signals and other systemically-engineered constructs can be used, for example, for a wide range of thin-client arrangements. the paradigm for both nanopore device and ntd kit implementations involve system-oriented interactions, where the kit implementation may operate on more of a data service/data repository level and thus need real-time system processing of data-service requests or data-analysis requests. although not as system-dependent on database-server linkages, the more self-contained device implementation will still typically have, for example, local networked data-warehousing, and fast-access, for distributed processing speedup on real-time experimental operations. for the general user and the ntd-kit user, the server interfaces for the ssa server facilities are being designed to provide services ranging from web-interface based services to an active, streaming, experimental calibration/troubleshooting effort. nanopore device and nanopore kit users will have the signal acquisition module, the tfsa, bundled with their hardware. this allows for bare-bones functionality with the bundling as a stand-alone device. when there is need for more refined signal processing, such as when using the weakness recovery protocol to acquire signal when the fsa fails, the ssa server will have the full panoply of methods available to get a lock on the signal, and examine it further. the transfer of data at this juncture will also be efficiently pre-processed and will thereby allow for much lower bandwidth and latency in the networked signal processing. biology has invented such structure repeatedly, such as with the retinal pre-processing in the human eye. *specialized enhancement packages to be made available from meta logos in future refinements to their nanoscope effort. discussion in what follows we describe efforts underway with ntd biosensing using y-aptamers; ntd-based assaying; and for molecular capture for preprocessing and biofouling prevention. ntd y-aptamer detection systems snp detection a test of dna snp annealing can be done with the y-shaped dna transduction molecule shown in fig. which is minimally altered from the y-annealing transducer studied in fig. fig. the y-snp with test complex is shown at the base-level specification and at the diagrammatic level, where a snp base is as indicated. if the snp is its variant form, then a base-pairing will not occur at the nexus of the y-snp shown. this allows discrimination between the annealed forms with high accuracy, while also discerning from the signals produced by the non-annealed y-snp, where there is no target-bound, or only non-specific molecular interactions imparting much less conformational structure as occurs with the matching annealing interaction. once the y-snp transducer has been tested on a single-species of short overhang length test molecules the next experimental challenge will be to detect snp variants using the y-snp transducer probe in the presence of a heterogeneous length mixture, with overhang as shown in figure fig. the y-snp test complex with dt length overhang is shown at the base-level specification, where a snp base is as shown. if the snp is its variant form, then a base-pairing will not occur at the nexus of the y-snp shown. this allows discrimination between the annealed forms with high accuracy, while also discerning from the signals produced by the non-annealed y-snp, where there is no target-bound, or only non-specific molecular interactions imparting much less conformational structure as occurs with the matching annealing interaction. the value of ‘t’s on the extension is to also match the approximate extension, with the same ‘y’-sequence as the previously blunt-ended annealed conformation. snp variant detection is reduced to resolving the signals of two y-shaped duplex dna molecules, one with mismatch at snp, one with watson-crick base-pairing match at snp. from the above, it is clear that the ntd method provides a viable prospect for snp variant detection to very high accuracy. snp detection via translocation-based methods, on the other hand, must discern between two snp variants according to the different dwell times of the complement-template annealed snps, until dissociation from the template allows translocation of the blockading dsdna annealed conformation. aptamer-based detection aptamers are especially appropriate for study by nanopore detection due to the fact they can be designed with an end to be captured by and modulate a nanopore, while other parts of the aptamer are designed to bind a specific target. this directly provides a ntd transducer if one or both of the bound/unbound states provides distinctive channel modulations. the binding statistics derived from the study of aptamers in a nanopore detector can also be used in the design of the aptamer itself, e.g., nadir selection instead of further selex-based selection. in fig. we see the first aptamer test case to be considered, where we seek to detect thrombin in one case, and igg in another. in one effort, we use the thrombin aptamer found by ikebukuro et al, which is selected via selex and ema and is a 31-mer,that we link by a dt spacer to the y-transducer. fig. the thrombin aptamer from is 5’-cactggtaggttggtgtggttggggccagtg-3’. antibody-based detection linkage of ssdna to antibody is commonly done in immuno-pcr preparations, so another path with rapid deployment is to make use of a linkage technology that is already commoditized, e.g., the molecules required for the antibody-based biosensing with this approach are simple molecular components. the core issue to be resolved here is whether a good ntd signal can be produced with immuno-pcr tagged antibodies that are designed to anneal to another dna molecule to form an ntd ‘y-transducer’. from previous efforts, with more complicated edc linkages between a modified thymine and an antibody, it is clear that there are strong prospects for success with this method. what is sought is not just further validation of the method, however, but a less expensive, accessible, platform from which to refine and develop ntd-based systems. ntd based assaying systems part of the complexity of glycoforms, and other modifications, of proteins such as hemoglobin and tsh, is that these glycoforms are present as a heterogeneous mixture, and it is the relative populations of the different glycoforms that may relate to clinical diagnosis or identification of disease. to this end, a protein’s heterogeneous mixture of glycations and other modified forms might be directly observable with a ntd nanoscope, and this constitutes the clinically relevant data of interest in many situations, not simply the concentration of some particular glycoform. furthermore, it is the transient, dynamic, changes of the glycoform profile that is often the data of interest, such that a real-time profile of glycoform populations may be of clinical relevance, and obtaining such real-time profiling of modified forms would be another area of natural advantage for the ntd approach. ntd assaying provides a new technology for characterization of transient glycoprotein complexes, antibody assays, and multicomponent enzyme assays. for further discussion see. molecular capture & terisa it is possible to couple ntd methods with antibody capture systems, or any specific-binding capture system. this allows for a pre-processing step that can concentrate the analyte of interest in a capture matrix, as well as provide a means to eliminate biofouling components from entering the nanopore detector and possibly damaging the bilayer. antibody capture in the terisa approach allows an ntd experiment to report on the presence of the target molecules via indirect observation of transduction molecule signal corresponding to uv cleaved ntd substrate molecules. commercially produced systems are available with matrices pre-loaded with immobilized fc-binding antibodies; the secondary antibody can then be introduced and bound by the fc-binding ab’s to establish the desired, immobilized, specific-binding matrix. if solution with target molecule is now repeatedly washed across the immunosorbant surface, an immobilized concentration of that target molecule can be obtained. we can now introduce our primary antibody that targets the immobilized antigen. the further novel aspect of this setup is to now have the primary antibody linked to an enzyme that acts on a ntd transducer substrate. by taking some of the methodology from the elisa approach, and merging it with unique aspects of the nanopore detection approach, we have the ‘transducer enzyme-release with immunoabsorbent assay’ approach,, where “sandwich terisa” is assumed to typically be the case, since specific immobilization is desired. analogous to real-time pcr, where a qualitative pcr result is self-calibrated according to real-time values to obtain quantitative pcr results, we can do the same with the terisa and tarisa biosensing methods outlined here. in other words, for all three methods with real-time observation, we can shift to a more quantitative footing. in our case this is trivially achieved, since the data-acquisition and signal processing is already in use and operating in real-time. this real-time tracking information helps to stabilize the method and complements the biosensing capability with a self-calibrating quantitative assaying capability. see for other applications of note, such as: ntd operation as a gel assay; ntd use as a nanopore processing unit; ntd use in pathogen detection; and ntd-based stationary statistics based dna sequencing approach.  ntd methods offer prospects for improved snp detection and improved detection of other localized dna regions in general, with a profound impact on how snp detections are done in diagnostics. ntd methods are shown to offer significant advantage in the aptamer and antibody based biosensing systems as well. currently there are a huge variety of tests for various forms of cancer or genetic disorders, for example, and the ntd approach offers a highly-accurate, inexpensive, and fast test result on a platform functionalized for all tests – where the tests may be done simultaneously on the same sample using a mixture of the appropriate ntd probes. nanopore transduction detection provides a highly discriminative method for biosensing, assaying and performing medical diagnostics using highly specific binding to some biomarker. kidney damage, for example, is often undiagnosed until significant damage has already occurred and preventive action is limited. it is hypothesized that ntd probes can also be developed for early stage kidney disease detection and other disease detection, via biomarker biosensing with high sensitivity, without loss of necessary specificity in the standard clinical tests of the future. the overall market impacted by this technology is very broad and includes the diagnostics, pharmaceutical, and biotechnology industries. we describe ntd nanoscope device deployments that provide an inexpensive, quick, accurate and versatile method for biosensing, assaying and performing medical diagnostics. we also describe the model systems examined on the ntd nanoscope. the ntd approach provides a significant new tool for biology, biotechnology, public health and biodefense. general methods experimental setup each experiment described in what follows was conducted using one alpha-hemolysin channel inserted into a diphytanoyl-phosphatidylcholine/hexadecane bilayer, where the bilayer was formed across a 20-micron diameter horizontal teflon aperture. the bilayer separates two seventy-microliter chambers containing m kcl buffered at ph. a completed bilayer between the chambers was indicated by the lack of ionic current flow when a voltage was applied across the bilayer. once the bilayer was in place, a dilute solution of α–hemolysin was added to the cis chamber. self-assembly of the α–hemolysin heptamer and insertion into the bilayer results in a stable, highly reproducible, nanometer-scale channel with a steady current of pa under an applied potential of mv at 23°c. once one channel formed, further pores were prevented from forming by thoroughly perfusing the cis chamber with buffer. molecular blockade signals were then observed by mixing analytes into the cis chamber. the experiment is performed on a vibration isolation table to keep the bilayer from rupturing. as mentioned earlier, the bilayer is the main weakness in the device operability, and it is primarily due to the problem of bilayer rupture, although permeability is another complicating factor in some circumstances as well. these are ongoing areas of research and refinements to the methods along these lines will not be described in this paper. channel current cheminformatics architecture a protocol has been developed for the discovery, characterization, and classification of localizable, approximately-stationary, statistical signal structures in stochastic sequential data, such as channel current data. the channel current cheminformatics pattern recognition informed signal processing architecture is described in. the processing is designed to rapidly extract useful information from noisy blockade signals using feature extraction protocols, wavelet analysis, hidden markov models and support vector machines. for blockade signal acquisition and simple, time-domain, feature-extraction, a finite state automaton approach is used that is based on tuning a variety of threshold parameters. a generic hmm is then used to characterize current blockades by identifying a sequence of sub-blockades as a sequence of state emissions. the parameters of the generic-hmm can then be estimated using a method called expectation/maximization, or em, to effect de-noising. the hmm method with em is part of the standard implementation used in what follows. classification of feature vectors obtained by the hmm for each individual blockade event is then done using svms. for the nanopore detector augmented with auxiliary molecules, much more data is usually needed to properly train the machine learning algorithms. the distributed training of these algorithms is a critical component in real-time signal processing. the ccc software helps in the discovery, characterization, and classification of localizable, approximately-stationary, statistical signal structures in channel current data, and changes between such structures. along the lines of previous work in channel current cheminformatics, the ccc real-time implementation is used for analysis of the data to be measured, and refined as needed. the ssa server the ssa protocol entails methods for signal acquisition, feature extraction, classification and clustering. for nanoscope device or nanoscope kit users, a further refinement in the standard web-interfaces to ssa web-server methods is to provide a streaming-data ssa-server access. the latter setting offers nanoscope calibration and troubleshooting capabilities in operational settings, as well as access to distributed real-time speedup on core computational methods. thus, the entire pipeline of signal processing needed to go from raw streaming data to concise results can be passed to the ssa-server. usually it is more efficient, however, to separate the acquisition module and bundle that with the device to enable a bare-bones operational capability locally for signal acquisition, then enable feature extraction and classification/clustering enhancements via centralized maintenance of an ssa-server algorithm toolset and data library. many of the latter analyses can be done in real-time, for the experiments shown in the methods for example, where a networked device is already set up as described. this same deployment is, thus, also sought in the nanoscope device or nanoscope kit development and is thereby already validated with the results on the existing system over the past several years. what this means for the signal processing needs is that the latency of a large raw data transfer is largely eliminated by the local pre-processing to perform rudimentary signal acquisition, which only passes acquired signal onto the network. for gene finding in mammals, for example, the gene regions amount to about 3% of the genome, so crude recognition of such would reduce acquisition and transmission of data by a factor of the drop in bandwidth needs in ntd channel current signal processing can be even more pronounced. competing interests the authors declare that they have no competing interests. 
meta logos inc. is developing both pre-packaged device platforms and component-level kit platforms. nanopore transduction detection offers prospects for a number of highly sensitive and discriminative applications, including: single nucleotide polymorphism detection; targeted dna re-sequencing; protein isoform assaying; and biosensing via antibody or aptamer coupled molecules. the ntd nanoscope method is shown to functionalize a single nanopore with a channel current modulator that is designed to transduce events, such as binding to a specific target. the ntd nanoscope has seen limited use in the scientific community, however, due to lack of information about potential applications, and lack of availability for the device itself. in this paper we provide an overview of the ntd nanoscope applications and implementations. the ntd nanoscope kit, in particular, is a component-level reproduction of the standard ntd device used in previous research papers. to expedite set-up in new lab settings, the calibration and troubleshooting for the ntd nanoscope kit components and signal processing software, the ntd nanoscope kit, is designed to include a set of test buffers and control molecules based on experiments described in previous ntd papers. the ntd nanoscope offers a means to obtain higher accuracy as it is a single-molecule method that does not inherently involve use of enzymes, using a functionalized nanopore instead. in both cases a lipid bi-layer workstation is first established, then augmentations and operational protocols are provided to have a nanopore transduction detector.
 nanopore transduction detection offers prospects for a number of highly sensitive and discriminative applications, including: single nucleotide polymorphism detection; targeted dna re-sequencing; protein isoform assaying; and biosensing via antibody or aptamer coupled molecules. nanopore event transduction involves single-molecule biophysics, engineered information flows, and nanopore cheminformatics. the ntd nanoscope has seen limited use in the scientific community, however, due to lack of information about potential applications, and lack of availability for the device itself. meta logos inc. is developing both pre-packaged device platforms and component-level kit platforms. in both cases a lipid bi-layer workstation is first established, then augmentations and operational protocols are provided to have a nanopore transduction detector. in this paper we provide an overview of the ntd nanoscope applications and implementations. the ntd nanoscope kit, in particular, is a component-level reproduction of the standard ntd device used in previous research papers.  the ntd nanoscope method is shown to functionalize a single nanopore with a channel current modulator that is designed to transduce events, such as binding to a specific target. to expedite set-up in new lab settings, the calibration and troubleshooting for the ntd nanoscope kit components and signal processing software, the ntd nanoscope kit, is designed to include a set of test buffers and control molecules based on experiments described in previous ntd papers. the description of the server-interfacing for advanced signal processing support is also briefly mentioned.  snp assaying, snp discovery, dna sequencing and rna-seq methods are typically limited by the accuracy of the error rate of the enzymes involved, such as methods involving the polymerase chain reaction enzyme. the ntd nanoscope offers a means to obtain higher accuracy as it is a single-molecule method that does not inherently involve use of enzymes, using a functionalized nanopore instead. 1- april eighth annual mcbios conference. computational biology and bioinformatics for a new decade college station, tx, usa 