U.S. patent application number 10/434897 was filed with the patent office on 2003-11-20 for stochastic sensing through covalent interactions.
Invention is credited to Bayley, Hagan, Cheley, Stephen, Luchian, Tudor, Shin, Seong-Ho.
Application Number | 20030215881 10/434897 |
Document ID | / |
Family ID | 29423678 |
Filed Date | 2003-11-20 |
United States Patent
Application |
20030215881 |
Kind Code |
A1 |
Bayley, Hagan ; et
al. |
November 20, 2003 |
Stochastic sensing through covalent interactions
Abstract
A system and method for stochastic sensing in which the analyte
covalently bonds to the sensor element or an adaptor element. If
such bonding is irreversible, the bond may be broken by a chemical
reagent. The sensor element may be a protein, such as the
engineered P.sub.SH type or .alpha.HL protein pore. The analyte may
be any reactive analyte, including chemical weapons, environmental
toxins and pharmaceuticals. The analyte covalently bonds to the
sensor element to produce a detectable signal. Possible signals
include change in electrical current, change in force, and change
in fluorescence. Detection of the signal allows identification of
the analyte and determination of its concentration in a sample
solution. Multiple analytes present in the same solution may be
detected.
Inventors: |
Bayley, Hagan; (College
Station, TX) ; Shin, Seong-Ho; (College Station,
TX) ; Luchian, Tudor; (College Station, TX) ;
Cheley, Stephen; (Bryan, TX) |
Correspondence
Address: |
BAKER BOTTS L.L.P.
2001 ROSS AVENUE
SUITE 600
DALLAS
TX
75201-2980
US
|
Family ID: |
29423678 |
Appl. No.: |
10/434897 |
Filed: |
May 9, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60379527 |
May 10, 2002 |
|
|
|
60450930 |
Feb 28, 2003 |
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Current U.S.
Class: |
435/7.1 ;
205/777.5; 435/287.2 |
Current CPC
Class: |
C12Q 1/001 20130101;
G01N 33/5438 20130101; G01N 33/5308 20130101; G01N 33/6872
20130101 |
Class at
Publication: |
435/7.1 ;
435/287.2; 205/777.5 |
International
Class: |
G01N 033/53; C12M
001/34; G01F 001/64; G01N 027/26; G01N 033/50 |
Claims
What is claimed is:
1. A system for sensing at least one reactive analyte in a solution
comprising: a sensing device separated into a trans chamber and a
cis chamber by a divider; a protein pore operably disposed in the
divider; a detection system operable to detect current between the
cis and trans chambers; and an ionic solution containing at least
one reactive analyte capable of covalently bonding to the protein
pore; wherein bonding of the reactive analyte to the protein pore
produces a change in current between the cis and trans chambers
detectable by the current detection system.
2. The system of claim 1, wherein the protein pore is an engineered
protein pore.
3. The system of claim 1, wherein the protein pore is an .alpha.HL
pore.
4. The system of claim 3, wherein the .alpha.HL pore comprises at
least one monomer having a Cys residue at amino acid 117.
5. The system of claim 1, wherein the ionic solution is a pH
buffered KCl solution.
6. The system of claim 1, wherein the reactive analyte is an
environmental toxin.
7. The system of claim 1, wherein the reactive analyte is a
chemical weapon.
8. The system of claim 1, wherein the reactive analyte is a
pharmaceutical.
9. The system of claim 1, wherein the reactive analyte comprises an
arsenical.
10. The system of claim 1, wherein the reactive analyte covalently
bonds to the protein pore within the lumen of the pore.
11. The system of claim 1, wherein the reactive analyte
irreversibly covalently bonds to the protein pore, further
comprising an ionic solution containing a chemical reagent capable
of breaking the covalent bond.
12. The system of claim 11, wherein the reactive analyte and
chemical reagent are substantially disposed in separate
chambers.
13. The system of claim 1, wherein the ionic solution contains a
plurality of species of chemically distinct reactive analytes
capable of covalently bonding with the protein pore.
14. The system of claim 13, wherein bonding of at least one species
of the reactive analytes to the protein pore produces a first
change in current between the cis and trans chambers and the
bonding of at least a second species of the reactive analytes to
the protein pore produces a second change in current between the
cis and trans chambers, wherein the first and second changes in
current are distinctly detectable by the detection system.
15. A system for sensing at least one reactive analyte in a sample
comprising: a sensor element; and a sample containing at least a
first reactive analyte, wherein the reactive analyte covalently
bonds to the sensor element.
16. The system of claim 15, wherein sensing comprises stochastic
sensing.
17. The system of claim 15, wherein the sensor element produces a
detectable signal when covalently bound to the reactive
analyte.
18. The system of claim 15, wherein the sensor element is a
protein.
19. The system of claim 18, wherein the sensor element is a protein
pore.
20. The system of claim 15, wherein the analyte directly covalently
bonds to the sensor element.
21. The system of claim 15, wherein the analyte covalently bonds to
an adaptor molecule that bonds to the sensor element.
22. The system of claim 15, wherein the reactive analyte is
selected from the group consisting of: chemical weapons,
environmental toxins and pharmaceuticals.
23. The system of claim 15 wherein the reactive analyte
irreversibly covalently bonds to the sensor element, further
comprising a chemical reagent capable of breaking the covalent
bond.
24. The system of claim 15, further comprising at least a second
reactive analyte, wherein the second reactive analyte covalently
bonds to the sensor element.
25. The system of claim 15, further comprising a detection system
operable to detect a signal produced by covalent bonding of the
reactive analyte to the sensor element.
26. The system of claim 25, wherein the signal is selected from the
group consisting of: a change in electrical current, a change in
force, and a change in fluorescence.
27. The system of claim 25, wherein the signal comprises a change
in the magnitude of an electrical current.
28. The system of claim 25, further comprising: at least a second
reactive analyte, wherein the second reactive analyte covalently
bonds to the sensor element; and a detection system operable to
detect a signal produced by covalent bonding of of a reactive
analyte to the sensor element, wherein the detection system is
capable of distinctly detecting the signals produced by covalent
bonding of at least the first and second reactive analytes to the
sensor element.
29. The system of claim 15, further comprising a plurality of
sensor elements.
30. The system of claim 25, wherein the first and second reactive
analytes covalently bond to two different binding sites on the
sensor element.
31. A method for sensing at least one analyte in a solution
comprising: providing a sensing device separated into a trans
chamber and a cis chamber by a divider, wherein a protein pore is
operably disposed in the divider; providing a detection system
operable to detect current between the cis and trans chambers;
providing an ionic solution containing at least one reactive
analyte capable of covalently bonding to the protein pore to the
cis or trans chamber; and detecting the current between the cis and
trans chambers.
32. The method of claim 31, wherein the pore is an engineered
protein pore.
33. The method of claim 31, wherein the pore is an .alpha.HL pore
comprising at least one monomer having a Cys residue at amino acid
117.
34. The method of claim 31, wherein the reactive analyte is
selected from the group consisting of: chemical weapons,
environmental toxins and pharmaceuticals.
35. The method of claim 31, wherein the reactive analyte comprises
an arsenical.
36. The method of claim 31, wherein the reactive analyte covalently
bonds to the protein pore within the lumen of the pore.
37. The method of claim 31, wherein the reactive analyte
irreversibly covalently bonds to the protein pore, further
comprising providing to the cis or trans chamber an ionic solution
containing a chemical reagent capable of breaking the covalent
bond.
38. The method of claim 37, wherein the reactive analyte and
chemical reagent are provided to separate chambers.
39. The method of claim 31, wherein the ionic solution comprises a
plurality of species of chemically distinct reactive analytes
capable of covalently bonding with the protein pore to produce
distinct current signals.
40. The method of claim 39, further comprising analyzing the
detected conductance between the cis and trans chambers for the
distinct current signals.
41. The method of claim 31, further comprising determining the
identity and concentration of at least one reactive analyte based
upon the current between the cis and trans chambers.
42. The method of claim 31, wherein the reactive analyte
irreversibly covalently bonds to the protein pore, further
comprising a chemical capable of reacting with the analyte to
produce a second signal.
43. A method for sensing at least one reactive analyte in a sample
comprising: providing a sensor element; providing a sample
containing at least a first reactive analyte capable of covalently
bonding to the sensor element; allowing the reactive analyte to
covalently bond to the sensor element to produce a first signal;
and detecting the signal.
44. The method of claim 43, wherein sensing comprises stochastic
sensing.
45. The method of claim 43, wherein the sensor element is a
protein.
46. The method of claim 43, wherein the sensor element is an
engineered protein.
47. The method of claim 43, wherein the reactive analyte is
selected from the group consisting of: chemical weapons,
environmental toxins, and pharmaceuticals.
48. The method of claim 43, wherein the reactive analyte
irreversibly covalently bonds to the sensor element, further
comprising allowing a chemical reagent to break the covalent
bond.
49. The method of claim 43, further comprising: allowing at least a
second reactive analyte to covalently bond to the sensor element to
produce a second signal, wherein the second signal is distinct from
the first signal; and detecting the second signal.
50. The method of claim 43, further comprising providing a
plurality of sensor elements.
51. The method of claim 43, further comprising comparing the
detected signal to a known set of signals to determine the identity
of the reactive analyte.
52. The method of claim 43, further comprising: determining the
frequency of detection of the signal; and calculating the
concentration of the reactive analyte based upon the frequency of
detection of the signal.
53. The method of claim 43, wherein the signal is selected from the
group consisting of: change in electrical current, change in force,
and change in fluorescence.
Description
BACKGROUND
[0001] Stochastic sensing is based on the detection of individual
binding events between analyte molecules and a single sensor
element. Upon binding, a property of the sensor element is altered.
This property or the effects of the changed property are
measured.
[0002] In a simple example, the sensor element is a protein that is
altered when it binds another molecule. The binding molecule to be
detected is referred to as the analyte. The alteration of the
sensor element that occurs upon binding is measured either directly
or indirectly. In simple systems the alteration produces a simple
signal, such as a difference in electrical current, force or
fluorescence. Measurements of the signal indicate whether the
analyte is bound and how long it remains bound. The frequency of
occurrence of binding events is determined by the concentration of
the analyte. The nature of the binding event is determined by the
binding properties of the analyte, which determine, for example,
the magnitude and duration of the resulting signal. Thus, a single
sensor element to which multiple analytes may bind either directly
may be used to determine which of those analytes are in a solution
and the concentration of each particular analyte.
[0003] Although in simpler systems the sensor element has one
binding site to which all analytes bind directly, it is possible
for the sensor to have multiple binding sites, with different sites
for different analytes. Additionally, a host or adaptor molecule
may be used to facilitate binding of the analyte to the sensor
element. The host molecule may merely facilitate the direct
interaction of the analyte and sensor element, or it may serve as
an adaptor that binds to both the analyte and the sensor element
and allows connection of the two.
[0004] Stochastic sensing may be accomplished with various sensing
elements, using various modes of detection. One simple model uses
an ion channel protein pore embedded in a membrane between a cis
chamber and a trans chamber. When the pore is fully open a large
ion flux occurs (e.g. 10.sup.8 ions/s) which constitutes an
electrical current that may be monitored by single channel
recording. When an analyte binds to the pore, ion flux is altered,
usually by decreasing the flow of ions. This generates a current
trace which shows conduction over time.
[0005] One particular pore that has been used in stochastic sensing
is Staph alpha hemolysin (.alpha.HL), which is actually an exotoxin
secreted by Staphylococcus aureus. The monomeric 293 amino acid
polypeptide can self-assemble on lipid bilayers, such as membranes,
to form a heptameric pore. Alternatively, pre-formed pores may be
inserted into a lipid bilayer. The pore is a mushroom-shaped
structure in which the lower half of the stem forms a transmembrane
channel. The interior of the pore is referred to as the "lumen" and
may be accessible from outside the pore. By convention, when the
pore is situated in a membrane, the side of the membrane on which
the top of the mushroom shape is located is designated as the "cis"
side of the membrane. The side of the membrane to which the stem
portion leads is designated the "trans" side of the membrane. The
pore essentially forms a hole in the membrane through which ions
will flow if an electric potential is generated between the two
chambers. (See FIG. 1.)
[0006] Stochastic sensing methods have been previously described in
a number of publications, including U.S. Pat. No. 6,426,231 to
Bayley et al. and a divisional application of that patent, U.S.
patent application Ser. No. 10/180,792, filed Jun. 25, 2002.
Protein pores for use in stochastic sensing and methods of using
such pores have also been described in U.S. patent application Ser.
No. 09/781,697 filed Feb. 12, 2001.
[0007] However, these previous manifestations of stochastic sensing
have utilized non-covalent interactions between the analyte and the
sensor element. There is considerable interest in the detection of
reactive molecules including chemical warfare agents, pesticides,
chemotherapeutic agents, and so on which will covalently bond to a
sensor element. The reactivity of such molecules may be utilized to
facilitate sensing and distinguish the reactive molecules from
unreactive molecules.
SUMMARY OF THE INVENTION
[0008] The present invention relates to methodologies for
stochastic sensing in which the analyte covalently bonds to a
sensor element. Stochastic sensing through such covalent
interactions permits the detection of reactive analytes by making
use of their reactivity.
[0009] Such covalent interactions may be reversible or
irreversible. Where the covalent reaction is reversible, individual
reaction events are be resolved by the sensing system within
reasonable time frame, and the distribution in time and the
character of these reaction events may be used to detect both the
concentration and identity of the analytes. Similarly, where the
reaction is normally irreversible, but is reversed due to the
present of a chemical reagent, both the concentration and identity
of the analytes may be readily detected. Where the covalent
reaction is irreversible and the analyte remains covalently bound
to the sensor element, the character of a the signal produced by
binding and its response to physical and chemical manipulations may
reveal the identity of a single analyte molecule.
[0010] Using the methodologies of the present invention, the
concentrations and identities of multiple analytes may be
determined simultaneously. Furthermore, these determinations may
even be observed on a millisecond time scale. Although these
abilities were previously observed using analytes that do not
covalently bond with sensor elements, the present invention allows
the stochastic sensing of analytes that do covalently bond with the
sensor element. Certain embodiments even allow detection of
concentration of analytes that irreversibly bond with the sensor
element by providing a chemical reagent that reverses the other
otherwise irreversible bonding. Furthermore, because the present
invention allows covalent bonding of the analyte, it allows
distinction between reactive and unreactive analytes.
[0011] The following abbreviations are used throughout the
specification and claims:
[0012] .alpha.HL--alpha hemolysin, a heptameric pore protein
[0013] BAL--British anti-Lewisite, 2,3-dimercaptopropanol
[0014] .beta.ME--2-mercaptoethanol
[0015] Cys--Cysteine, an amino acid
[0016] DTNB--5,5'-dithiobis(2-nitrobenzoic acid)
[0017] DTT--dithiothreitol
[0018] EDTA--ethylenediamine tetra-acetic acid, a metal chelating
agent.
[0019] "Irreversible"--As used in the present application,
"irreversible" designates a reaction which, under the specified
conditions, has an extremely low dissociation rate or forms very
stable covalent bond. In specific embodiments of the invention, an
irreversible bond or reaction forms an association between at least
two molecules that is too long-lived for stochastic sensing using
that system or method, absent the presence of a specific chemical
reagent that directly or indirectly causes dissociation or breaks
the covalent bond.
[0020] KCl--Potassium chloride, a salt which dissociates to form
K.sup.+ and Cl.sup.- ions in water. These ions may be moved through
an open pore in an electrical potential.
[0021] P.sub.SH--(T117C-D8).sub.1(RL2).sub.6, an engineered
.alpha.HL pore formed from six wild-type-like monomers and one
mutated monomer in which Thr-117 is replaced with Cys, providing a
sulfhydryl group within the lumen of the pore.
[0022] MOPS--a buffer used to maintain constant pH.
[0023] MTSES--(2-sulfonatoethyl)methane thiosulfonate to maintain
constant pH.
[0024] MES--a buffer used in various solutions.
[0025] Thr--Threonine, an amino acid.
[0026] Tris--a buffer used to maintain constant pH.
[0027] In certain exemplary embodiments of the present invention, a
system for sensing at least one reactive analyte in a sample is
provided. The system includes a sensor element and a sample
containing at least one reactive analyte. The reactive analyte
covalently bonds to the sensor element. This covalent bond between
the analyte and the sensor element may be a direct covalent bond
between the analyte and the sensor element or it may be a covalent
bond between the analyte and an adaptor molecule that then
interacts with the sensor element, for instance through a covalent
or non-covalent bond. Other host molecules that facilitate covalent
bonding of the analyte to the sensor element may also be
present.
[0028] The system may be used for stochastic sensing, where the
sensor element produces a detectable signal when covalently bound
to the reactive analyte. The sensor element may be a protein,
including engineered proteins such as a protein pore. Because of
the covalent bonding aspect of the system, it is capable of
detecting reactive analytes among similar unreactive molecules.
More than one sensor element may be used in a sensing system.
Examples of reactive analytes that may be detected with the system
include chemical weapons, environmental toxins, pharmaceuticals and
food contaminants.
[0029] Signals produced upon analyte bonding include changes in
electrical current, changes in force, and changes in fluorescence.
In many embodiments with protein pore sensors the signal may be a
change in the magnitude of an electrical current. In some
embodiments of the invention, the signal may simply register a
change in the condition. In other embodiments the nature of the
change may be recorded and used in greater detail.
[0030] In some embodiments the reactive analyte may irreversibly
covalently bond to the sensor element. In these systems, the sensor
may simply detect bonding alone. It may also detect other
properties of the analyte while it is bound to the sensor. For
example, other chemicals maybe added after initial detection of the
analyte which produce additional signals to further identify or
characterize the analyte. Additionally, in some embodiments of the
invention the system may also include a chemical reagent capable of
breaking the irreversible covalent bond between the analyte and the
sensor element. This may allow detection of, for instance,
concentration of the analyte, which may not be detectable if the
irreversible covalent bond is not broken.
[0031] These systems may also be used to detect multiple analytes
that may bond to the sensor element. In certain embodiments the
sample tested contains at least a second reactive analyte that
forms a covalent bond with the sensor element. As with the single
analyte example, this may be a direct covalent bond or a covalent
bond to an adaptor molecule. Different adaptor molecules, if
present, may be provided for different analytes, or the system may
be able to detect some analytes that require an adaptor and other
that do not. Similarly, multiple analytes need not all bind to the
same binding site of the sensor element.
[0032] Multiple analytes may be distinguished by differences in the
signal produced when different analytes bond to the sensor
element.
[0033] In more specific embodiments, a sensing devices is provided
that contains a cis and a trans chamber connected by a protein
pore. A detection system is set up so as to detect current between
the chambers. If a potential is applied between the chambers while
an ionic solution such as KCl is in them, the ions will move
through the open protein pore, resulting in a detectable current.
Bonding of a reactive analyte to the protein pore produces a change
in the current. This is generally effected by causing a restriction
of the pore. In many examples, the reactive analyte actually bonds
within the lumen of the pore. Although the analyte may generally be
on either side of the pore, the rate of covalent bond formation may
be influenced by whether it is placed in the cis or trans chamber
because the lumen may be more accessible from one side or the
other. Generally the analyte is proved in only one chamber,
although in certain embodiments of the invention it may be placed
in both chambers.
[0034] The pore in these embodiments may be an .alpha.HL pore,
particularly. Pores may be engineered proteins such as an .alpha.HL
pore with at least one monomer having a Cys residue at amino acid
117. Such an engineered pore may be used to detect arsenicals.
[0035] If the analyte is such a system irreversibly bonds to the
protein pore, a chemical reagent may be used to break the
irreversible covalent bond. The chemical reagent is generally
provided in one chamber, while the analyte is in the other chamber.
However, other configurations in which the reagent and analyte may
be together in one or both chambers are possible.
[0036] Current detection systems as described above may be used to
detect and identify analytes. Different analytes tend to remain in
the lumen for a different period of time. Thus the duration of any
current change may be used to identify the analyte. The frequency
of any current change caused by a specific analyte may be used to
determine its concentration in the sample. A compilation of such
current change signatures may be produced and used to quickly
identify specific analytes within a sample containing multiple
analytes.
[0037] The invention also includes methods for producing and using
systems as described above. In certain methods the analyte is
supplied in a sample to the sensor element, which remains in
contact with the element for a given time period. In a sensor
designed to be used multiple times, the sample may be removed and
replaced with another sample after testing of the first sample is
completed. Additionally, in some embodiments a constant sample
stream may be provided to the sensor element.
[0038] For a better understanding of the invention and its
advantages, reference may be had to the following drawings and
description of exemplary embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 illustrates a molecular graphics rendition of the
P.sub.SH version of the .alpha.HL heptamer pore as used in certain
embodiments of the present invention. In P.sub.SH, one of the seven
.alpha.HL subunits has been replaced with a mutated subunit
containing a Cys residue in place of the naturally occurring
Thr-117.
[0040] FIG. 2 illustrates the interaction of 4-sulfophenylarsine
oxide with the P.sub.SH pore, according to certain embodiments of
the present invention. To obtain the data presented, single channel
current traces were carried out for 1 second at -50 mV with 2 M
KCl, 80 mM MOPS and 100 .mu.M EDTA at pH 8.4 in both chambers. FIG.
2A shows current traces in the presence of 0, 10 and 50 .mu.M
4-sulfophenylarsine oxide in the trans chamber. Current levels of
the unoccupied and occupied pore are indicated in the figure. FIG.
2B is a plot of P.sub.bound (probability that the arsenical is
attached to the pore) versus concentration of 4-sulfophenylarsine
oxide. FIG. 2C plots the reciprocal of the mean residence time
(.tau..sub.off) and the mean inter-event intervals (.tau..sub.on)
versus 4-sulfophenylarsine oxide concentration. Values of
.tau..sub.off and .tau..sub.on were obtained by fitting dwell-time
histograms to single exponential functions. 1/.tau..sub.off is
represented with downward-pointing triangles. 1/.tau..sub.on is
represented with upward-pointing triangles.
[0041] FIG. 3 illustrates the interaction of arsenicals with the
P.sub.SH pore under conditions similar to those used in FIG. 2,
according to embodiments of the present invention. FIG. 3A is a 10
second current trace using 500 .mu.M arsenate. FIG. 3B is a 10
second current trace using 500 .mu.M arsenite. FIG. 3C is a 10
second current trace using 500 .mu.M cacodylate. FIG. 3D is a 10
second current trace using 10 .mu.M phenylarsine oxide.
[0042] FIG. 4 illustrates the chemical reaction cycle of certain
embodiments of the present invention in which DTT is used as a
chemical reagent to reverse an irreversible covalent bond between
the P.sub.SH pore and DTNB. Specifically, Cys-117 reacts with DTNB
in a bimolecular reaction, with a rate constant k.sub.1-obs to form
a mixed disulfide (1) ("Step 1"). Disulfide 1 then reacts with
D,L-DTT in a second biomolecular reaction, with a rate constant
k.sub.2-obs, to form an unstable disulfide (2) ("Step 2"), which
breaks down in a unimolecular reaction, with a rate constant
k.sub.3-obs, in which P.sub.SH is regenerated ("Step 3").
[0043] FIG. 5 illustrates a 0.5 second current trace of stochastic
sensing using a P.sub.SH pore, according to an embodiment of the
present invention. 50 .mu.M DTNB was used as the analyte and 50
.mu.M DTT was present in the cis chamber. The transmembrane
potential was -50 mV with 2 M KCl, 50 mM Tris.HCl and 100 .mu.M
EDTA at pH 8.5 in both chambers. The region of the current trace
corresponding to the reaction of Cys-117 in the P.sub.SH pore with
DTNB to form mixed disulfide 1 is labeled as "Step 1". The region
corresponding to the reaction of the mixed disulfide (1) with DTT
is to form unstable disulfide 2 is labeled as "Step 2". The region
corresponding to the breakdown of unstable disulfide 2 to
regenerate unbound P.sub.SH is labeled as "Step 3."
[0044] FIG. 6A illustrates the dependence of 1/.tau..sub.1 on
[DTNB]trans, where .tau..sub.1 is the time interval between Step 3
and Step 1 during the interaction of DTNB and DTT with a P.sub.SH
pore (i.e. the lifetime of free P.sub.SH before the bonding of
DTNB), according to certain embodiments of the present invention.
The data were obtained using the indicated concentrations of DTNB
on the trans side of the pore. All measurements were for a 10
second interval at -50 mV with 2 M KCl, 30 mM MOPS, 100 .mu.M EDTA
at pH 8.5 in both chambers and and 50 .mu.M DTT in the cis chamber.
The current level corresponding to a single, unmodified pore was
-82.2.+-.2.1 pA. .sub.K1-obs was obtained from the slope of the
straight fit line as illustrated.
[0045] FIG. 6B illustrates the effects of various concentrations of
DTT of the cis side of a P.sub.SH pore, according to certain
embodiments of the present invention. Measurements were made using
the system described for FIG. 6A, but with 100 .mu.M DTNB and
varying concentrations of DTT. The effects of DTT concentration on
1/.tau..sub.1 are indicated.
[0046] FIG. 7A illustrates the dependence of 1/.tau..sub.2 on
[DTT].sub.trans, where .tau..sub.2 is the time interval between
Step 1 and Step 2 during the interaction of DTNB and DTT with a
P.sub.SH pore (i.e. the lifetime of the state with DTNB covalently
bound to P.sub.SH as a mixed disulfide 1 and the reaction of the
mixed disulfide 1 with DTT), according to certain embodiments of
the present invention. The data were obtained using the indicated
concentrations of DTT on the trans side of the pore. All
measurements were for a 10 second interval at -50 mV with 2 M KCl,
30 mM MOPS, 100 .mu.M EDTA at pH 8.5 in both chambers and 50 .mu.M
DTNB in the cis chamber. .sub.K2-obs was obtained from the slope of
the straight fit line as illustrated.
[0047] FIG. 7B illustrates the effects of various concentrations of
DTNB on the cis side of a P.sub.SH pore, according to certain
embodiments of the present invention. Measurements were made using
the system described for FIG. 7A, but with 50 .mu.M DTT and varying
concentrations of DTNB. The effects of DTNB concentration on
1/.tau..sub.2 are indicated.
[0048] FIG. 8A illustrates k.sub.3-obs as a function of
[DTT].sub.cis in certain embodiments of the present invention. All
measurements were for a 10 second interval at -50 mV with 2 M KCl,
30 mM MOPS, 100 .mu.M EDTA at pH 8.5 in both chambers and 50 .mu.M
DTNB in the trans chamber. [DTT] is as indicated. FIG. 8B
illustrates k.sub.3-obs as a function of [DTNB].sub.trans in
certain embodiments of the present invention. All measurements were
for a 10 second interval at -50 mV with 2 M KCl, 30 mM MOPS, 100
.mu.M EDTA at pH 8.5 in both chambers and 50 .mu.M DTT in the cis
chamber. [DTNB] is as indicated.
[0049] FIG. 9 illustrates the pH dependence of the rate of
breakdown of the unstable disulfide 2 formed during the interaction
of DTNB, DTT and the P.sub.SH pore, according to certain
embodiments of the present invention. 50 .mu.M DTNB was used as the
analyte in the trans chamber and 50 .mu.M DTT was present in the
cis chamber. The transmembrane potential was -50 mV with 2 M KCl,
30 mM MOPS and 100 82 M EDTA. pH in both chambers is as indicated.
k.sub.3-obs is shown as a function of pH. The data were fitted to
k.sub.3-obs=(k.sub.3K.sub.a)/(K.sub.a+[H.sup.+]).
[0050] FIG. 10 illustrates a 30 second current trace of stochastic
sensing using a P.sub.SH pore, according to an embodiment of the
present invention. 2.5 mM MTSES was used as the analyte in the
trans chamber and DTT was present in the cis chamber. The
transmembrane potential was -50 mV with 2 M KCl, 50 mM Tris.HCl and
100 .mu.M EDTA at pH 8.5 in both chambers.
DETAILED DESCRIPTION
[0051] The present invention relates to methodologies of stochastic
sensing in which the analyte covalently bonds to the sensor
element. The sensor element may be an isolated or engineered
protein pore, such as an ion channel. It may also be other
engineered or isolated macromolecules such as proteins,
oligonucleotides, inorganic and organic host molecules and
imprinted surfaces.
[0052] Additionally, a variety of signals and bonding detection
procedures may be used. Signals include conductance, fluorescence,
force and any other effects of analyte binding to the chosen sensor
element. These signals may be detected using electrical and optical
measurements, force measurements, chemical techniques and any
combination of the above.
[0053] Analytes may be reactive chemicals that covalently bond to
the sensor element under the stochastic sensing conditions.
Covalent bonds may be reversible or irreversible. The analytes may
be any type of chemical including toxins, environmental indicators,
chemical process products or by-products and contaminants. Chemical
warfare agents, which can be arsenicals, organophosphates, mustard
gasses, etc. Chemicals in foodstuffs, such as onion and garlic,
pesticides such as organophosphates. Naturally occurring
environmental toxins. Reactive pharmaceuticals. Many physiological
molecules such as nitric oxide, endo and exo peroxides are
extremely reactive messengers. Potential analytes that are not
reactive are generally unable to covalently bond with the sensor
element. Therefore, reactive and unreactive species of a similar
chemical may be distinguished.
[0054] Analytes may covalently bond to the sensor element alone or
in combination with a second molecule, such as a host or adaptor
molecule. The analyte may covalently bond to the second molecule,
which then covalently or non-covalently bonds to the sensor element
or the second molecule may covalently bond to the analyte.
Alternatively, the second molecule may simply affect the reaction
kinetics of analyte bonding. If used to affect the reaction
kinetics, the second molecule may be used to slow down or speed up
rate of bonding or duration of the bound state to facilitate
detection of the identity, concentration, or other properties of
the analyte. The second molecule may also exert an effect on
analyte bonding reaction kinetics by interacting with the analyte
while not bound to the sensor element.
[0055] Other chemicals which act upon the bound analyte or are
otherwise affected by the analyte/sensor element combination may
also be added to the sensing system to augment sensing. For
instance, the an analyte bound to an .alpha.HL pore that causes a
change in ion flux, but is not easily distinguishable from another
analyte by current trace may be distinguished by introduction of a
chemical that cleaves one of the indistinguishable analytes, but
not the other. Additionally, added chemicals may be used to further
explore the chemical identity properties of the analyte using
sensing methods other than the original sensor element. Such
methods are used in exemplary embodiments with analytes that
irreversibly bond to the sensor element.
[0056] Additionally, in stochastic sensing methodologies in which
the analyte irreversibly covalently bonds to the sensor element, a
chemical reagent may be added which breaks the covalent bond. The
chemical reagent may exert this breaking effect through direct
action on the bond or through indirect action such as binding to
the analyte, an adapter molecule, or the sensor element. Such
indirect action may result in a physical change of the analyte,
adaptor or sensor element, such as a conformational change, or it
may destroy the analyte or adapter. For example, the chemical
reagent may actually be an enzyme that cleaves the analyte when it
is bound to the sensor element.
[0057] The stochastic sensing methodology of the present invention
may utilize a variety of physical arrangements. For example,
systems using protein pore arrangements may have the pore embedded
in a membrane, located between separate cis and trans chambers. It
may be recommended to place the analyte in either the cis or the
trans chamber exclusively, or the system may be readily functional
regardless of the chamber in which the analyte is placed. The same
is true for chamber placement of any adapter or chemical reagent
for breaking of irreversible bonds. In most systems the chemical
reagent will be placed in one chamber and the analyte in the other
chamber. Other systems, such as those not relying upon electrical
detection methods, may merely be a single chamber in which all
elements of the system are combined.
[0058] It is also possible to prepare a sensing system in which
portions of the system are introduced in a time-dependent manner or
varied over time. For example, in a system for sensing an analyte
that irreversibly bonds to the sensor element, a chemical reagent
capable of breaking the bond may be introduced into the system only
after irreversible bonding has occurred. It may even be removed
after breaking the bond, for instance by replacing the solution on
one side of a membrane.
[0059] In other embodiments the sample to be tested for analyte may
be discharged and replaced over time. This may result from periodic
introduction of new samples or by flowing a continuous stream of
sample through the sensor. For example, a sensor system to detect
the presence of a reactive chemical in industrial effluent may
contain a chamber through which a small diverted stream of effluent
flows. In such examples it may be necessary to regulate flow so
that the sample remains in proximity with the sensor element for a
sufficient amount of time to statistically allow detection of a
particular analyte at or above a selected concentration.
[0060] The systems and methodologies of the present invention may
be used in sensors for detection of various reactive molecules.
These sensors may be fixed or portable and they may be designed for
single-use applications or any number of multiple uses. Remote
application sensors that may be placed or dropped in a target
location and then transmit sensor information may also be used.
Solutions or other chemicals for use in the sensors may be supplied
with the sensors in a kit, or supplied independently for use with
the sensors.
[0061] In certain embodiments of the present invention, an
.alpha.HL protein pore may be embedded in a membrane which
separates a cis chamber from a trans chamber. Interaction of the
analyte, such as an organoarsenic compound, with the .alpha.HL
blocks the of ions through the pore. This change in ion
concentration in the cis and trans chambers modulates a current
flow through a conductor connecting the two chambers. Measurement
of this current to produce a current trace allows determination of
when the analyte bonds with the .alpha.HL and when it becomes
unbound.
[0062] When using the .alpha.HL pore, most analytes may be added to
either the cis or the trans side, although analytes can more
readily reach the pore from the trans side. The same is true for
many adapters or chemical reagents for reversal of irreversible
bonds.
[0063] The following examples are provided only to illustrate
certain aspects of the invention and are not intended to embody the
total scope of the invention or the totality of any aspect thereof.
Variations of the exemplary embodiments of the invention below will
be apparent to one skilled in the art and are intended to be
included within the scope of the invention.
EXAMPLES
Example 1
[0064] Arsenic Compounds
[0065] Many arsenic compounds are poisonous and cause an array of
serious to fatal disorders ranging from skin disease to cancer.
Both man-made and artificial arsenic compounds are also problematic
environmental contaminants. Arsenic compounds have also been made
into chemical weapons. Stockpiles of one chemical weapon, Lewisite
(2-chlorovinyldichloroarsine), are still in existence. Lewisite
hydrolyzes in water resulting in a toxic arsenous acid that likely
exerts its biological effects through reaction with thiols.
4-sulfophenylarsine oxide, an organoarsenic (III) reagent, reacts
with thiols in a manner similar to Lewisite. Other organoarsenic
compounds are also potential chemical weapons or environmental
contaminants, or mimic the biological action of arsenic-based
weapons and contaminants.
[0066] Sodium 4-sulfophenylarsonic acid used in these Examples was
prepared by dissolving 7.62 g of Sulfanilic acid in 40 mL of water
to create a 44 mmol solution also containing 2.8 g of sodium
carbonate. Diazotization was performed by adding, simultaneously,
10 mL concentrated HCl and 40 mL of 46.6 mmol sodium nitrite
solution (formed from 3.2 g sodium nitrite in 40 mL water) with two
dropping funnels. The resulting solution, which contained some
precipitate, was dropped into an ice-chilled 100 mL of solution of
100 mmol arsenic trioxide, 200 mmol sodium hydroxide, and 0.75 mmol
copper sulfate (formed from adding 9.82 g arsenic trioxide, 8.08 g
sodium hydroxide, and 120 mg copper sulfate to 100 mL water). The
color of the solution changed from blue to dark green and large
amounts of N.sub.2 gas were released. After three days at room
temperature, the volume was reduced to approximately 25 mL with a
rotary evaporator. A precipitate was removed by filtration. The
filtrate was boiled briefly, the acidified with concentrated HCl
resulting in an immediate precipitate. After 4 days at room
temperature, the solid was collected by filtration and washed in
turn with saturated KCl, 80% ethanol, then acetone. After drying
under a vacuum, a 5.21 g of a light brown solid was recovered. NMR
analysis showed the following results: .sup.1H NMR 300 MHz
(D.sub.2O): 8.03(d, 2H, J=8.4 Hz), 7.97(d, 2H, J=8.7 Hz).
[0067] Sodium 4-sulfophenyldiiodoarsine used in these Examples was
formed by adding 3 mL of 57% hydroiodic acid in water to 10 mL of a
55-60.degree. C. solution of 3.39 mmol sodium 4-sulfophenylarsonic
acid (formed by solubilizing 1.03 g in 10 mL water). After 5
minutes, the dark solution was cooled on ice and the precipitated
solid was collected and washed with acetic acid, followed by
acetone. The bright yellow solid was dried under vacuum. It
weighted 1.05 g (61% yield). This crude product was recrystallized
from 80% acetic acid, yielding yellow needles. The .sup.1H NMR
spectrum in D.sub.2O showed that sodium p-sulfophenyldiiodoarsine
is hydrolyzed to sodium 4-sulfophenylarsine oxide. .sup.1H NMR 300
MHz (D.sub.2O):7.88(d, 2H, J=8.7 Hz), 7.84(d, 2H, J=8.4 Hz). HRMS
(of hydrolysis) calculated for C.sub.6H.sub.4O.sub.4SAs (M.sup.-)
246.9046 found 246.9041. Microanalysis (Atlantic Microlab, Inc.)
calculated for C.sub.6H.sub.4O.sub.3I.sub.2SAsNa; C: 14.19, H:
0.79, O: 9.45, I: 49.97, S: 6.31, found C: 15.33, H: 1.13, O:
10.90, I: 49.54, S: 5.79. A microanalysis after additional
recrystallization also suggested that the crystals retained solvent
or that slight hydrolysis of the diiodide occurred.
[0068] 4-aminophenylarsine oxide used in these Examples was formed
by adding 10.9 g of p-Arsanilic acid to a solution 30 mL methanol,
24 mL concentrated hydrochloric acid, and 100 mg potassium iodide.
Sulfur dioxide was bubbled into the solution for 25 minutes at room
temperature. The color of the solution changed from bright brown to
white, followed by precipitation of 4-aminophenyldichloroarsine as
the HCl salt. The mixture was cooled in an ice bath and the
precipitate was collected and washed with dry ethyl ether. 500 mg
of the precipitate was dissolved in 65 mL of 10% ammonium
hydroxide. 4-aminophenylarsine oxide slowly precipitated overnight
at room temperature. The product was collected, washed with diethyl
ether and dried in a vacuum. A white solid (325 mg) was recovered.
Microanalysis (Atlantic Microlab, Inc.) calculated for
C.sub.6H.sub.6NOAs; C: 39.37, H: 3.30, N: 7.65, O: 8.74 found C:
39.44, H: 3.21, N: 7.73, O: 8.83.
[0069] Arsenite solutions used in these Examples were formed by
dissolving 100 mM sodium metaarsenite in water.
[0070] Arsenate solutions used in these Examples were formed by
dissolving 100 mM arsenic acid in water.
[0071] Dimethylarsinate solutions used in these Examples were
formed by dissolving 100 mM cacodylic acid, sodium salt in
water.
[0072] Phenylarsine oxide solutions used in these Examples were
formed by dissolving phenylarsine oxide at 1 mM in DMSO. This
solution was diluted with water for make a 100 .mu.M stock.
[0073] 4-sulfophenylarsine oxide solutions used in these Examples
were formed by dissolving 100 mM 4-sulfophenylarsine in water with
adjustment to pH 7 using 1 M sodium hydroxide.
[0074] 4-aminophenylarsine oxide solutions used in these Examples
were formed by dissolving 4-aminophenylarsine in 50 mM MES at pH
5.5 (titrated with HCl) to make a 1 mM solution.
[0075] DTT solutions used in these Examples were formed from
preweighed samples of sold D,L-DTT stored at -20.degree. C. At the
time of the experiment, buffer was added to the sample in a tube to
generate a stock of 1M DTT, which was kept on ice. Fresh stock
solutions of DTT were prepared every 2 hours.
[0076] DTNB solutions used in these Examples were formed from 100
mM DTNB dissolved in 200 mM Na phosphate, pH 8.5. This stock
solution was diluted as necessary. Because DTNB decomposes in basic
solution, fresh DTNB stocks were made daily.
Example 2
[0077] Preparation of the PSH .alpha.HL Pore Protein
[0078] The P.sub.SH variant of the .alpha.HL pore contains a
Cys-117 mutation, in which Cys replaces the usual Thr. This places
the thiol side chain of Cys projecting into the lumen of the
.alpha.HL pore. To prepare P.sub.SH pores, first [.sup.35S] Met
labeled monomeric polypeptides were prepared by in vitro
transcription and translation (IVTT). These monomeric IVTT products
were used to make the .alpha.HL heptamers.
[0079] 50 .mu.L of a 50 .mu.g/mL solution of wild-type-like
.alpha.HL (RL2) were mixed with 20 .mu.L of a 50 .mu.g/mL mutated
.alpha.HL (T117C-D8) solution 30 .mu.L of a suspension of rabbit
red blood cell membranes (at 3 mg membrane protein/mL) was diluted
with 500 .mu.L MBSA (10 mM MOPS, 150 mM NaCl, pH 7.4, titrated with
HCl). The membranes were centrifuged for 5 minutes at 21,000.times.
g and the supernatant was removed. The washed membranes were then
resuspended with the protein mixture. After 70 minutes at
37.degree. C., the membranes were pelleted by centrifugation for 5
minutes at 21,000.times. g and resuspended in 100 .mu.L of MBSA
containing 2 mM DTT. The membranes were then recovered again by
centrifugation.
[0080] The washed membrane pellet containing the assembled
heptamers was solubilized in 50 .mu.L of sample buffer and loaded
into one lane of a 5% SDS polyacrylamide gel, which was run at 30V
overnight. The D8 (Asp.sub.8) tail allowed the separation of
heptamers with different combinations of mutated .alpha.HL
subunits. The gel was dried at 50.degree. C. for 4 hours and then
exposed to X-ray film for 2 hours. The band corresponding to
P.sub.SH (a heptamer with 6 wild-type-like monomers and one mutant
monomer) was excised.
[0081] The excised gel portion was then hydrated in 400 .mu.L of 10
mM Tris.HCl, pH 7.5 containing 2 mM DTT and the paper was removed.
The gel was then crushed using a plastic pestle and the resulting
suspension was rotated at 4.degree. C. overnight. The material was
then filtered through a 0.2 .mu.M cellulose acetate filter. The
filtrate, which contained P.sub.SH was stored at -80.degree. C.
[0082] Although the example above is intended for small-scale
preparations of .alpha.HL sensor elements and includes
radioactivity, which may be unwanted in industrial applications,
the large-scale production and assembly of complex proteins without
the use of radioactivity or other hazardous materials is well known
in the art. Accordingly, one skilled in the art should be able to
produce commercial scale batches of the .alpha.HL pore with little
difficultly.
[0083] For example, wild-type-like and mutated portion of the pore
may be grown separately in bioreactors and purified then combined
in proportions similar to those described above to produce
self-assembled pores.
Example 3
[0084] Electrophysiology Apparatus
[0085] Experiments using .alpha.HL pores in these Examples were
carried out using folded bilayer membranes. To prepare such
membranes, a 25 .mu.m thick Teflon septum was clamped between two
Teflon chambers each of 1 mL volume. A bilayer was formed on a 100
.mu.m diameter aperture in the septum. The septum was pretreated
with 10% (v/v) hexadecane in highly purified n-pentane. Both
chambers contained an electrolyte solution containing 2M KCl, 100
.mu.M EDTA and a selected amount of MOPS, Tris or another
buffer.
[0086] Initially the chambers were filled with electrolyte to a
level slightly below the aperture. A bilayer material 1% (w/v)
1,2-diphytanoyl-sn-glycerophosphocholine in 6 .mu.L pentane was
spread on the surface of each chamber. After about 3 minutes,
during which time the pentane solvent in the bilayer material
evaporated, the electrolyte level in each chamber was raised above
the aperture. The formation of a bilayer was monitored by observing
the increase in membrane capacitance to a value of approximately
130 pF.
[0087] A single .alpha.HL pore was inserted into the bilayer by
adding a solution of the pore to the cis chamber to produce a final
concentration of about 0.5 ng/mL. The cis chamber, which was kept
at ground, was stirred until electrical recordings indicated that a
single protein channel appeared in the bilayer.
[0088] The test arsenical or arsenicals were added to the trans
chamber. Electrical current disruption from blocking of the
.alpha.HL pore upon arsenical binding was detected through two
Ag/AgCl electrodes and amplified with a patch-clamp amplifier
(Axopatch 200B; Axon Instruments, Union City, Calif.), filtered
with a low-pass Bessel filter (80 dB/decade) with a corner
frequency of 1 kHz, and then digitized with a DigiData 1200
A/D/converter (Axon Instruments) at a sampling frequency of 5 kHz.
Data samples were stored electronically and then current traces
were filtered digitally at 100 Hz for further analysis and display.
Event data files used to generate dwell-time (durations of various
states of the pore) histograms were constructed using the Fetchan
program (Axon Instruments). Processed data were plotted using
Origin 6.1 (OriginLab Corp., Northampton, Mass.). Individual values
were presented as the mean .+-. standard deviation.
Example 4
[0089] Interaction of 4-sulfophenylarsine oxide with P.sub.SH
[0090] The interaction of P.sub.SH with 4-sulfophenylarsine oxide
was examined by single-channel electrical recording. In the absence
of 4-sulfophenylarsine oxide P.sub.SH produced a quiet single
channel current of 1.54.+-.0.03 nS (n=7) (See FIG. 2A.) In the
presence of both 10 .mu.M and 50 .mu.M 4-sulfophenylarsine oxide,
steps were observed in which the current was reduced to
1.49.+-.0.04 nS (n=7). (See FIG. 2A.) The steps had a mean duration
of 702.+-.38 msec (n=6) and represent individual couplings of
4-sulfophenylarsine oxide to the Cys residue in the P.sub.SH pore.
As FIG. 2A shows, an increase in 4-sulfophenylarsine oxide
concentration also resulted in an increase in the frequency of such
couplings.
[0091] These events were not observed when 4-sulfophenylarsine
oxide was added to a bilayer containing a completely wild type
.alpha.HL pore, which contains no lumen Cys residues. Additionally,
the coupling events were eliminated by either replacing the
arsenical solution with buffer or by the addition of 1.5 mM BAL to
10 .mu.M arsenical. BAL is a dithiol which is know to react with
As.sup.III compounds and therefore was expected at sufficient
concentration to react with all available arsenical and prevent its
bonding to the P.sub.SH pore.
[0092] FIG. 2B was constructed using data obtained as described
above for various concentrations of 4-sulfophenylarsine oxide.
P.sub.bound is the probability that a molecule of the arsenical is
attached to the pore. The increase of P.sub.bound with arsenical
concentration indicates that the reaction frequency does increase
with increasing arsenical concentration.
[0093] FIG. 2C was also constructed using data obtained as
described above for various concentrations of 4-sulfophenylarsine
oxide. .tau..sub.off represents the mean residence time of the
arsenical in the pore. .tau..sub.on is the mean inter-event
(arsenical-pore coupling) interval. The association constant
(k.sub.on) for the analyte/pore coupling is 1/.tau..sub.on[A] where
[A] is the concentration of analyte, in this case
4-sulfophenylarsine oxide. Accordingly, the constant relationship
of 1/.tau..sub.on with [A], as shown in FIG. 2C by the line with
upward pointing triangles, indicates that the rate of pore coupling
correlates in a linear fashion with the concentration of analyte.
This is consistent with a simple bimolecular interaction where
concentration of one molecule (the pore) is held constant. In such
an instance, the rate at which the interaction occurs should
increase linearly with the concentration of the other molecule (the
analyte). k.sub.on is the slope of the line. For the interaction of
4-sulfophenylarsine oxide with P.sub.SH, k.sub.on was calculated to
be 20.+-.3.times.10.sup.3 M.sup.-1s.sup.-1.
[0094] The dissociation constant (k.sub.off) for the analyte/pore
coupling is 1/.tau..sub.off. k.sub.off is shown by the line with
downward pointing triangles in FIG. 2C. Because k.sub.off in a
unimolecular interaction is not influenced by concentrations of the
molecules, but rather by the nature of the interaction, one would
expect it to remain essentially constant despite changing analyte
concentration. FIG. 2C shows just this effect. For the interaction
of 4-sulfophenylarsine oxide with P.sub.SH, k.sub.off was
calculated to be 1.4.+-.0.1 s.sup.-1.
[0095] The dissociation constant for the interaction, K.sub.d,
which indicates the overall strength of the interaction and how
easily the bimolecular complex can be separated, is
75.+-.15.times.10.sup.-6 M at 24.degree. C. (n=4).
K.sub.d=dissociation rate (k.sub.off)/association rate (k.sub.on).
Accordingly, lower K.sub.d values indicate stronger binding.
Example 5
[0096] Interaction of Various Arsenicals with P.sub.SH
[0097] The interaction of P.sub.SH with various other arsenicals
was examined by single-channel electrical recording. In the absence
of arsenicals P.sub.SH produced a quiet single channel current (See
FIG. 3.) In the presence of 500 .mu.M arsenate, arsenite, or
cacodylate or 10 .mu.M phenylarsine oxide steps were observed in
which the current was reduced. (See FIGS. 3A-D, respectively.) The
amplitudes of current reduction and mean dwell times varied
depending upon the arsenical. This indicates that different
arsenicals result in detectably different signals when bound to the
P.sub.SH pore. Therefore, it is possible to sort signals from the
binding of different arsenicals with a P.sub.SH pore to determine
individual arsenical identity and concentration even if the
arsenicals are mixed in a solution.
Example 6
[0098] Reaction Mechanism for Reversal of an Irreversible Covalent
Interaction
[0099] The P.sub.SH .alpha.HL pore may also be used to investigate
the interaction of non-arsenical compound with Cys-117. For
example, DTNB forms a disulfide bond with Cys-117. DTNB is a
non-arsenical model reactive analyte. This bond between DTNB and
the pore is irreversible, but may be cleaved by DTT to regenerate
the Cys residue and open the pore. In the overall reaction,
(diagrammed in FIG. 4) Cys-117 in the lumen of the pore reacts with
DTNB in a bimolecular reaction ("Step 1") with rate constant
k.sub.1-obs to form a mixed disulfide 1. In mixed disulfide 1 the
sulfur atom proximal to the protein wall is activated for reaction
with free thiolates because the aromatic thiolate is a good leaving
group. Therefore, mixed disulfide 1 reacts with DTT in a
bimolecular reaction ("Step 2") with a rate constant k.sub.2-obs to
form an unstable disulfide 2. Unstable disulfide 2 breaks down in a
unimolecular reaction with rate constant k.sub.3-obs in which
P.sub.SH is regenerated. As a result, P.sub.SH is able to undergo
multiple cycles of reaction.
[0100] The steps of the above reaction are clearly visible in a
current trace of a single pore system. To produce the current trace
of FIG. 5, a P.sub.SH pore in a bilayer membrane was prepared as
described in the previous examples. DTNB was added to the trans
chamber and DTT was added to the cis chamber. In the single channel
recording shown, three separate current levels representing unbound
P.sub.SH, mixed disulfide 1 and unstable disulfide 2 were observed.
The current trace at Step 3 is consistent with the existence and
unimolecular breakdown of unstable disulfide 2.
Example 7
[0101] Kinetics of the Interaction of DTNB, DTT and P.sub.SH
[0102] The rate of reaction of DTNB with Cys-117 of P.sub.SH was
measured with DTNB in the trans chamber and 50 .mu.M DTT in the cis
chamber at an applied potential of -50 mV with a solution of 2 M
KCl, 30 mM MOPS and 100 .mu.M EDTA, pH 8.5 in both chambers.
Because the reaction of DTNB with P.sub.SH is a bimolecular
reaction and the concentration of P.sub.SH is constant, the
reaction rate, k.sub.1-obs is constant and may be calculated as
1/.tau..sub.1[DTNB], where .tau..sub.1 is the mean amount of time
P.sub.SH remains unbound in the presence of DTNB. As FIG. 6A
illustrates, there is a linear relationship between [DTNB] and
1/.tau..sub.1, consistent with the assumption that the reaction is
bimolecular. Using the data of FIG. 6A, k.sub.1-obs was calculated
to be 4.9.+-.0.5.times.10.sup.3 M.sup.-1s.sup.-1 (n=5).
[0103] In the system described above, it was uncertain whether DTT
might leak from the cis side of the chamber to the trans side and
react there with DTNB, thereby reducing the concentration of DTNB
available to interact with P.sub.SH and producing inaccurate DTNB
concentration measurements using the system. FIG. 6B illustrates
that at concentrations of DTT below approximately 60 .mu.M, the
rate constant for the interaction of DTNB with P.sub.SH is not
significantly affected. At DTT concentrations below approximately
40 .mu.M the effect is nearly unnoticeable.
[0104] Similarly, it was plausible that the reaction rate of DTT
with mixed disulfide 1 to form unstable disulfide 2 and ultimately
free the P.sub.SH pore might be influenced by the rate of diffusion
of DTT from the cis chamber through the pore. This rate might be
decreased by the movement of DTNB from the trans chamber to the cis
chamber, where it would interact with DTT and thereby deplete DTT
levels. To investigate the possibility of this event, a reverse
arrangement from that described above was used with 50 .mu.M DTNB
on the cis side of the membrane and DTT on the trans side. Because
of the shape of the .alpha.HL pore, from the trans side chemicals
have free access to the lumen, but are considerably more restricted
from the cis side. Because the interaction of DTT with the
DTNB/P.sub.SH complex is a biomolecular interaction with a constant
concentration of DTNB/P.sub.SH, the rate constant, k.sub.2-obs is
equal to 1/.tau..sub.2 [DTT], where .tau..sub.2 is the length of
time between binding of DTNB to P.sub.SH and the beginning of Step
2. As FIG. 7A illustrates, there is a linear relationship between
[DTT] and 1/.tau..sub.2, consistent with the assumption that the
reaction is bimolecular. Using the data of FIG. 7A, k.sub.2-obs was
calculated to be 1.1.+-.0.1.times.10.sup.4 M.sup.-1s.sup.-1.
Additionally, FIG. 7B illustrates that, in fact, at low
concentrations of [DTNB] in the cis chamber the lifetime of mixed
disulfide 1 is not affected. In particular, at concentrations below
60 .mu.M little effect is observed and below 40 .mu.M almost no
detectable effect can be seen. This confirms that movement of DTNB
from the cis chamber to the trans chamber is minimal at low
concentrations.
[0105] The breakdown of unstable mixed disulfide 2 is a
unimolecular reaction. Therefore, k.sub.3-obs=1/.tau..sub.3, where
.tau..sub.3 is the time between formation of the mixed disulfide 2
at the end of Step 2 and its breakdown at the end of Step 3. To
illustrate the unimolecular nature of Step 3 and determine
k.sub.3-obs, DTNB was added to the trans chamber of a single pore
system while DTT was added to the cis chamber. A solution of 2 M
KCl, 30 mM MOPS and 100 .mu.M EDTA at pH 8.5 was used in both
chambers and the applied potential was -50 mV. In the tests
depicted in FIG. 8A, the concentration of DTNB was held constant at
50 .mu.M while concentration of DTT was varied as shown. The linear
relationship of k.sub.3-obs with [DTT] is apparent in the figure.
Similarly, in FIG. 8B, test were performed with the concentration
of DTT fixed at 50 .mu.M while the concentration of DTNB was varied
as shown. The linear relationship of k.sub.3-obs with [DTNB] is
apparent in the figure. In both tests, k.sub.3-obs was calculated
to be 23.+-.1 s.sup.-1.
[0106] To verify that the intermediate unstable disulfide 2 was
formed from DTT, several control experiments were performed. In
addition, the intermediate structures of unstable disulfide 2 were
investigated at various pH values. A plot of k.sub.3-obs as a
function of pH is shown in FIG. 9. The data related to the plot
could be fitted to the equation
k.sub.3-obs=(k.sub.3K.sub.a)/(K.sub.a+[H.sup.+]). This suggests
that the reactive form of 2 is a thiolate. The data indicate that
the pK.sub.a of 2 is 9.5.+-.0.2 and .sub.K3 is 250.+-.80 s.sup.-1
in the deprotonated form. In comparison, the first pK.sub.a of DTT
is 9.2 and the pK.sub.a of mercaptoethanol is 9.5.
Example 8
[0107] Interaction of MTSES, DTT and P.sub.SH
[0108] A single pore system as described above was prepared and
supplied with a solution of 2 M KCl, 50 mM Tris.HCl and 100 .mu.M
EDTA at pH 8.5 in both chambers. 50 .mu.M DTT was supplied to the
cis chamber. 2.5 mM MTSES was provided in the trans chamber. A
transmembrane potential of -50 mV was applied. As shown in FIG. 10,
MTSES bound to the P.sub.SH pore, thereby blocking it. Interaction
of DTT with MTSES broke the irreversible disulfide bond and opened
the P.sub.SH pore.
Example 9
[0109] Other Engineered Pores and Alternative Systems
[0110] The above examples indicate that the conditions inside the
P.sub.SH pore at least approximate the conditions in solution. In
order to mimic solution conditions even more closely, additional
mutations may be made to the wild-type .alpha.HL monomer. For
example, hydrophobic side chains in the channel may be may be
replaced with hydrophilic side chains such as Ser, Thr, Asn and
Gln. Surface charge effects in the pore can also be minimized by
using high salt concentrations, as in these examples.
[0111] Other sensors may be used in the present invention that do
not require solution conditions within a pore or other area to
function well.
[0112] The .alpha.HL system described above may also be used to
study the effects of protein environment on chemistry. Residues
that bind substrates or take part in the chemistry may be
introduced onto on the wall of the channel by mutagenesis, targeted
chemical modification of non-natural amino acid substitution. For
example, histidine residues may be used for catalysis. Through
selected substitutions, it may be possible to improve turnover
rates or couple catalysis transmembrane transport.
[0113] All of the systems and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the systems and methods of
this invention have been described in terms of specific
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the systems and/or methods and in the
steps or in the sequence of steps of the methods described herein
without departing from the concept, spirit and scope of the
invention. All such similar substitutes and modifications apparent
to those skilled in the art are deemed to be within the spirit,
scope and concept of the invention.
* * * * *