U.S. patent application number 17/248197 was filed with the patent office on 2021-05-06 for electrochemical cell with increased current density.
The applicant listed for this patent is Roche Sequencing Solutions, Inc.. Invention is credited to John Foster, Morgan Mager.
Application Number | 20210130945 17/248197 |
Document ID | / |
Family ID | 1000005333127 |
Filed Date | 2021-05-06 |
![](/patent/app/20210130945/US20210130945A1-20210506\US20210130945A1-2021050)
United States Patent
Application |
20210130945 |
Kind Code |
A1 |
Foster; John ; et
al. |
May 6, 2021 |
ELECTROCHEMICAL CELL WITH INCREASED CURRENT DENSITY
Abstract
A nanopore cell is disclosed. The nanopore cell includes an
electrolyte well having a bottom base, a surrounding sidewall, and
a hydrophobic surface above the surrounding sidewall. The nanopore
cell further includes a first layer of electrode material disposed
on the bottom base of the electrolyte well. The nanopore cell
further includes a second layer of electrode material disposed on
the surrounding sidewall of the electrolyte well and electrically
connected to the first layer of electrode material. The first layer
of electrode material and the second layer of electrode material
are configured to jointly provide capacitive coupling when an
electrolyte is placed in the electrolyte well.
Inventors: |
Foster; John; (Mountain
View, CA) ; Mager; Morgan; (Sunnyvale, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Roche Sequencing Solutions, Inc. |
Pleasanton |
CA |
US |
|
|
Family ID: |
1000005333127 |
Appl. No.: |
17/248197 |
Filed: |
January 13, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14841120 |
Aug 31, 2015 |
10920312 |
|
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17248197 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6874 20130101;
C23C 14/35 20130101; C23C 14/3414 20130101; C23C 14/0641 20130101;
G01N 33/48721 20130101 |
International
Class: |
C23C 14/35 20060101
C23C014/35; C12Q 1/6874 20060101 C12Q001/6874; C23C 14/06 20060101
C23C014/06; C23C 14/34 20060101 C23C014/34; G01N 33/487 20060101
G01N033/487 |
Claims
1. A method of constructing a nanopore cell, comprising:
constructing an electrolyte well having a bottom base, a
surrounding sidewall, and a hydrophobic surface above the
surrounding sidewall; disposing a first layer of electrode material
on the bottom base of the electrolyte well; and disposing a second
layer of electrode material on the surrounding sidewall of the
electrolyte well, and wherein the second layer of electrode
material is electrically connected to the first layer of electrode
material; and wherein the first layer of electrode material and the
second layer of electrode material are configured to jointly
provide capacitive coupling when an electrolyte is placed in the
electrolyte well.
2. The method of claim 1, wherein the first layer of electrode
material and the second layer of electrode material form a single
working electrode of the nanopore cell.
3. The method of claim 2, and wherein the first layer of electrode
material forms a substantially planar portion of the working
electrode, and wherein the second layer of electrode material forms
a surrounding wall of the working electrode, and wherein the
surrounding wall of the working electrode extends perpendicular to
or at an angle from the substantially planar portion and along the
periphery of the substantially planar portion of the working
electrode.
4. The method of claim 3, and wherein an aspect ratio of the
working electrode comprises a ratio between a height of the
surrounding wall of the working electrode and a width of the
substantially planar portion of the working electrode, and wherein
the aspect ratio is selected in part based on a desired ratio of a
capacitance associated with a membrane and a capacitance associated
with the working electrode, and wherein the membrane is formed atop
the electrolyte well and spans across an opening of the well.
5. The method of claim 3, and wherein a width of the substantially
planar portion of the working electrode is between 1 to 100
microns.
6. The method of claim 3, and wherein a height of the surrounding
wall of the working electrode is between 100 nm to 20 microns.
7. The method of claim 2, wherein the working electrode comprises a
spongy and porous TiN working electrode that is deposited by a
deposition technique with conditions tuned to deposit
sparsely-spaced TiN columnar structures or columns of TiN
crystals.
8. The method of claim 1, wherein the hydrophobic surface above the
surrounding sidewall provides a surface for facilitating a membrane
to form atop the electrolyte well, and wherein the membrane spans
across an opening of the electrolyte well.
9. The method of claim 8, wherein the hydrophobic surface extends
downward partially into the electrolyte well and covers part of the
surrounding sidewall of the electrolyte well to further facilitate
a membrane to form atop the electrolyte well and to span across the
opening of the electrolyte well.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 14/841,120, filed Aug. 31, 2015, which is
herein incorporated by reference in its entirety for all
purposes.
BACKGROUND OF THE INVENTION
[0002] Advances in micro-miniaturization within the semiconductor
industry in recent years have enabled biotechnologists to begin
packing traditionally bulky sensing tools into smaller and smaller
form factors, onto so-called biochips. It would be desirable to
develop techniques for biochips that make them more robust,
efficient, and cost-effective.
BRIEF DESCRIPTION OF THE DRAWINGS
[0003] Various embodiments of the invention are disclosed in the
following detailed description and the accompanying drawings.
[0004] FIG. 1 illustrates an embodiment of a cell 100 in a nanopore
based sequencing chip.
[0005] FIG. 2 illustrates an embodiment of a cell 200 performing
nucleotide sequencing with the Nano-SBS technique.
[0006] FIG. 3 illustrates an embodiment of a cell about to perform
nucleotide sequencing with pre-loaded tags.
[0007] FIG. 4 illustrates an embodiment of a process 400 for
nucleic acid sequencing with pre-loaded tags.
[0008] FIG. 5 illustrates an embodiment of a circuitry 500 in a
cell of a nanopore based sequencing chip.
[0009] FIG. 6 illustrates an embodiment of a circuitry 600 in a
cell of a nanopore based sequencing chip, wherein the voltage
applied across the nanopore can be configured to vary over a time
period during which the nanopore is in a particular detectable
state.
[0010] FIG. 7A illustrates an additional embodiment of a circuitry
700 in a cell of a nanopore based sequencing chip, wherein the
voltage applied across the nanopore can be configured to vary over
a time period during which the nanopore is in a particular
detectable state.
[0011] FIG. 7B illustrates an additional embodiment of a circuitry
701 in a cell of a nanopore based sequencing chip, wherein the
voltage applied across the nanopore can be configured to vary over
a time period during which the nanopore is in a particular
detectable state.
[0012] FIG. 7C illustrates a double layer that is formed at any
interface between a conductive electrode and an adjacent liquid
electrolyte. In the example shown, the electrode surface is
negatively charged, resulting in the accumulation of positively
charged species in the electrolyte. In another example, the
polarity of all charges shown may be opposite to the example
shown.
[0013] FIG. 7D illustrates a pseudocapacitance effect that can be
formed, simultaneously with the formation of a double-layer as in
FIG. 7C, at an interface between a conductive electrode and an
adjacent liquid electrolyte.
[0014] FIG. 8 illustrates an embodiment of a process 800 for
analyzing a molecule inside a nanopore, wherein the nanopore is
inserted in a membrane.
[0015] FIG. 9 illustrates an embodiment of a plot of the voltage
applied across the nanopore versus time when process 800 is
performed and repeated three times.
[0016] FIG. 10 illustrates an embodiment of the plots of the
voltage applied across the nanopore versus time when the nanopore
is in different states.
[0017] FIG. 11 illustrates an embodiment of an electrochemical cell
1100 in a nanopore based sequencing chip.
[0018] FIG. 12 illustrates an embodiment of an electrochemical cell
1200 for nucleic acid sequencing that has a bowl-shaped or lidless
box-shaped working electrode that can provide an increased current
in the cell.
[0019] FIG. 13 illustrates an embodiment of a process for
constructing an electrochemical cell of a nanopore based sequencing
chip that includes a bowl-shaped or cup-shaped working electrode
that can provide an increased current in the cell.
[0020] FIG. 14 illustrates another embodiment of a process for
constructing an electrochemical cell of a nanopore based sequencing
chip that includes a bowl-shaped or cup-shaped working electrode
that can provide an increased current in the cell.
[0021] FIG. 15 illustrates a top view of a plurality of circular
openings 1502 of a plurality of wells in a nanopore based
sequencing chip.
DETAILED DESCRIPTION
[0022] The invention can be implemented in numerous ways, including
as a process; an apparatus; a system; a composition of matter; a
computer program product embodied on a computer readable storage
medium; and/or a processor, such as a processor configured to
execute instructions stored on and/or provided by a memory coupled
to the processor. In this specification, these implementations, or
any other form that the invention may take, may be referred to as
techniques. In general, the order of the steps of disclosed
processes may be altered within the scope of the invention. Unless
stated otherwise, a component such as a processor or a memory
described as being configured to perform a task may be implemented
as a general component that is temporarily configured to perform
the task at a given time or a specific component that is
manufactured to perform the task. As used herein, the term
`processor` refers to one or more devices, circuits, and/or
processing cores configured to process data, such as computer
program instructions.
[0023] A detailed description of one or more embodiments of the
invention is provided below along with accompanying figures that
illustrate the principles of the invention. The invention is
described in connection with such embodiments, but the invention is
not limited to any embodiment. The scope of the invention is
limited only by the claims and the invention encompasses numerous
alternatives, modifications and equivalents. Numerous specific
details are set forth in the following description in order to
provide a thorough understanding of the invention. These details
are provided for the purpose of example and the invention may be
practiced according to the claims without some or all of these
specific details. For the purpose of clarity, technical material
that is known in the technical fields related to the invention has
not been described in detail so that the invention is not
unnecessarily obscured.
[0024] Nanopore membrane devices having pore sizes on the order of
one nanometer in internal diameter have shown promise in rapid
nucleotide sequencing. When a voltage potential is applied across a
nanopore immersed in a conducting fluid, a small ion current
attributed to the conduction of ions across the nanopore can be
observed. The size of the current is sensitive to the pore
size.
[0025] A nanopore based sequencing chip may be used for DNA
sequencing. A nanopore based sequencing chip incorporates a large
number of sensor cells configured as an array. For example, an
array of one million cells may include 1000 rows by 1000 columns of
cells.
[0026] FIG. 1 illustrates an embodiment of a cell 100 in a nanopore
based sequencing chip. A membrane 102 is formed over the surface of
the cell. In some embodiments, membrane 102 is a lipid bilayer. The
bulk electrolyte 114 containing protein nanopore transmembrane
molecular complexes (PNTMC) and the analyte of interest is placed
directly onto the surface of the cell. A single PNTMC 104 is
inserted into membrane 102 by electroporation. The individual
membranes in the array are neither chemically nor electrically
connected to each other. Thus, each cell in the array is an
independent sequencing machine, producing data unique to the single
polymer molecule associated with the PNTMC. PNTMC 104 operates on
the analytes and modulates the ionic current through the otherwise
impermeable bilayer.
[0027] With continued reference to FIG. 1, analog measurement
circuitry 112 is connected to an electrode 110 covered by a thin
film of electrolyte 108. The thin film of electrolyte 108 is
isolated from the bulk electrolyte 114 by the ion-impermeable
membrane 102. PNTMC 104 crosses membrane 102 and provides the only
path for ionic current to flow from the bulk liquid to working
electrode 110. The cell also includes a counter electrode (CE) 116.
The cell also includes a reference electrode 117, which acts as an
electrochemical potential sensor.
[0028] In some embodiments, a nanopore array enables parallel
sequencing using the single molecule nanopore-based sequencing by
synthesis (Nano-SBS) technique. FIG. 2 illustrates an embodiment of
a cell 200 performing nucleotide sequencing with the Nano-SBS
technique. In the Nano-SBS technique, a template 202 to be
sequenced and a primer are introduced to cell 200. To this
template-primer complex, four differently tagged nucleotides 208
are added to the bulk aqueous phase. As the correctly tagged
nucleotide is complexed with the polymerase 204, the tail of the
tag is positioned in the barrel of nanopore 206. The tag held in
the barrel of nanopore 206 generates a unique ionic blockade signal
210, thereby electronically identifying the added base due to the
tags' distinct chemical structures.
[0029] FIG. 3 illustrates an embodiment of a cell about to perform
nucleotide sequencing with pre-loaded tags. A nanopore 301 is
formed in a membrane 302. An enzyme 303 (e.g., a polymerase, such
as a DNA polymerase) is associated with the nanopore. In some
cases, polymerase 303 is covalently attached to nanopore 301.
Polymerase 303 is associated with a nucleic acid molecule 304 to be
sequenced. In some embodiments, the nucleic acid molecule 304 is
circular. In some cases, nucleic acid molecule 304 is linear. In
some embodiments, a nucleic acid primer 305 is hybridized to a
portion of nucleic acid molecule 304. Polymerase 303 catalyzes the
incorporation of nucleotides 306 onto primer 305 using single
stranded nucleic acid molecule 304 as a template. Nucleotides 306
comprise tag species ("tags") 307.
[0030] FIG. 4 illustrates an embodiment of a process 400 for
nucleic acid sequencing with pre-loaded tags. At stage A, a tagged
nucleotide (one of four different types: A, T, G, or C) is not
associated with the polymerase. At stage B, a tagged nucleotide is
associated with the polymerase. At stage C, the polymerase is in
close proximity to the nanopore. The tag is pulled into the
nanopore by an electrical field generated by a voltage applied
across the membrane and/or the nanopore.
[0031] Some of the associated tagged nucleotides are not base
paired with the nucleic acid molecule. These non-paired nucleotides
typically are rejected by the polymerase within a time scale that
is shorter than the time scale for which correctly paired
nucleotides remain associated with the polymerase. Since the
non-paired nucleotides are only transiently associated with the
polymerase, process 400 as shown in FIG. 4 typically does not
proceed beyond stage B.
[0032] Before the polymerase is docked to the nanopore, the
conductance of the nanopore is .about.300 pico Siemens (300 pS). At
stage C, the conductance of the nanopore is about 60 pS, 80 pS, 100
pS, or 120 pS corresponding to one of the four types of tagged
nucleotides. The polymerase undergoes an isomerization and a
transphosphorylation reaction to incorporate the nucleotide into
the growing nucleic acid molecule and release the tag molecule. In
particular, as the tag is held in the nanopore, a unique
conductance signal (e.g., see signal 210 in FIG. 2) is generated
due to the tag's distinct chemical structures, thereby identifying
the added base electronically. Repeating the cycle (i.e., stage A
through E or stage A through F) allows for the sequencing of the
nucleic acid molecule. At stage D, the released tag passes through
the nanopore.
[0033] In some cases, tagged nucleotides that are not incorporated
into the growing nucleic acid molecule will also pass through the
nanopore, as seen in stage F of FIG. 4. The unincorporated
nucleotide can be detected by the nanopore in some instances, but
the method provides a means for distinguishing between an
incorporated nucleotide and an unincorporated nucleotide based at
least in part on the time for which the nucleotide is detected in
the nanopore. Tags bound to unincorporated nucleotides pass through
the nanopore quickly and are detected for a short period of time
(e.g., less than 10 ms), while tags bound to incorporated
nucleotides are loaded into the nanopore and detected for a long
period of time (e.g., at least 10 ms).
[0034] FIG. 5 illustrates an embodiment of a circuitry 500 in a
cell of a nanopore based sequencing chip. As mentioned above, when
the tag is held in nanopore 502, a unique conductance signal (e.g.,
see signal 210 in FIG. 2) is generated due to the tag's distinct
chemical structures, thereby identifying the added base
electronically. The circuitry in FIG. 5 maintains a constant
voltage across nanopore 502 when the current flow is measured. In
particular, the circuitry includes an operational amplifier 504 and
a pass device 506 that maintain a constant voltage equal to V.sub.a
or V.sub.b across nanopore 502. The current flowing through
nanopore 502 is integrated at a capacitor n.sub.cap 508 and
measured by an Analog-to-Digital (ADC) converter 510.
[0035] However, circuitry 500 has a number of drawbacks. One of the
drawbacks is that circuitry 500 only measures unidirectional
current flow. Another drawback is that operational amplifier 504 in
circuitry 500 may introduce a number of performance issues. For
example, the offset voltage and the temperature drift of
operational amplifier 504 may cause the actual voltage applied
across nanopore 502 to vary across different cells. The actual
voltage applied across nanopore 502 may drift by tens of millivolts
above or below the desired value, thereby causing significant
measurement inaccuracies. In addition, the operational amplifier
noise may cause additional detection errors. Another drawback is
that the portions of the circuitry for maintaining a constant
voltage across the nanopore while current flow measurements are
made are area-intensive. For example, operational amplifier 504
occupies significantly more space in a cell than other components.
As the nanopore based sequencing chip is scaled to include more and
more cells, the area occupied by the operational amplifiers may
increase to an unattainable size. Unfortunatly, shrinking the
operational amplifier's size in a nanopore based sequencing chip
with a large-sized array may raise other performance issues. For
example, it may exacerbate the offset and noise problems in the
cells even further.
[0036] FIG. 6 illustrates an embodiment of a circuitry 600 in a
cell of a nanopore based sequencing chip, wherein the voltage
applied across the nanopore can be configured to vary over a time
period during which the nanopore is in a particular detectable
state. One of the possible states of the nanopore is an
open-channel state when a tag-attached polyphosphate is absent from
the barrel of the nanopore. Another four possible states of the
nanopore correspond to the states when the four different types of
tag-attached polyphosphate (A, T, G, or C) are held in the barrel
of the nanopore. Yet another possible state of the nanopore is when
the membrane is ruptured. FIGS. 7A and 7B illustrate additional
embodiments of a circuitry (700 and 701) in a cell of a nanopore
based sequencing chip, wherein the voltage applied across the
nanopore can be configured to vary over a time period during which
the nanopore is in a particular detectable state. In the above
circuits, the operational amplifier is no longer required.
[0037] FIG. 6 shows a nanopore 602 that is inserted into a membrane
612, and nanopore 602 and membrane 612 are situated between a cell
working electrode 614 and a counter electrode 616, such that a
voltage is applied across nanopore 602. Nanopore 602 is also in
contact with a bulk liquid/electrolyte 618. Note that nanopore 602
and membrane 612 are drawn upside down as compared to the nanopore
and membrane in FIG. 1. Hereinafter, a cell is meant to include at
least a membrane, a nanopore, a working cell electrode, and the
associated circuitry. In some embodiments, the counter electrode is
shared between a plurality of cells, and is therefore also referred
to as a common electrode. The common electrode can be configured to
apply a common potential to the bulk liquid in contact with the
nanopores in the measurements cells. The common potential and the
common electrode are common to all of the measurement cells. There
is a working cell electrode within each measurement cell; in
contrast to the common electrode, working cell electrode 614 is
configurable to apply a distinct potential that is independent from
the working cell electrodes in other measurement cells.
[0038] In FIGS. 7A and 7B, instead of showing a nanopore inserted
in a membrane and the liquid surrounding the nanopore, an
electrical model 702 representing the electrical properties of the
nanopore and the membrane and an electrical model 714 representing
the electrical properties of the working electrode are shown. Note
in FIGS. 7A and 7B that the respective circuitry does not require
an extra capacitor (e.g., n.sub.cap 508 in FIG. 5) to be fabricated
on-chip, thereby facilitating the reduction in size of the nanopore
based sequencing chip.
[0039] Electrical model 702 includes a capacitor 706 that models a
capacitance associated with the membrane (C.sub.membrane) and a
resistor 704 that models a resistance associated with the nanopore
in different states (e.g., the open-channel state or the states
corresponding to having different types of tags or molecules inside
the nanopore). Electrical model 714 includes a capacitor 716 that
models a capacitance associated with the working electrode. The
capacitance associated with the working electrode is also referred
to as an electrochemical capacitance (C.sub.electrochemical). The
electrochemical capacitance C.sub.electrochemical associated with
the working electrode includes a double-layer capacitance and may
further include a pseudocapacitance.
[0040] FIG. 7C illustrates a double layer that is formed at any
interface between a conductive electrode and an adjacent liquid
electrolyte. If a voltage is applied, electronic charges (positive
or negative) accumulate in the electrode at the interface between
the conductive electrode and adjacent liquid electrolyte. The
charge in the electrode is balanced by reorientation of dipoles and
accumulation of ions of opposite charge in the electrolyte near the
interface. The accumulation of charges on either side of the
interface between electrode and electrolyte, separated by a small
distance due to the finite size of charged species and solvent
molecules in the electrolyte, acts like a dielectric in a
conventional capacitor. The term "double layer" refers to the
ensemble of electronic and ionic charge distribution in the
vicinity of the interface between the electrode and electrolyte.
FIG. 7D illustrates a pseudocapacitance effect that can be formed,
simultaneously with the formation of a double-layer as in FIG. 7C,
at an interface between a conductive electrode and an adjacent
liquid electrolyte. A pseudocapacitor stores electrical energy
faradaically by electron charge transfer between the electrode and
the electrolyte. This is accomplished through electrosorption,
reduction-oxidation reactions, or intercalation processes.
[0041] FIG. 8 illustrates an embodiment of a process 800 for
analyzing a molecule inside a nanopore, wherein the nanopore is
inserted in a membrane. Process 800 may be performed using the
circuitries shown in FIG. 6, 7A, or 7B. FIG. 9 illustrates an
embodiment of a plot of the voltage applied across the nanopore
versus time when process 800 is performed and repeated three times.
The voltage across the nanopore changes over time. The rate of the
voltage decay (i.e., the steepness of the slope of the voltage
across the nanopore versus time plot) depends on the cell
resistance (e.g., the resistance of resistor 704 in FIG. 7A). More
particularly, as the resistances associated with the nanopore in
different states (e.g., the states corresponding to having
different types of molecules inside the nanopore) are different due
to the molecules' distinct chemical structure, different
corresponding rates of voltage decay may be observed and thus may
be used to identify the molecule in the nanopore.
[0042] FIG. 10 illustrates the plots of the voltage applied across
the nanopore versus time when the nanopore is in different states.
Curve 1002 shows the rate of voltage decay during an open-channel
state. In some embodiments, the resistance associated with the
nanopore in an open-channel state is in the range of 100 Mohm to 20
Gohm. Curves 1004, 1006, 1008, and 1010 show the different rates of
voltage decay corresponding to the four capture states when the
four different types of tag-attached polyphosphate (A, T, G, or C)
are held in the barrel of the nanopore. In some embodiments, the
resistance associated with the nanopore in a capture state is
within the range of 200 Mohm to 40 Gohm. Note that the slope of
each of the plots is distinguishable from each other.
[0043] Allowing the voltage applied across the nanopore to decay
over a time period during which the nanopore is in a particular
detectable state has many advantages. One of the advantages is that
the elimination of the operational amplifier, the pass device, and
the capacitor (e.g., n.sub.cap 508 in FIG. 5) that are otherwise
fabricated on-chip in the cell circuitry significantly reduces the
footprint of a single cell in the nanopore based sequencing chip,
thereby facilitating the scaling of the nanopore based sequencing
chip to include more and more cells (e.g., incorporating millions
of cells in a nanopore based sequencing chip). The capacitance in
parallel with the nanopore includes two portions: the capacitance
associated with the membrane and the capacitance associated with
the integrated chip (IC). Due to the thin nature of the membrane,
the capacitance associated with the membrane alone can suffice to
achieve the required RC time constant without the need for
additional on-chip capacitance, thereby allowing significant
reduction in cell size and chip size.
[0044] Another advantage is that the circuitry of a cell does not
suffer from offset inaccuracies because V.sub.pre is applied
directly to the working electrode without any intervening
circuitry. Another advantage is that since no switches are being
opened or closed during the measurement intervals, the amount of
charge injection is minimized.
[0045] Furthermore, the technique described above operates equally
well using positive voltages or negative voltages. Bidirectional
measurements have been shown to be helpful in characterizing a
molecular complex. For example, they can be used to correct for
baseline drift arising from AC-non-faradaic operation.
[0046] The ratio of the capacitance associated with the membrane
(see C.sub.membrane 706 of FIGS. 7A and 7B) and the capacitance
associated with the working electrode (see C.sub.electrochemical
716 of FIGS. 7A and 7B) may be adjusted to achieve optimal overall
system performance. Increased system performance may be achieved by
reducing C.sub.membrane while maximizing C.sub.electrochemical. For
example, C.sub.membrane is adjusted to achieve the required RC time
constant without the need for additional on-chip capacitance,
thereby allowing a significant reduction in cell size and chip
size. C.sub.electrochemical is maximized such that the impedance
associated with C.sub.electrochemical is close to an AC
(alternating current) short circuit compared with the impedance
associated with C.sub.membrane. C.sub.electrochemical is also
maximized such that the information signal measured by the
circuitries shown in FIG. 6, 7A, or 7B becomes more stable and that
the spurious signal convoluted on top of the information signal is
minimized.
[0047] FIG. 11 illustrates an embodiment of an electrochemical cell
1100 in a nanopore based sequencing chip. In this embodiment, the
ratio of the C.sub.membrane and C.sub.electrochemical may be
adjusted by increasing C.sub.electrochemical, as will be described
in greater detail below.
[0048] Cell 1100 includes a conductive or metal layer 1101. Metal
layer 1101 connects cell 1100 to the remaining portions of the
nanopore based sequencing chip. In some embodiments, metal layer
1101 is the metal 6 layer (M6). Cell 1100 further includes a
dielectric layer 1104 above conductive layer 1101. Dielectric layer
1104 forms the wall surrounding a well 1105 in which a working
electrode 1102 is located at the bottom. Dielectric material used
to form dielectric layer 1104 includes glass, oxide, silicon
mononitride (SiN), silicon dioxide (SiO.sub.2), and the like. In
some embodiments, working electrode 1102 is made of materials that
are resistant to corrosion and oxidation for non-faradaic
conduction. In some embodiments, elcctrode material should be
moderately hydrophilic to facilitate wetting, thereby improving
electrical contact. The top surface of dielectric layer 1104 may be
silanized. Silanization forms a hydrophobic layer 1120 above the
top surface of dielectric layer 1104. Well 1105 formed by the
dielectric layer wall 1104 further includes a film of salt solution
1106 above working electrode 1102. Salt solution 1106 may include
one of the following: lithium chloride (LiCl), sodium chloride
(NaCl), potassium chloride (KCl), lithium glutamate, sodium
glutamate, potassium glutamate, lithium acetate, sodium acetate,
potassium acetate, calcium chloride (CaCl.sub.2), strontium
chloride (SrCl.sub.2), Manganese chloride (MnCl.sub.2), and
magnesium chloride (MgCl.sub.2).
[0049] As shown in FIG. 11, a membrane is formed on top of
dielectric layer 1104 and spans across well 1105. For example, the
membrane includes a lipid monolayer 1118 formed on top of
hydrophobic layer 1120. As the membrane reaches the opening of well
1105, the lipid monolayer transitions to a lipid bilayer 1114 that
spans across the opening of the well. A bulk electrolyte 1108
containing protein nanopore transmembrane molecular complexes
(PNTMC) and the analyte of interest is placed directly above the
well. Bulk electrolyte 1108 may further include one of the
following: lithium chloride (LiCl), sodium chloride (NaCl),
potassium chloride (KCl), lithium glutamate, sodium glutamate,
potassium glutamate, lithium acetate, sodium acetate, potassium
acetate, calcium chloride (CaCl.sub.2), strontium chloride
(SrCl.sub.2), manganese chloride (MnCl.sub.2), and magnesium
chloride (MgCl.sub.2). A single PNTMC/nanopore 1116 is inserted
into lipid bilayer 1114 by electroporation. Nanopore 1116 crosses
lipid bilayer 1114 and provides the only path for ionic flow from
bulk electrolyte 1108 to working electrode 1102.
[0050] Cell 1100 includes a counter electrode (CE) 1110. Cell 1100
also includes a reference electrode 1112, which acts as an
electrochemical potential sensor. In some embodiments, counter
electrode 1110 is shared between a plurality of cells, and is
therefore also referred to as a common electrode. The common
electrode can be configured to apply a common potential to the bulk
liquid in contact with the nanopores in the measurements cells. The
common potential and the common electrode are common to all of the
measurement cells.
[0051] As discussed above, the ratio of C.sub.membrane and
C.sub.electrochemical in cell 1100 may be adjusted by increasing
C.sub.electrochemical. The electrochemical capacitance
(C.sub.electrochemical) associated with working electrode 1102 may
be increased by increasing the thickness of working electrode 1102.
In some embodiments, the thickness of working electrode 1102 ranges
from 10 nanometers to 1 micron.
[0052] C.sub.electrochemical may also be increased by maximizing
the specific surface area of the electrode. The specific surface
area of working electrode 1102 is the total surface area of the
electrode per unit of mass (e.g., m.sup.2/kg), per unit of volume
(e.g., m.sup.2/m.sup.3 or m.sup.-1), or per unit of base area
(e.g., m.sup.2/m.sup.2) As the specific surface area increases, the
electrochemical capacitance (C.sub.electrochemical) increases, and
a greater amount of ions can be displaced with the same applied
potential before the capacitor becomes charged. For example, the
specific surface area of the working electrode may be increased by
making the electrode "spongy."
[0053] Another way to increase C.sub.electrochemical is by
increasing the base surface area of working electrode 1102. For
example, if the working electrode has a cylindrical shape, then the
base surface area of the cylinder may be increased. In another
example, if the working electrode has a rectangular prism shape,
then the base surface area of the rectangular prism may be
increased. However, cell 1100 has a number of drawbacks. Working
electrode 1102 and lipid bilayer 1114 have the same (or similar)
base surface area or cross sectional area. When the base surface
area of working electrode 1102 is increased, the base surface areas
of the opening of well 1105 and lipid bilayer 1114 are both
increased as well. As a result, both C.sub.membrane and
C.sub.electroechemical are increased simultaneously. In other
words, to optimize the overall system performance, C.sub.membrane
cannot be reduced while maximizing C.sub.electrochemical by
adjusting the base area of well 1105 alone. In addition, the
current that can be supported by working electrode 1102 is limited
when the dimensions of cell 1100 are shrunk to allow greater device
density. Typical electrodes can support 1-10 mA/cm.sup.2 of current
density. Given this current density limitation, a nanopore array
with a 1 .mu.m pitch or 100 nm pitch can supply a maximum of 100 pA
current per cell or 1 pA current per cell, respectively. Resolving
signals at such low current levels is challenging because the
signals can be severely distorted by noise.
[0054] In the present application, an electrochemical cell for
nucleic acid sequencing that has a bowl-shaped (can also be lidless
box-shaped, cup-shaped or bucket-shaped) working electrode that can
support an increased current in the cell is disclosed. The
bowl-shaped working electrode has a planar portion at the bottom,
forming the base of the bowl. The bowl-shaped working electrode
further includes a surrounding wall extending perpendicular to (or
at an angle from) the planar portion and along the periphery of the
planar portion. Both the upper surface of the planar portion and
the interior surface of the surrounding wall provide an electrode
surface area that is exposed to the electrolyte 1106. As a result,
the base surface area of the opening to the well (which is the same
as the base surface area of the lipid bilayer) and the surface area
of the working electrode that is exposed to the electrolyte can be
adjusted independently of each other. Therefore, the base surface
area of the opening to the well and the surface area of the
bowl-shaped electrode may be adjusted independently to provide the
desired ratio between C.sub.membrane and C.sub.electrochemical for
optimized cell performance.
[0055] FIG. 12 illustrates an embodiment of an electrochemical cell
1200 for nucleic acid sequencing that has a bowl-shaped or
cup-shaped working electrode that can provide an increased current
in the cell. Cell 1200 is one of the cells in a nanopore based
sequencing chip. Cell 1200 includes a plurality of similar
components as cell 1100, as indicated by identical numerals in
FIGS. 11 and 12.
[0056] One difference between cell 1100 and cell 1200 is the shape
and construction of their respective working electrodes (1102 and
1202). Working electrode 1102 of cell 1100 is a planar electrode
located at the bottom of well 1105. Working electrode 1202 of cell
1200 is a bowl-shaped electrode; it can also be lidless box-shaped,
cup-shaped or bucket-shaped. The bowl-shaped working electrode 1202
has a planar portion 1202A at the bottom, forming the base of the
bowl. The base surface area may be circular or octagonal in shape.
The bowl-shaped working electrode 1202 further includes a
surrounding wall 1202B extending perpendicular to (or at an angle
from) the planar portion and along the periphery of the planar
portion. Both the upper surface of the planar portion 1202A and the
interior surface of the surrounding wall 1202B provide an electrode
surface area that is exposed to the electrolyte 1106. The
surrounding wall 1202B takes advantage of the vertical device real
estate, i.e., the space orthogonal to the substrate plane. The
width (or diameter) of the planar portion 1202A is indicated by
1203A of FIG. 12, and the height of the surrounding wall 1202B is
indicated by 1203B of FIG. 12. In some embodiments, width 1203A is
between 1 to 100 microns, and height 1203B is between 100 nm to 20
microns. In one embodiment, width 1203A is about 5.5 microns and
height 1203B is about 3.5 microns. The ratio between 1203B and
1203A is referred to as the aspect ratio of working electrode 1202.
The aspect ratio may be smaller or greater than one.
[0057] Working electrode 1202 can provide an increased current in
cell 1200 as compared to working electrode 1102 in cell 1100. In
the case of cell 1100, assuming that the nanopore based sequencing
chip has a 100 nm pitch, the working electrode 1102 has a
cylindrical shape, and the current density is 10 mA/cm.sup.2, cell
1100 can generate about 0.8 pA of current. In the case of cell
1200, assuming a 10:1 aspect ratio, a height 1203B of 1 micron, and
a total electrode surface area of 40 times that of electrode 1102,
cell 1200 can generate about 32 pA of current, thereby increasing
the current by a factor of 40 or above. In some embodiments, the
current that can be provided to cell 1200 may be tuned by adjusting
the aspect ratio of working electrode 1202.
[0058] Working electrode 1202 can also provide an increased
C.sub.electrochemical as compared to working electrode 1102 in cell
1100. Both the upper surface of planar portion 1202A and the
interior surface of surrounding wall 1202B of electrode 1202
provide an electrode surface area that is exposed to the
electrolyte 1106, thereby increasing C.sub.electrochemical
associated with working electrode 1202. In some embodiments, the
ratio of the capacitance associated with the membrane (see
C.sub.membrane 706 of FIGS. 7A and 7B) and the capacitance
associated with the working electrode (see C.sub.electrochemical
716 of FIGS. 7A and 7B) may be adjusted to achieve optimal overall
system performance by adjusting the aspect ratio of working
electrode 1202.
[0059] FIG. 13 illustrates an embodiment of a process for
constructing an electrochemical cell of a nanopore based sequencing
chip that includes a bowl-shaped or cup-shaped working electrode
that can provide an increased current in the cell.
[0060] At step A, a layer of dielectric 1304 (e.g., SiO.sub.2 or
Si.sub.3N.sub.4) is disposed on top of a conductive layer 1302
(e.g, M6). The conductive layer includes circuitries that deliver
the signals from the cell to the rest of the chip. For example, the
circuitries deliver signals from the cell to an integrating
capacitor. In some embodiments, the layer of dielectric 1304 has a
thickness of about 400 nm. A photoresist mask 1303 is deposited
above dielectric layer 1304. Photoresist mask 1303 includes a
pattern for etching a well.
[0061] At step B, the layer of dielectric 1304 is etched to create
a well 1306. For example, the well may be etched by chemically
selective reactive-ion etching (RIE). The well 1306 provides a
space for growing a spongy and porous electrode, e.g., a spongy and
porous titanium nitrite (TiN) electrode or platinum electrode.
[0062] At step C, a spongy and porous TiN layer 1308 is deposited
to fill a portion of well 1306 created at step B. TiN layer 1308
further covers the side vertical wall 1304A of dielectric 1304. TiN
layer 1308 also covers the top surface of photoresist mask 1303. In
some embodiments, the spongy and porous TiN layer is grown and
deposited in a manner to create rough, sparsely-spaced TiN columnar
structures or columns of TiN crystals that provide a high specific
surface area that can come in contact with an electrolyte. The
layer of spongy and porous TiN layer can be deposited using
different deposition techniques, including atomic layer deposition,
chemical vapor deposition, physical vapor deposition (PVD)
sputtering deposition, and the like. The TiN layer may also be
deposited by PVD sputtering deposition. For example, titanium can
be reactively sputtered in an N.sub.2 environment or directly
sputtered from a TiN target. The conditions of each of the
deposition methods may be tuned in such a way to deposit
sparsely-spaced TiN columnar structures or columns of TiN crystals.
For example, when the TiN layer is deposited by DC (direct current)
reactive magnetron sputtering from a titanium (Ti) target, the
deposition system can be tuned to use a low temperature, low
substrate bias voltage (the DC voltage between the silicon
substrate and the Ti target), and high pressure (e.g., 25 mT) such
that the TiN can be deposited more slowly and more gently to form
columns of TiN crystals.
[0063] At step D, the photoresist mask 1303 is removed by a
lift-off process. The lift-off process removes the portion of TiN
layer 1308 that covers the top surface of photoresist mask 1303,
but not the portions of TiN layer 1308 in the interior of the well.
The remaining portions of TiN layer 1308 form a bowl-shaped
electrode 1310. At the bottom of well 1306, there is a planar
portion of the bowl-shaped electrode 1310. Coated on the
surrounding wall of well 1306 (which is also the side wall of
dielectric layer 1304) is the surrounding wall portion of the
electrode 1310. The empty space in bowl-shaped electrode 1310 holds
an electrolyte. Well 1306 has an opening 1312. A lipid bilayer can
be formed to span across opening 1312. In some embodiments, the top
surface of dielectric layer 1304 may be silanized. Silanization
forms a hydrophobic layer above the top surface of dielectric layer
1304. The hydrophobic layer provides a surface for facilitating a
membrane to form atop well 1306 and to span across the opening 1312
of the well. In some embodiments, the hydrophobic layer extends
downward partially into well 1306 and covers part of the sidewall
of the well to further facilitate a membrane to form atop the well
and to span across the opening 1312 of the well.
[0064] Electrode 1310 provides a capacitavely-coupled non-faradaic
electrode, as TiN has a high capacitance and large working window
in aqueous solution. However, if charge transfer is required, step
C can be modified to deposit another material, such as platinum,
that is more suitable for faradaic reactions to form the working
electrode.
[0065] FIG. 14 illustrates another embodiment of a process for
constructing an electrochemical cell of a nanopore based sequencing
chip that includes a bowl-shaped or cup-shaped working electrode
that can provide an increased current in the cell.
[0066] At step A, a layer of dielectric 1404 (e.g., SiO.sub.2 or
Si.sub.3N.sub.4) is disposed on top of a conductive layer 1402
(e.g, M6). The conductive layer includes circuitries that deliver
the signals from the cell to the rest of the chip. For example, the
circuitries deliver signals from the cell to an integrating
capacitor. In some embodiments, the layer of dielectric 1404 has a
thickness of about 400 nm. A photoresist mask 1403 is deposited
above dielectric layer 1404. Photoresist mask 1403 includes a
pattern for etching a well.
[0067] At step B, the layer of dielectric 1404 is etched to create
a well 1406. For example, the well may be etched by chemically
selective reactive-ion etching (RIE). The well 1406 provides a
space for growing a spongy and porous electrode, e.g., a spongy and
porous TiN electrode or platinum electrode. Photoresist mask 1403
is removed.
[0068] At step C, a spongy and porous TiN layer 1408 is deposited
to fill a portion of well 1406 created at step B. TiN layer 1408
further covers the vertical wall 1404A of dielectric 1404 and well
1406. TiN layer 1408 also covers the top surface of dielectric
layer 1404. In some embodiments, the spongy and porous TiN layer is
grown and deposited in a manner to create rough, sparsely-spaced
TiN columnar structures or columns of TiN crystals that provide a
high specific surface area that can come in contact with an
electrolyte. The layer of spongy and porous TiN layer can be
deposited using different deposition techniques, including atomic
layer deposition, chemical vapor deposition, physical vapor
deposition (PVD) sputtering deposition, and the like. The TiN layer
may also be deposited by PVD sputtering deposition. For example,
titanium can be reactively sputtered in an N.sub.2 environment or
directly sputtered from a TiN target. The conditions of each of the
deposition methods may be tuned in such a way to deposit
sparsely-spaced TiN columnar structures or columns of TiN crystals.
For example, when the TiN layer is deposited by DC (direct current)
reactive magnetron sputtering from a titanium (Ti) target, the
deposition system can be tuned to use a low temperature, low
substrate bias voltage (the DC voltage between the silicon
substrate and the Ti target), and high pressure (e.g., 25 mT) such
that the TiN can be deposited more slowly and more gently to form
columns of TiN crystals. A layer of sacrifical layer 1409 is
deposited. In some embodiments, sacrifical layer 1409 is a layer of
tungsten or nickel. Sacrifical layer 1409 fills well 1406 and
covers the portion of TiN layer 1408 (1408A) that covers the top
surface of dielectric layer 1404.
[0069] At step D, the excess sacrifical layer 1409 and the excess
TiN layer 1408A are removed, for example by chemical mechanical
polishing (CMP) techniques, to create a coplanar surface. The
remaining bottom TiN portion 1408C and the remaining vertical wall
TiN portion 1408B form a spongy and porous TiN working electrode
1410. The diameter or width of working electrode 1410 is between 20
nm to 100 microns.
[0070] At step E, sacrificial layer 1409 is isotropically etched to
open up well 1406 with an opening 1412. The remaining portions of
TiN layer 1408 (1408B and 1408C) form a bowl-shaped electrode 1410.
Sacrificial layer 1409 is made of a material that is differentially
etchable from dielectric layer 1404 and electrode 1410. In some
embodiments, sacrificial layer 1409 is made of tungsten, and a
selective etchant such as hot H.sub.2O.sub.2 is used as the wet
etchant. The wet etchant selectively etches sacrificial layer 1409
without damaging dielectric layer 1404 and electrode 1410. The
advantage of using a sacrificial layer 1409 is that the etching
process is more robust and the process requires less tuning because
over etching does not damage dielectric layer 1404 or working
electrode 1410.
[0071] At the bottom of well 1406, there is a planar portion of the
bowl-shaped electrode 1410. Coated on the surrounding wall of well
1406 (also the side wall of dielectric layer 1304) is the
surrounding wall portion of the electrode 1410. The empty space in
bowl-shaped electrode 1410 holds an electrolyte. Well 1406 has an
opening 1412. A lipid bilayer can be formed to span across opening
1412.
[0072] FIG. 15 illustrates a top view of a plurality of circular
openings 1502 of a plurality of wells in a nanopore based
sequencing chip. In some embodiments, opening 1502 above the
working electrode is octogonal in shape.
[0073] Although the foregoing embodiments have been described in
some detail for purposes of clarity of understanding, the invention
is not limited to the details provided. There are many alternative
ways of implementing the invention. The disclosed embodiments are
illustrative and not restrictive.
* * * * *