Download 8_Nanopores-A Brilliant Future!_VChopra

NANOPORES-A BRILLIANT FUTURE!
Vimlarani Chopra, PhD
MNT 2015
CARBON NANOPORES
Carbon nanotube “porins” have significant implications for future health
care, biophysics, and bioengineering applications.
The passage of individual molecules through nanosized pores
“nanopores” in membranes is central to many biological processes.
Nanopores have the potential to be used for targeted drug delivery, as
novel biosensors in DNA sequencing applications, and also as components
of synthetic cells.
Experiments are not restricted to naturally occurring nanopores, but
artificial solid-state nanopores can be fabricated in insulating membranes
to monitor ion currents and forces as molecules pass through them for
various applications.
NANOPORE FABRICATION
 Nanopore fabrication and sophistication in biological applications can revolutionize medicine
in terms of speed, cost, and quality.
 DNA / Protein mapping
 Bimolecular structure analysis
 DNA sequencing
 Target detection
 Genetics / Genomics
 Proteomics (protein detection)
 Biomarker identification / Detecting harmful chemicals (anthrax)
 Drug discovery
 Detecting emerging environmental threats
NANOPORE TECHNOLOGY
Many biological processes depend on the ability of ion channels to regulate the flow
of ions through cellular membranes.
Nanopores are nanometer scale holes formed naturally by proteins in an insulating
membrane on cells.
Nanopore sequencing can be used for early disease detection using biomarker
identification.
Nanopore technology employs a nanoscale hole to stochastically sense with high
throughput individual biomolecules in solution.
The generality of the nanopore detection principle and the ease of single-molecule
detection suggest many potential applications of nanopores in biotechnology.
Nanopores are single-molecule detectors with exceptional sensitivity and versatility.
Nanopore-based DNA sequencing can revolutionize healthcare by reducing both
sequencing costs and time.
NANOPORE SEQUENCING
CHARACTERISTIC DISRUPTION IN CURRENT
 This diagram shows a protein nanopore set in an
electrically resistant membrane bilayer.
 An ionic current is passed through the nanopore by setting
a voltage across this membrane.
 If an analyte passes through the pore or near its
aperture, this event creates a characteristic disruption in
current.
 Measurement of that current makes it possible to identify
the molecule in question.
 This system can be used to distinguish between the four
standard DNA bases G, A, T and C, and also modified
bases.
 It can be used to identify target proteins and small
molecules, or to gain rich molecular information, for
example to distinguish between the enantiomers of
ibuprofen or study molecular binding dynamics.
ADVANTAGES
Eliminate the need to amplify DNA or use expensive reagents, work by passing a single
strand of DNA through a protein pore created in a membrane. An electric current flows
through the pore; different DNA bases disrupt the current in different ways, letting the
machine electronically read out the sequence.
Most use fluorescent reagents to identify bases or require chopping up the DNA molecule
and amplifying the fragments.
Can avoid errors that can creep in during these steps.
Being able to read DNA molecules directly also means that longer segments of a genome can
be read at a time.
This makes it easier for researchers to see large-scale patterns such as translocations, in which
chunks of DNA are displaced from one part of the genome to another, and copy number
variations, in which DNA sequences are repeated several times or more. (Translocations:
cancers; copy number variations: neurological and developmental disorders).
Analyte molecules are sampled rapidly in solution, and no chemical labeling/tagging is
required for attachment to optical probes or surfaces.
Nanopores can also be used to probe subtle changes to the internal structure of nucleic acids.
Largest strand of DNA roughly 48,000 bases long can be read at a stretch. (Oxford
Nanopores).
COMPUTER SIMULATION_VIRUSES
Electrophysical Properties of DNA
Solid State Nanopores
Discriminate between different rod like virus strains.
Current blockages caused by three different virus strains going through
a nanopore.
All three virus strains have the same diameter, so they block more or
less the same amount of current.
M13 virus has less charge than fd virus, and so tends to go through the
pore more slowly.
Similarly, pf1 virus, while having the same charge density of fd virus, is
approximately twice as long as fd virus and so tends to take longer to
go through the pore.
A nanoscale device that translocates polymer molecules in sequential
monomer order through a very small volume of space, a small pore in an
electrically biased membrane.
A single molecule detector that is also a very high throughput device. A
nanopore can probe thousands of different molecules or thousands of
identical molecules in a few minutes.
A detector that directly converts characteristic features of the
translocating polymer into an electrical signal. Transduction and
recognition occur in real time, on a molecule-by-molecule basis.
A device that can probe very long lengths of DNA.
ROLE
IN
BIOPHYSICS
As nanopores have sparked the imagination of scientists as tools that can help in solving problems in
biophysics.
Similar to atomic force microscopy, optical/magnetic tweezers, and fluorescence microscopy, nanopores
are taking stage as one of a handful of main tools for investigating individual protein and DNA
molecules.
Some considerations:
The inability of nanopores to provide any spectroscopic information about the identity of a molecule.
Ion current from a nanopore cannot determine the identity of a molecule, and therefore, properties
of the molecule are inferred.
Translocation of subunits occurs with constant speed.
Nanopores inherently have commercial value because of single-molecule detection capabilities.
While nanopores do detect individual molecules, far more than a handful of molecules are required
for detection in solution, hence all molecules can not be detected with same precision.
SOLID STATE PORES
Solid-state pores allow a tunable pore size, shape, stability, and integration with
compact electronic/optical sensor modalities.
Biophysical studies carried out using solid-state nanopores would not have been
possible using protein pores.
Experiments with solid-state devices are easier than handling fragile lipid bilayers
and waiting for single-channel reconstitution.
Experiments with α-hemolysin have shown that the atomic-level precision and control
over functionality is a winning combination (protein channels are superior).
The main drawback here is the relatively low precision with which pores are
fabricated, as compared with the atomic precision of proteins.
Questions: Can we design protein channels with a more flexible size, and can we
design more stable bilayers? When evaluating the state-of-the-art in nanopore
fabrication, there's no comparison to protein channels.
OTHER APPLICATIONS_SOLID STATE NANOPORES
Day-to-day
Vacuum Insulation
Thermal Insulation
Insulated Shipping
Specialty Materials
Faster Computers
Microchips
Flexible Touchscreens
Solar Cells
Desalination Membranes
Graphene is the thinnest substance ever made: a single sheet of
carbon atoms arranged in a hexagonal honeycomb pattern.
Stiff as diamond and hundreds of times stronger than steel,
extremely flexible, even stretchable.
It conducts electricity faster at room temperature than any other
known material, and it can convert light of any wavelength into a
current.
Biotechnology
FUTURE
 Improve nanopore surfaces to reduce non-specific adsorption, pore clogging, and
electrical noise.
Fabricate and test a nanopore detector articulated with integrated probes for molecular
identification.
Investigate and optimize the electronic properties of probe - DNA interactions to control
DNA translocation, orientation, and nucleotide contrast.
Develop new enzymatic methods to better control and limit the rate of DNA translocation
through articulated nanopores.
Develop algorithms for signal feature detection and base identification from articulated
nanopores.
Demonstrate single base sensitivity and resolution on single-stranded DNA translocating
through a nanopore.
Integrating the two approaches holds significant potential for rapid electronic sequencing
(single molecule /electric current transport).
CONCLUSION
A nanopore is a very small hole, created by a pore-forming protein or a hole in synthetic
materials such as silicon or graphene.
Nanopores may be formed by pore-forming proteins, typically a hollow core passing
through a mushroom-shaped protein molecule (alpha-hemolysin), inserted into a lipid bilayer
membrane and single-channel.
A nanopore present in an electrically insulating membrane is used as a single molecule
detector: biological protein channel, a high electrical resistance lipid bilayer, a pore in a
solid-state membrane or a hybrid of a protein channel set in a synthetic membrane.
The detection principle is based on monitoring the ionic current passing through the
nanopore as a voltage is applied across the membrane.
When the nanopore is of molecular dimensions, passage of molecules (DNA) cause
interruptions of the "open" current level, leading to a "translocation event" signal.
The passage of RNA or single-stranded DNA molecules through the membrane-embedded
alpha-hemolysin channel (1.5 nm diameter), causes a ~90% blockage of the current
(measured at 1 M KCl solution).
Solid-state nanopores are generally made in silicon (silicon nitride) compound membranes.
REFERENCES
http://nanopore.com/
Deamer, D.W. & Akeson, M. Nanopores and nucleic acids:
prospects for ultrarapid sequencing. Trends in Biotechnology 18,
131-180 (2000).
Chen, P., Gu, J., Brandin, E., Kim, Y.-R., Wang, Q. & Branton, D.
Probing single DNA molecule transport using fabricated
nanopores. Nano Letters 4, 2293-2298 (2004).
http://labs.mcb.harvard.edu/branton/projectsNanoporeSequencing.htm
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3780799/