U.S. patent application number 16/525269 was filed with the patent office on 2020-03-26 for monitoring enzymatic process.
The applicant listed for this patent is GENALYTE, INC.. Invention is credited to Lawrence Cary Gunn, III.
Application Number | 20200096451 16/525269 |
Document ID | / |
Family ID | 40756091 |
Filed Date | 2020-03-26 |
United States Patent
Application |
20200096451 |
Kind Code |
A1 |
Gunn, III; Lawrence Cary |
March 26, 2020 |
MONITORING ENZYMATIC PROCESS
Abstract
Techniques, apparatus and systems are described for performing
label-free monitoring of processes. In one aspect, a label-free
monitoring system includes an array of label-free optical sensors
to detect an optical signal in response to synthesis of one or more
target genetic structures. Each label-free optical sensor is
functionalized with a respective target genetic structure. The
system also includes a fluid flow control module that includes
fluid receiving units to provide paths for different fluids to flow
into the fluid flow control module and at least one switch
connected to the fluid receiving units to selectively switch among
the fluid receiving units to receive a select sequence of the
fluids through the fluid receiving units. The select sequence of
the fluids includes at least a dNTP or base. A fluid channel is
connected between the fluid flow control module and the array of
sensors to allow the select sequence of the fluids to flow from the
fluid flow control module to the array of label-free optical
sensors.
Inventors: |
Gunn, III; Lawrence Cary;
(Encinitas, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENALYTE, INC. |
SAN DIEGO |
CA |
US |
|
|
Family ID: |
40756091 |
Appl. No.: |
16/525269 |
Filed: |
July 29, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12746747 |
Nov 8, 2010 |
10365224 |
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PCT/US2008/085988 |
Dec 8, 2008 |
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16525269 |
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61005372 |
Dec 6, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/68 20130101; C12Q
1/6869 20130101; G01N 21/7746 20130101; G01N 33/54373 20130101;
G01N 33/574 20130101; G01N 21/84 20130101 |
International
Class: |
G01N 21/77 20060101
G01N021/77; G01N 21/84 20060101 G01N021/84; C12Q 1/68 20060101
C12Q001/68; G01N 33/543 20060101 G01N033/543; G01N 33/574 20060101
G01N033/574 |
Claims
1-22. (canceled)
23. A method comprising: causing a known sequence of nucleotides to
continuously flow by a label-free optical sensor having a surface
functionalized with an unknown species of nucleic acid; measuring
changes in an output signal of the optical sensor to detect
synthesis reactions between the unknown species of nucleic acid and
the known sequence of nucleotides; and identifying a sequence of
nucleotides in the unknown species of nucleic acid based on the
measured changes in the output signal and the known sequence of
nucleotides.
24. The method of claim 23, further comprising discouraging
comingling of different nucleotides in the known sequence of
nucleotides.
25. The method of claim 24, wherein discouraging comingling of
different nucleotides in the known sequence of nucleotides
comprises providing buffer solutions in between the different
nucleotides.
26. The method of claim 24, wherein discouraging comingling of
different nucleotides in the known sequence of nucleotides
comprises providing air bubbles in between the different
nucleotides.
27. The method of claim 23, further comprising detecting repeated
incorporation of a nucleotide, from the known sequence of
nucleotides, in the unknown species of nucleic acid based on
magnitude of change in the output signal of the optical sensor.
28. The method of claim 27, further comprising detecting a double
incorporation of a nucleotide, from the known sequence of
nucleotides, in the unknown species of nucleic acid based on a
doubled change in the output signal of the optical sensor.
29. A device comprising: a fluid flow control module configured to
cause a known sequence of nucleotides to continuously flow by a
label-free optical sensor having a surface functionalized with an
unknown species of nucleic acid; wherein the device is configured
to measure changes in an output signal of the optical sensor to
detect synthesis reactions between the unknown species of nucleic
acid and the known sequence of nucleotides; and wherein the device
is configured to identify a sequence of nucleotides in the unknown
species of nucleic acid based on the measured changes in the output
signal and the known sequence of nucleotides.
30. The device of claim 29, wherein the fluid flow control module
is configured to discourage comingling of different nucleotides in
the known sequence of nucleotides.
31. The device of claim 30, wherein the fluid flow control module
is configured to provide buffer solutions in between the different
nucleotides in the known sequence of nucleotides.
32. The device of claim 30, wherein the fluid flow control module
is configured to provide air bubbles in between the different
nucleotides in the known sequence of nucleotides.
33. The device of claim 29, wherein the device is configured to
detect repeated incorporation of a nucleotide, from the known
sequence of nucleotides, in the unknown species of nucleic acid
based on magnitude of change in the output signal of the optical
sensor.
34. The device of claim 33, wherein the device is configured to
detect a double incorporation of a nucleotide, from the known
sequence of nucleotides, in the unknown species of nucleic acid
based on a doubled change in the output signal of the optical
sensor.
Description
INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/746,747, filed Nov. 8, 2010, and entitled
"LABEL-FREE OPTICAL SENSORS," which is a 371 National Phase of
International Patent Application PCT/US2008/085988, filed Dec. 8,
2008, and entitled "MONITORING ENZYMATIC PROCESS," which claims
priority to U.S. Provisional Patent Application 61/005,372, filed
Dec. 6, 2007, and entitled "METHOD AND APPARATUS FOR CLOCKED
SYNTHESIS OF GENETIC MATTER." The foregoing applications and any
and all applications for which a foreign or domestic priority claim
is identified in the Application Data Sheet as filed with the
present application are hereby incorporated by reference in their
entireties under 37 CFR 1.57.
BACKGROUND
Field
[0002] This document relates to label-free sensing of chemical and
biological materials and applications of such label-free
sensing.
Description of the Related Art
[0003] Various sequencing techniques use a label to attach to a
molecule and the labeled molecule is monitored and interrogated to
identify which base has been added or removed from a strand of
nucleic acid (NA). Such labeling can be achieved by various
labeling techniques, including molecular labeling based on
radioactivity, fluorescence, and chemiluminescence. However, a
label may cause undesired effects, such as altering the molecular
binding kinetics, interfering with the accuracy of the reaction,
and limiting the length of a contiguous readout, and may require
multiple readouts to construct a high confidence sequence. In
addition, molecular labeling may require numerous processing steps
such as label attachment, washing, label removal, scanning, etc.
and thus could complicate the process, require extended time for
processing and add significant cost.
SUMMARY
[0004] Techniques, apparatus and system are described to provide
label free sensors used to monitor enzymatic processes. Such label
free sensors can be used to detect sequencing of nucleic acid, for
example.
[0005] In one aspect, a label-free enzymatic process monitoring
system includes an array of label-free optical sensors to detect an
optical signal in response to modification of one or more target
genetic structures by addition of a base by synthesis. Each
label-free optical sensor is functionalized with a respective
target genetic structure. The system includes a fluid flow control
module that includes fluid receiving units to provide paths for
different fluids to flow into the fluid flow control module. The
fluid flow control module includes at least one switch connected to
the fluid receiving units to selectively switch among the fluid
receiving units to receive a select sequence of the fluids through
the fluid receiving units. The select sequence the fluids includes
at least a nucleotide base or deoxyribonucleoside 5'-triphosphate
(dNTP). A fluid channel is connected between the fluid flow control
module and the array of label-free sensors to allow the select
sequence of the fluids to flow from the fluid flow control module
to the array of label-free optical sensors.
[0006] Implementations can optionally include one or more of the
following features. The array of label-free optical sensors can
include an optical evanescent field sensor to hold the respective
target genetic structure within an evanescent field. The label-free
optical evanescent field sensor can include a resonant cavity. The
resonant cavity can include a ring resonator cavity. The array of
label-free optical sensors can measure a shift in a resonant
frequency of the resonant cavity. The array of label-free optical
sensors can measure a change in a complex refractive index of the
resonant cavity. The fluid flow control module can provide a single
species of dNTP or nucleotide to the array of label-free optical
sensors. The fluid flow control module can provide a reagent for
modifying the target genetic structure to the array of sensors. The
array of label-free optical sensors can detect the optical signal
while adding the nucleotide base.
[0007] In another aspect, sequencing nucleic acids includes
functionalizing a surface of a label-free optical sensor with
unknown species of nucleic acid. A reagent comprising synthesis
materials and a known nucleotide base is selectively introduced to
the unknown species of nucleic acid. A change in an output signal
of the label-free optical sensor is measured to detect synthesis of
the nucleic acid when a nucleotide base in the unknown species of
nucleic acid reacts with the known dNTP or nucleotide base. A next
nucleotide base in the unknown nucleic acid to react is identified
based on the introduced known dNTP or nucleotide base and the
measured change in the output signal.
[0008] Implementations can include one or more of the following
features. A magnitude of the output signal can be measured to
determine a number of the introduced known nucleotide base
incorporated during the detected synthesis. The label-free optical
sensor that includes an optical resonator can be used to monitor
the synthesis process occurring within an optical field of the
resonator. The unknown species of nucleic acid can be amplified
using a selectively bound primer and hybridization sequences. Solid
phase amplification and hybridization of the unknown species of
nucleic acid can be performed in parallel. An amount of the unknown
species of nucleic acid can be measured based on the output signal
of the optical sensor before and after functionalization. A known
sequence of nucleotide bases can be applied and inadvertently or
non-selectively bound materials can be removed by applying a
washing agent between the nucleotide bases. The surface of the
optical sensor can be functionalized with a single species of
nucleic acid based on the output signal of the optical sensor.
Measuring a change in an output signal of the label-free optical
sensor can include: measuring an output signal of the label-free
optical sensor before introducing the known nucleotide base;
measuring another output signal of the label-free optical sensor
after introducing the known nucleotide base; and identifying a
difference between the measured output signals. The unknown species
of nucleic acid can be held within an evanescent field.
[0009] Yet in another aspect, monitoring an enzymatic process
within an optical field of a label-free optical resonator includes
detecting an optical signal from the label-free optical resonator
in response to an application of one or more enzymes to identify an
enzymatic process that results in a modification of the nucleic
acid. The enzymatic process can include one of the following
reactions: polymerase driven base extension; polymerase repair
activity driven base excision; reverse transcriptase driven DNA
extension; reverse transcriptase driven RNA exonuclease activity;
DNA cleavage driven by site specific endonuclease activity;
annealing driven by ligase enzyme activity and or topoisomerase
action and or recombination enzymes; phosphorylation driven by
kinase; dephosphorylation driven by phosphatase; RNA Splicing
driven by splicing enzymes and or catalytic RNA splicing fragments;
and cleavage through miRNA driven DICER complex.
[0010] Yet in another aspect, a label-free enzymatic process
monitoring system can include an array of label-free optical
sensors to detect an optical signal in response to modification of
one or more target genetic structures. Each label-free optical
sensor holds a respective target genetic structure within an
evanescent field. The system includes a fluid flow control module
to receive one or more fluids comprising a reagent to modify the
one or more target genetic structure. A fluid channel is connected
between the fluid flow control module and the array of label-free
sensors to allow the one or more fluids to flow from the fluid flow
control module to the array of label-free optical sensors.
[0011] The techniques, apparatus and system as described in this
specification can potentially provide one or more of the following
advantages. For example, the amount of target material on a sensor
can be measured to allow accurate calibration of the amount on the
sensor. Also, the system can evaluate when the addition of a base
has run to completion. To speed the synthesis reaction rate, real
time monitoring of the reaction can be performed. In addition, the
real time incorporation of bases in a sequence extension reaction
can be performed based on small sensor size and high
sensitivity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 illustrates a cross-section of an exemplary optical
evanescent field sensor suitable for sequencing applications.
[0013] FIG. 2 illustrates a perspective cross section of another
example of an optical sensor having a ring resonator cavity and a
coupling waveguide, formed on a silicon substrate.
[0014] FIG. 3a illustrates a top down view of another example of an
optical sensor that includes a ring resonator cavity and two
coupling waveguides in evanescent coupling to the ring resonator
cavity.
[0015] FIGS. 3b-3d illustrate examples of non-circular shaped ring
resonant cavities.
[0016] FIG. 4a illustrates a schematic of an example of a synthesis
system with a fluid flow control module and a sensor array based on
label-free sensors.
[0017] FIG. 4b illustrates another example of a synthesis
system.
[0018] FIGS. 5a-d illustrate several options for sequence of the
different solutions which can be applied over the sensors.
[0019] FIG. 6 shows an example process for synthesizing a nucleic
acid.
[0020] FIG. 7 shows an example process for monitoring an enzymatic
process within an optical field of a label-free optical
resonator.
[0021] These fluid options and sequences are for illustrative
purposes, and can be combined in ways that employ one or more of
these approaches in a variety of different orders and
combinations.
DETAILED DESCRIPTION
[0022] Label-free techniques can provide molecular sensing and
detection without using a molecular label. Such label-free
techniques can be used to mitigate certain undesired effects in
molecular labeling. For example, time consuming and potentially
side-effect causing label-related process steps can be eliminated.
In particular, label-free techniques can be used to run a synthesis
reaction at a substantially higher rate than that of a synthesis
reaction based on molecular labeling. As such, the processing time
of a label-free technique may be reduced to a time determined by
the kinetics of the synthesis reaction. For example, a label-free
technique may be used to reduce the time from 10s of minutes per
read in a system based on molecular labeling down to seconds, or
even milliseconds per base call.
[0023] Techniques, systems and apparatus as described in this
specification can be used to provide label-free sensors for
monitoring enzymatic processes, such as synthesis of genetic
material. A resonant cavity with an evanescent field can be used to
sequence an unknown nucleic acid sequence without labels. In one
aspect, genetic material is held within the evanescent field of the
resonant cavity and chemical precursors for the extension of
nucleic acid base pairs are added repetitively in sequence. The
sensor is interrogated synchronously with the addition of each
subsequent nucleic acid based. A change in the resonant cavity
properties that corresponds to the addition of a particular base
indicates incorporation into the synthesis product and indicates
the next corresponding base.
[0024] Examples of label-free techniques, systems and apparatus are
described below for sequencing a nucleic acid. For example, a
label-free sequencing apparatus can include one or more label-free
sensors for sensing a biological and chemical material, a mechanism
for holding a nucleic acid in interaction with a label-free sensor,
a means for controllably introducing a reagent and components for
modification of the nucleic acid, and a label-free means for
interrogating the one or more label-free sensors to obtain output
from the one or more label-free sensors and evaluating whether a
nucleic acid in interaction with a sensor is modified. Such a
label-free sensor may be implemented to achieve a limit of
detection at or below the addition of a single base.
[0025] As a specific example, such a label-free sensor can be
implemented by using an optical sensor that monitors the physical
presence of a base via detection of the optical evanescent wave to
determine synthesis, and does not require a label attached to the
base. In the above exemplary apparatus, a nucleic acid is placed in
the optical evanescent field of a label-free optical sensor. In
another example, a method for sequencing nucleic acids can be
implemented based on one or more label-free optical sensors. In
this method, a nucleic acid species is placed within the range of
an optical evanescent field sensor and a reagent containing
synthesis materials and a known dNTP or base are introduced to the
bound species. The output from the optical evanescent field sensor
is monitored to measure a change and the measured change is used to
determine whether synthesis has occurred. This method also includes
determining the next base in sequence based on knowledge of which
dNTP or base is present at the time a sensor signal from the
optical evanescent field sensor indicates the presence of an
additional bound matter.
[0026] In one implementation of a label-free sequencing apparatus,
an optical sensor is placed on a substrate in such a manner that
the optical sensor can be interrogated while simultaneously
allowing a reaction to occur in the sensing region of the optical
sensor. The optical sensor can be implemented using an evanescent
field sensor. Examples of an evanescent field sensor include:
resonant cavities, Mach-Zehnder interferometers, or other
applicable interferometers with a sensing mechanism that involves a
change in the complex refractive index in the optical path. One
example is a ring resonator, which can be addressed using
waveguides that are routed out of the sensing region.
[0027] An optical ring resonant cavity forms a closed-loop
waveguide. In the optical ring resonant cavity, light propagates in
the form of whispering gallery modes (WGMs) that result from the
total internal reflection of the light along the curved surface of
the ring. The WGM is a surface mode that circulates along the ring
resonator surface and interacts repeatedly with any material (e.g.,
target genetic material) on the surface through the WGM evanescent
field. Unlike a straight waveguide sensor, the effective
light-material interaction length of a ring resonator sensor is no
longer determined by the sensor's physical size, but rather by the
number of revolutions of the light supported by the resonator,
which is characterized by the resonator quality factor, or the
Q-factor. The effective length L.sub.eff is related to the Q-factor
by equation 1 below.
L eff = Q .lamda. 2 .pi. n ( 1 ) ##EQU00001##
Where .lamda. is wavelength and n is the refractive index of the
ring resonator. Due to the large Q-factor, the ring resonant cavity
can provide sensing performance superior to a straight waveguide
sensor while using orders of magnitude less surface area and sample
volume. In addition, the small size of the ring resonator allows an
implementation of a larger number of ring resonant cavities in an
array of sensors.
[0028] An optical sensor on a substrate can be fabricated using a
lithographic technique. Bounding the optical sensor to a substrate
can provide a convenient means to handle the optical sensor and to
fabricate multiple sensors in arrays. In other designs, an optical
sensor may be detached from a substrate and be free floating.
[0029] FIG. 1 illustrates a cross-section of an exemplary optical
evanescent field sensor suitable for sequencing applications. This
sensor includes an optical resonator or an optical interferometric
structure that includes a waveguide 102 formed on a substrate 106
which may be, for example, a silicon substrate. A first, lower
cladding layer 101 with an index less than that of the waveguide
102 is formed on the substrate 106 and is located beneath the
waveguide 102. A second, upper cladding layer 103 is formed over
the waveguide 102 and has an index less than that of the waveguide
102. The upper cladding layer 103 is patterned to have one or more
regions 103A in which the cladding material for the upper cladding
layer 103 is removed to form a sensing region 103A. The sensing
region is structured to either completely expose a section of the
waveguide 102 or to have a thin layer of the cladding material, to
allow a sufficient amount of the optical evanescent field of the
guided light in the waveguide 102 to be present in the sensing
region 103A. A genetic material 104 (e.g., DNA, RNA, LNA, etc.) is
deposited on a surface via a functionalizing process in the sensing
region 103A in proximity to the waveguide 102, in such a manner
that the evanescent field of the waveguide 102 can interact with
the genetic material 104. Cladding regions in the upper cladding
layer 103 are shown to define one exemplary sensing region 103A
that determines which portion of the waveguide 102 is to be
functionalized with the genetic material 104. A flow channel or
fluidic cavity 105 is formed on top of the sensor and a fluidic
control mechanism is provided to direct different solutions into
the flow channel or fluidic cavity 105 during a sequencing process
for synthesizing a target genetic structure, such as a single
species nucleic acid in a sensing region 103A. In addition, the
fluidic control mechanism can direct the solutions into the flow
channel or fluidic cavity 105 for other enzymatic processes.
[0030] FIG. 2 illustrates a perspective cross section of another
example of an optical evanescent field sensor having a ring
resonator cavity 203 and a coupling waveguide 202, formed on a
silicon substrate 106. The waveguides 202 and 203 are displaced
from the substrate via a buried insulator layer 101 as the lower
cladding layer, which may be, for example, silicon dioxide.
Functionalization can occur in proximity to the surface(s) of the
ring resonator cavity 203. In one implementation, similar to the
design in FIG. 1, an upper cladding layer over the ring resonator
cavity 203 can be patterned to form sensing regions in proximity to
the surface of the ring resonator cavity 203 for the synthesis of a
target genetic structure, such as a substantially single species
nucleic acid.
[0031] FIG. 3a illustrates a top down view of another example of an
optical evanescent field sensor that includes a ring resonator
cavity 203 and two coupling waveguides 301 and 302 in evanescent
coupling to the ring resonator cavity 203. An upper cladding layer
103 is formed over the first waveguide 301 and is patterned to
define one or more sensing regions above the first waveguide 301 as
shown in FIG. 1. The cladding layer 103 can be used to confine the
interaction of the genetic material in each sensing region to be
solely to the immediate proximity of the ring 203. The second
waveguide 302 is an optical waveguide and may be used to guiding
light in connection with the evanescent sensing at a sensing region
in the first waveguide 301.
[0032] The ring resonant cavity 203 of FIGS. 2 and 3 can be formed
by a waveguide in a closed loop in various configurations. In FIG.
3a, the ring resonator cavity is a closed waveguide loop of a
circular shape. This circular closed waveguide loop can support one
or more whispering gallery modes along the circular path of the
closed waveguide loop at and around the outer surface of the
circular waveguide and may be independent of the inner surface of
the circular waveguide because the whispering gallery mode exists
at and around the outer surface of the circular waveguide. The
optical input to the ring resonant cavity 203 can be achieved via
evanescent coupling between the waveguide 301 and the ring resonant
cavity 203 which are spaced from each other. In other
implementations, the closed waveguide loop may be in a non-circular
shape that does not support a whispering gallery mode. FIGS. 3b, 3c
and 3d show example shapes of non-circular ring resonant cavities
which operate based on the waveguide modes rather than whispering
gallery modes. A waveguide mode is supported by the waveguide
structure including both the outer and inner surfaces as the
waveguide boundaries and thus is different from a whispering
gallery mode. Each ring resonant cavity is spaced from the
waveguide 201 by a distance d that is selected to provide desired
evanescent coupling. The evanescent coupling configuration is
indicated by the numeral 320. One aspect of such a non-circular
closed waveguide loop forming the ring resonant cavity is to
provide the same evanescent coupling configuration 320 while
providing different closed loop waveguides. FIG. 3b and FIG. 3c
show a ring resonant cavity in an elliptical shape in a waveguide
mode in two different orientations 310 and 320. The specific
geometries of the closed waveguide loop can be selected based on
the need of a specific sensor design. Race-track shaped closed
waveguide loop, for example, may be used. FIG. 3d shows an example
where the closed waveguide loop 340 has an irregular shape that can
be designed to fit on a chip. A ring resonant cavity may be used to
achieve a high Q factor in the ring resonant cavity in part due to
re-circulation of the guided optical signal and such a high Q
factor can be exploited to achieve a high detection sensitivity in
detecting a minute amount of a material on the surface of the ring
resonant cavity in a label-free enzymatic process based on optical
sensing and monitoring.
[0033] FIG. 4a illustrates a schematic of a monitoring system with
a fluid flow control module 420 and a sensor array 409 based on
label-free sensors. The fluid flow control module 420 includes
fluid receiving units, such as ports 402, 403, 404, 405, 406 and
407 to receive various fluid types into the fluid flow control
module. Also, one or more switches 401 are provided in the fluid
flow control module to selectively switch-in or receive one or more
of the fluid types into the fluid flow control module. The sensor
array 409 includes a matrix of label-free sensors 411 arranged in
various configurations. For example, the label-free sensors 411 can
be arranged in a square or rectangular configuration with N number
of rows and M number of columns of sensors. The label-free sensors
411 can be arranged in other configurations, such as a circle or a
triangle. The label-free sensors 411 may be optical sensors based
on the sensor examples in FIGS. 1-3b and other sensor designs.
[0034] The fluid flow control module 420 is connected to the sensor
array 409 using a flow channel 408. Solutions in the fluid flow
control module 420 can flow through the flow channel 408 and arrive
at the sensor array 409. Different solutions can be obtained in the
fluid flow control module 420 by receiving the various fluid types
by using the switch 401, and mixing the received fluids. For
example, a mix of the various nucleic acids and the associated
synthesis compounds can be added through ports 402-405. In
addition, various washing and cleaning solutions, such as buffers
can be switched in through ports 406 and 407. The amount and type
of fluids to receive and mix in the fluid flow control module 420
can be controlled using the one or more of the switches 401. After
the fluids are combined and mixed in a junction region in the fluid
flow control module 420, the resultant solution can be applied
through the fluid channel 408 and over the sensor array 409. In
this configuration, each label-free sensor could have a different
unknown sequence attached.
[0035] The solution from the fluid flow control module 420 flows
over the sensor array 409 and exits the system through the fluid
exit 410. Thus, a continuous flow of solutions can be provided
across the sensor array 409. In some implementations, the solution
can be held static in the sensor array 409 by stopping the
flow.
[0036] FIG. 4b shows another example of a monitoring system with a
fluid flow control module 420 and a sensor array 409 based on
label-free sensors. Each of the fluid input units 402, 403, 404,
405, 406 and 407 is connected to a respective switch 401. To
selectively input a fluid type through one of the fluid input units
402, 403, 404, 405, 406 and 407, the respective switch is used.
Remaining components of the monitoring system are similar to the
system shown in FIG. 4a.
[0037] In the label-free sensors of the sensor array 409, the
sensor surface can be functionalized to have a target genetic
structure, such as a nucleic acid sequence held within an optical
mode, for example by attachment to the sensor surface.
Functionalizing the sensor surface can be accomplished by various
surface chemistry techniques. A single strand or a small number of
strands can be attached to the sensor surface. Once the sensor
surface is functionalized with a strand or strands of the target
nuclei acid sequence, solid phase synthesis can be performed.
[0038] In some implementations, the monitoring system of FIGS. 4a
and 4b can be used to functionalize the sensor surface by attaching
the species of the target nucleic acid sequence in large numbers.
To achieve large numbers of the species, the desired species can be
purified and amplified, as needed. The NA can be covalently linked,
hybridized to a template, or held by binding to a protein. The NA
can further be held directly on the surface, or held in a porous
film, such as a gel, hydrogel or sol-gel.
[0039] For solid phase synthesis, the monitoring system of FIGS.
4a-b can be used to amplify the target sequence only in the active
sensing region of the sensor. To amplify the target sequence only,
selectively bound primer and hybridization sequences can be used.
This can be achieved using synthesis in situ, photopatterning, or
masking techniques for example. Photopatterning of the primer could
be achieved using ultraviolet (UV) sensitive binding chemistry. The
desired selectivity could be achieved by using a mask layer formed
out of a material that does not allow surface binding, such as a
Teflon based material. This material itself can be patterned using
lithographic approaches and other techniques.
[0040] The monitoring systems of FIGS. 4a-b can be used in
applications where the target sequence is bound in sufficiently
large numbers, and an active surface is provided for binding only
in the region of the sensor. This surface treatment can be more
generic and may not need to contain primers or specific
hybridization sequences. However, for some applications, providing
a hybridization sequence for a known portion of a target molecule
may be advantageous.
[0041] For example, in the case of a single nucleotide polymorphism
assay, most of the target sequence is known, and a hybridization
probe could be designed to pull down the particular piece of
nucleic acid of interest. Then, sequencing can take place on the
unknown region to expose additions, deletions, substitutions, and
other mutations of interest.
[0042] The monitoring system of FIGS. 4a-b can be used to provide a
combination of multi-strand attachment and an amplification process
for the multi-strand attachment. For example, hybridization of a
sample could be obtained using known probe sequences, and then
solid phase amplification could be performed to boost the
numbers.
[0043] In some implementations, the solid phase amplification could
be performed in solution, perhaps in real time, while hybridization
is occurring on the sensors. For example, the sensors in the
synthesis system can be placed in a solution undergoing standard
Polymerase Chain Reaction (PCR) that performs amplification.
[0044] By using these techniques for target NA attachment to the
sensor, the amount of target material can be measured. This allows
accurate calibration of the target material amount on the sensors.
Such accurate calibration of the target material amount allows
normalization of the subsequent synthesis reactions, and allows a
user to determine when an appropriate quantity of target sequence
has been accumulated to proceed with synthesis. This can be
observed in real time by making a rapid succession of measurements
during the target NA attachment and/or amplification process. Also,
measurements can be made at significant points in the process, such
as between PCR cycles. With knowledge of the sensor response prior
to attachment and during/after attachment, the amount of target
material attached can be determined.
[0045] In the monitoring systems of FIGS. 4a-b, any inadvertently
or non-selectively bound materials can be removed from the sensor
surface. The removal can be achieved by any of a number of
well-known techniques, such as washing and modification of the
astringency of the sensor, for example but not limited to heating
and/or changing the salt concentration or pH of the ambient
solution.
[0046] Real-time examination of the sensors can provide information
regarding the progress of the removal process. In addition,
feedback information can be provided to determine when to stop
washing, or when to stop heating the target material. For example,
the ambient solution can be heated until the non-selectively bound
material has been melted off the sensors. However, the temperature
is kept below a point where the target sequence would be completely
removed. By watching the rate at which the non-desired material is
removed, an assessment of which temperature to use, and when to
stop heating could be accomplished. A similar approach could be
used to regulate the number of wash cycles.
[0047] The monitoring systems of FIGS. 4a-b can be used in
applications where a large number of different target species are
present in the same analyte. The system can be used to control the
surface attachment conditions to deposit only one species on each
sensor. For example, the sensor array 409 can be designed to
include multiple sensors with each sensor masked in such a manner
to provide surface attachment on the sensor. The concentration,
reaction time, temperature, etc., can be modified to statistically
allow only a single target molecule to deposit within each sensor
region. Subsequent solid phase amplification can increase the
number of target molecules up to an appropriate level for
observation of synthesis.
[0048] The potential issues with this single species attachment
approach include the possibilities that more than one species is
deposited on a single sensor, or no deposition occurs on a
particular sensor. Both of the non-single species cases can be
screened for during synthesis. For example, when more than one
species attach to a single sensor, an irregular sensor response is
obtained because only a fractional proportion of sites is available
for the addition of a particular base. This reduction of available
sites can result in a fractional sensor response in comparison to a
uniformly hybridized sensor. Sensors with no target molecules
provides little to no response. In such manners, a good sensor
(single species attachment) can be distinguished from a bad sensor
(multiple of no species attachment) during the course of the
synthesis process.
[0049] After target molecule attachment, the remainder of active
surface binding sites can be removed or blocked to prevent the
accumulation of non-selectively bound material. Because the
non-selectively bound material can inhibit the accuracy of
measurement, blocking the remaining active surface binding sites
can provide a more accurate result.
[0050] The monitoring systems of FIGS. 4a-b can be used to perform
NA synthesis using a number of techniques. For example, a
polymerase and its associated buffer solutions can be used to
perform the synthesis. When the sensors of the synthesis system
include an evanescent field sensor, the polymerase and buffer
solutions can cause a measurable offset to the sensor response.
This offset can be addressed in a number of ways. The synthesis can
be performed in conditions that encourage a steady state polymerase
attachment condition. The steady state polymerase attachment
results in a steady offset, and the desired synthesis signal is the
delta off of this baseline offset. The baseline offset may change
as the synthesis processes.
[0051] Also, the polymerase can be driven off of the target
sequence to perform the read, and then reattached when proceeding
to subsequent base addition. When the polymerase is detached, the
polymerase can remain in the ambient solution or washed away from
the sensor region and then subsequently replaced. For a single
synthesis step, such as that needed in a single nucleotide
polymorphism (SNP) reaction, removing the polymerase is not
critical because subsequent reattachment is not necessarily
required. When large numbers of bases are to be synthesized, then
it is advantageous to keep the polymerase attached, if
possible.
[0052] The monitoring systems of FIGS. 4a-b can be used to detect a
genetic variation called Single Nucleotide Polymorphisms (SNPs).
SNPs are commonly determined through use of hybridization arrays
containing each of the 4 possible variants as part of a 25-mer
strand of DNA. To detect SNPs, appropriately prepared DNA is
exposed to the hybridization arrays. An array element with the
strongest binding indicates the SNP type present. In the monitoring
systems of FIGS. 4a-b, a hybridization array is prepared on a
label-free sensor, where the hybridization sequence is designed to
bind to the DNA proximal to the SNP location, but leaving the SNP
base exposed for sequencing by synthesis. The hybridization array
can be designed on the label-free sensor such that the SNP is the
next base in sequence. Also, the hybridization array can be
designed on the label-free sensor such that the SNP is a known
number of bases from the termination of the hybridization sequence.
In either case, sequencing by synthesis is performed as described
above, and the identity of the SNP can be determined based on
sensor response.
[0053] The monitoring systems of FIGS. 4a-b can be implemented to
allow different bases to flow sequentially over the sensors. The
monitoring systems of FIGS. 4a-b can be designed to pump different
bases in sequence over the sensors. The flow can be continuous, or
it can be stopped once the desired mixture is over the sensor
region. Between the different solutions containing the different
bases, a number of other functional solutions can be added.
[0054] FIGS. 5a-d illustrate examples of a sequence of different
solutions which can be applied over the sensors in a sensor array.
FIG. 5a shows a basic configuration of a synthesis mixture that
allows addition of dNTPs or NA bases in sequential order. In this
example, the dATP or base A is applied over the sensors (e.g.,
sensors in the sensor array 409 of the monitoring systems) and a
measurement is made. After the bases or dATP, dCTP, dTTP, dGTP,
etc. are sequentially applied over the sensors. The synthesis
enzyme (such as polymerase) may be optionally added with each dNTP
or base. The synthesis enzyme can be added prior to the dNTP or
base if the synthesis enzyme maintains a steady or otherwise
predictable behavior. Also, the synthesis enzyme can be included
with all solutions to guarantee the presence of the enzyme.
[0055] The sequence of solution in FIG. 5b shows a buffer solution,
B, applied between the different dNTPs or bases. For example, the
sensors can be washed between dNTPs or bases. A washing solution,
such as a buffer, B, can be added to the sequence of reagents
applied over the sensors, potentially between each successive dNTP
or base nucleotide solution. The buffer solution, B, is applied to
ensure that the previous dNTP or base has been swept or washed from
the reaction region prior to addition of the next dNTP or base. For
example, the buffer solution is applied between application of dATP
or base A and buffer B to prevent or reduce commingling of dATP and
B (or bases A and B). This ensures that each base addition is
separated by time and solution and thus can be isolated. The buffer
solution can also be used to remove non-selective binding.
[0056] In addition, non-mixing regions can be added in the
synthesis system to prevent different base solutions from
intermixing. This could be accomplished by applying a bubble of air
or other non-mixing fluid injected in series with the reagents.
Also, a sufficiently large amount of the new type of dNTP or base
solution can be applied to guarantee removal of the previous dNTP
or base solution.
[0057] Even small amounts of the previous base solution remaining
in the sensor can become an issue, despite the fact that the
previous synthesis step should have been fully reacted. If the new
dNTP or base reacts, it is possible that the next subsequent dNTP
or base will be next in line to react, and thus, a small number of
strands will have skipped ahead one base. This anomaly can be
minimized by thorough removal of the old base solution (e.g., by
using the washing solution) prior to introduction of the next
one.
[0058] The sequence of solutions in FIG. 5c shows adding and
removing a synthesis enzyme prior to each base inquiry. For
example, a synthesis enzyme, such as polymerase, P, can be added
prior to each dNTP or base. The added polymerase, P, is removed
before the next dNTP or base is added using a solution that
encourages the disassociation of the polymerase with the target
strand, RP. Also, after removing the polymerase, another polymerase
is added before the next dNTP or base. For example, FIG. 5c shows
the addition of polymerase, P, before the addition of dATP or base
A. Then, after adding the dATP or base A and before adding the next
base, dCTP, the added polymerase, P, is removed using RP. Then
another polymerase, P, is added before the dCTP or base. This
adding and removing of the polymerase is repeated before addition
of each dNTP or base.
[0059] The sequence of fluids in FIG. 5d shows a polymerase and
associated dNTP or base that are added to obtain a synthesis
reaction (A+P). The added polymerase is removed using a solution
that disassociation of the polymerase with the target strand, RP.
Also, a known buffer that facilitates more accurate measurement, M,
of the sensor is added. The addition and removal of the polymerase
and the addition of the buffer for measurement are repeated before
addition of each dNTP or base.
[0060] In some implementations, these fluid sequence options of
FIGS. 5a-d are combined in ways that employ one or more of these
approaches in a variety of different orders and combinations.
[0061] Also, a particular dNTP or base can bind a number of times
in a row during a particular sequence. For example, the sequence of
A, A-A or A-A-A can react. Such repeated incorporation by a single
dNTP or base can be determined by measuring the amplitude of the
sensor response. If the complete incorporation of a single base
results in a certain response, the complete incorporation of two
bases will approximately double the response and three bases will
approximately triple the response, etc. Thus by knowing the
magnitude of the response quantitatively, multiple additions of a
single base type can be determined.
[0062] Also, bases that terminate the reaction after the addition
of a single nucleotide can be used. However, the use of terminating
bases needs additional chemistry to process a series of bases in
order to allow the reaction to proceed.
[0063] In addition, the monitoring systems of FIGS. 4a-b can be
used to evaluate when the addition of a dNTP or base has run to
completion. Because it is advantageous to speed up the synthesis
reaction rate, real time monitoring of the reaction can be
performed. When it is determined that the reaction is complete, or
that no reaction is going to occur, the process of introducing the
next dNTP or base can be started.
[0064] As more and more bases are added to the synthesis product,
the location for the additional base may be moving either closer or
further from the sensor surface, depending on what primer
configuration is employed and how the target sequence is attached
to the sensors. For an evanescent field sensor of the monitoring
systems shown in FIGS. 4a-b, a non-uniform response can be obtained
as a function of proximity to the sensor surface. Thus, the sensor
signal can be corrected for this change. The expected signal
baseline response can be adjusted as a function of time because the
response of the signal has been calibrated for the known location
of polymerization reaction or because the signal response is being
fit to the known field decay.
[0065] This approach is applicable for DNA, RNA or any type of
nucleic acid complex which can by sequentially synthesized. In the
case of RNA, a conversion to cDNA might be needed prior to any
amplification process, but is not absolutely necessary.
[0066] This approach can be scaled to high degrees of parallelism
by incorporating a large number of sensors and/or scanning systems.
For example, many sensors can be employed on a single chip of a
synthesis system. Also, many chips can be implemented in a
synthesis system to achieve scaling.
[0067] In some implementations, the monitoring systems of FIGS.
4a-b can be implemented to include a microfluidic switching
manifold in immediate proximity to the sensor(s) to allow more
rapid fluidic switching times. Such a microfluidic switching
manifold can accelerate the sequencing process.
[0068] In some implementations, monitoring systems of FIGS. 4a-b
can be used to monitor an enzymatic process, other than synthesis
described above, within an optical field of an optical resonator.
An enzymatic process can result in a modification of the nucleic
acid when one or more enzymes are applied. Examples of the
enzymatic process can include one or the following reactions: (1)
polymerase driven base extension; (2) polymerase repair activity
driven base excision; (3) reverse transcriptase driven DNA
extension; (4) reverse transcriptase driven RNA exonuclease
activity; (5) DNA cleavage driven by site specific endonuclease
activity; (6) annealing driven by ligase enzyme activity and or
topoisomerase action and or recombination enzymes; (7)
phosphorylation driven by kinase; (8) dephosphorylation driven by
phosphatase; (9) RNA Splicing driven by splicing enzymes and or
catalytic RNA splicing fragments; and (10) cleavage through miRNA
driven DICER complex.
[0069] FIG. 6 shows an example process for synthesizing a nucleic
acid. Label-free optical sensors are functionalized with a single
species of an unknown nucleic acid (602). A reagent comprising
synthesis materials and a known dNTP or base is selectively
introduced to the unknown species of nucleic acid (604). A change
in an output signal of the label-free optical sensor is measured to
detect synthesis of the nucleic acid when a nucleotide base in the
unknown species of nucleic acid matches with the known dNTP base
(606). A next nucleotide base in the unknown nucleic acid to react
is identified based on the introduced known dNTP or base and the
measured change in the output signal (608). This process can be
repeated by applying a sequence of dNTPs or base as shown in FIGS.
5a-d.
[0070] Also, a magnitude of the output signal can be measured to
determine a number of the introduced known dNTP or base
incorporated during the detected synthesis. The unknown species of
nucleic acid can be amplified using a selectively bound primer and
hybridization sequences. Solid phase amplification and
hybridization of the unknown species of nucleic acid can be
performed in parallel. An amount of the unknown species of nucleic
acid can be measured based on the output signal of the optical
sensor before and after functionalization. Not just one but a
sequence of known dNTPs or bases can be applied and inadvertently
or non-selectively bound dNTPs or bases can be removed by applying
a washing agent between the dNTPs or bases. The surface of the
optical sensor can be functionalized with a single species of
nucleic acid based on the output signal of the optical sensor.
Further, measuring a change in an output signal of the label-free
optical sensor can include: measuring an output signal of the
label-free optical sensor before introducing the known dNTP or
base; measuring another output signal of the label-free optical
sensor after introducing the known dNTP or base; and identifying a
difference between the measured output signals. The unknown species
of nucleic acid can be held within an evanescent field.
[0071] FIG. 7 shows an example process for monitoring an enzymatic
process within an optical field of a label-free optical resonator.
Label-free optical sensors can be used to hold a single species
nucleic acid within an evanescent field (702). A reagent for
modifying the single species nucleic acid is applied to the single
species of nucleic acid (704). An optical signal from the
label-free optical resonator is measured in response to the applied
reagent to identify an enzymatic process that results in a
modification of the nucleic acid (706). The enzymatic process can
include one of the following reactions: polymerase driven base
extension; polymerase repair activity driven base excision; reverse
transcriptase driven DNA extension; reverse transcriptase driven
RNA exonuclease activity; DNA cleavage driven by site specific
endonuclease activity; annealing driven by ligase enzyme activity
and or topoisomerase action and or recombination enzymes;
phosphorylation driven by kinase; dephosphorylation driven by
phosphatase; RNA Splicing driven by splicing enzymes and or
catalytic RNA splicing fragments; and cleavage through miRNA driven
DICER complex.
[0072] In some implementations, one or more evanescent wave sensors
includes multiple nucleic acid bound to the surface of the sensors.
The sensors can include a means for introducing a reagent
containing all necessary components for synthesis and a base of
choice. The sensors can include a means for interrogating the
sensors and evaluating if matter is bound.
[0073] A method for sequencing can include binding multiple known
species of nucleic acid within the range of an evanescent field
sensor. A reagent containing synthesis materials and a known base
are introduced to the bound species. The change in output from the
evanescent field sensor is observed to determine if synthesis has
occurred. The next base in sequence is determined based on
knowledge of which base was present at the time the sensor signal
indicates the presence of additional bound material. The sensor can
be a resonant cavity. The resonant cavity can be a ring resonator.
The ring resonator can be made primarily of silicon and the ring is
disposed on a silicon-on-insulator wafer.
[0074] One or more evanescent wave sensors can include a plurality
of a substantially single species nucleic acid held within the
evanescent field, a means for controllably introducing a reagent
and components for modification of the nucleic acid, and a
label-free means for interrogating the sensor and evaluating if the
nucleic acid is modified. One or more evanescent wave sensors can
include a plurality of a substantially single species nucleic acid
bound to the surface, a means for introducing a reagent containing
all necessary components for synthesis, and a base of choice, and a
label-free means for interrogating the sensor and evaluating if
matter is bound.
[0075] A method for sequencing nucleic acids can include holding a
plurality of an unknown species of nucleic acid within the range of
an evanescent field sensor, introducing a reagent containing
synthesis materials and a known base to the bound species,
observing the change in output from the evanescent field sensor to
determine if synthesis has occurred, and determining the next base
in sequence based on knowledge of which base was present at the
time the sensor signal indicates the present of additional bound
matter.
[0076] An optical resonator can be used to monitor an enzymatic
process occurring within the optical field of said resonator. The
enzymatic process can result in a modification of the nucleic acid,
such as: polymerase driven base extension, polymerase repair
activity driven base excision, reverse transcriptase driven DNA
extension, reverse transcriptase driven RNA exonuclease activity,
DNA cleavage driven by site specific endonuclease activity,
annealing driven by ligase enzyme activity and or topoisomerase
action and or recombination enzymes, phosphorylation, driven by
kinase, dephosphorylation driven by phosphatase, RNA Splicing
driven by splicing enzymes and/or catalytic RNA splicing fragments,
or cleavage through miRNA driven DICER complex.
[0077] While this specification contains many specifics, these
should not be construed as limitations on the scope of an invention
or of what may be claimed, but rather as descriptions of features
specific to particular embodiments of the invention. Certain
features that are described in this specification in the context of
separate embodiments can also be implemented in combination in a
single embodiment. Conversely, various features that are described
in the context of a single embodiment can also be implemented in
multiple embodiments separately or in any suitable subcombination.
Moreover, although features may be described above as acting in
certain combinations and even initially claimed as such, one or
more features from a claimed combination can in some cases be
excised from the combination, and the claimed combination may be
directed to a subcombination or a variation of a
subcombination.
[0078] Only a few implementations are disclosed. Variations and
enhancements of the described implementations and other
implementations may be made based what is described and
illustrated.
* * * * *