U.S. patent number RE35,317 [Application Number 08/321,649] was granted by the patent office on 1996-08-27 for potentiostatic preparation of molecular adsorbates for scanning probe microscopy.
This patent grant is currently assigned to The Arizona Board of Regents. Invention is credited to Stuart M. Lindsay.
United States Patent |
RE35,317 |
Lindsay |
August 27, 1996 |
Potentiostatic preparation of molecular adsorbates for scanning
probe microscopy
Abstract
.[.A method of.]. .Iadd.An apparatus and method for
.Iaddend.preparing molecular adsorbates for scanning probe
microscopy by potentiostatic methods. Negatively charged molecules
are deposited upon and held to a substrate with an electrochemical
cell having a gold substrate, a platinum wire counter electrode and
a silver wire reference electrode. The polymer to be observed is
dissolved into a buffer solution which is non-reactive with the
substrate which is gold (111).
Inventors: |
Lindsay; Stuart M. (Tempe,
AZ) |
Assignee: |
The Arizona Board of Regents
(Tempe, AZ)
|
Family
ID: |
24958493 |
Appl.
No.: |
08/321,649 |
Filed: |
October 11, 1994 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
Reissue of: |
736095 |
Jul 26, 1991 |
05155361 |
Oct 13, 1992 |
|
|
Current U.S.
Class: |
250/307; 204/400;
204/403.01; 204/409; 204/412; 205/780.5; 205/794.5; 250/304;
250/440.11; 361/234 |
Current CPC
Class: |
G01Q
30/14 (20130101); Y10S 977/852 (20130101) |
Current International
Class: |
G01N
1/28 (20060101); H01J 37/26 (20060101); H01J
37/20 (20060101); H01J 037/26 (); H01J 037/20 ();
G01N 001/28 () |
Field of
Search: |
;250/307,304,440.1,306
;361/234 ;204/153.1,400,403 |
References Cited
[Referenced By]
U.S. Patent Documents
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|
Primary Examiner: Berman; Jack I.
Attorney, Agent or Firm: D'Alessandro & Ritchie
Government Interests
INTRODUCTION
.[.This.]. .Iadd.The .Iaddend.present invention relates generally
to scanning probe microscopy and more particularly to .Iadd.an
apparatus and method for .Iaddend.the potentiostatic preparation of
molecular adsorbates for study with scanning probe microscopes.
.Iadd.This invention was made with Government support under
contract No. N00014-90-J-1655 awarded by the Department of the Navy
and grant DIR 89-20053 awarded by the National Science Foundation.
The Government has certain rights in the invention..Iaddend.
Claims
Accordingly, what is claimed is:
1. A method of preparing molecular adsorbates for scanning probe
microscopy comprising: loading a substrate into a cell; placing the
cell on a scanning probe microscope; placing a clean reference
electrode in said cell; placing a clean counter electrode in said
cell in spaced relationship to said reference electrode; dissolving
a polymer containing negatively charged molecules into a buffer
solution that is inert relative to said substrate; filling said
cell with said polymer .[.contained.]. .Iadd.containing
.Iaddend.buffer solution; activating said reference electrode and
said counter electrode and applying a potential to said substrate
to deposit and secure said polymer onto said substrate for
examination by said microscope.
2. A method according to claim 1 in which said substrate is gold
(111).
3. A method according to claim 1 in which said reference electrode
is silver wire.
4. A method according to claim 1 in which said counter electrode is
platinum wire.
5. A method according to claim 1 in which said buffer solution is
NaH.sub.2 PO.sub.4 at a pH of 6.
6. A method according to claim 3 in which said electrodes are
activated to a voltage of from about -1.2 up to about .Iadd.+
.Iaddend.1.3.
7. A method according to claim 5 in which said electrodes are
activated to a voltage of from about -1.2 up to about .Iadd.+
.Iaddend.1.3.
8. A method according to claim 2 in which said reference electrode
is silver wire.
9. A method according to claim 8 in which said counter electrode is
platinum wire.
10. A method according to claim 9 in which said buffer solution is
NaH.sub.2 PO.sub.4 at a pH of 6.
11. A method according to claim 10 in which said electrodes are
activated to a voltage of from about -1.2 up to about .Iadd.+
.Iaddend.1.3. .Iadd.
12. A method of preparing molecular adsorbates for scanning probe
microscopy comprising:
loading a substrate into a cell;
placing the cell on a scanning probe microscope;
placing a clean reference electrode in said cell;
placing a clean counter electrode in said cell in spaced
relationship to said reference electrode;
dissolving a polymer containing charged molecules into a buffer
solution that is inert relative to said substrate;
filling said cell with said polymer containing buffer solution;
activating said reference electrode and said counter electrode;
and
applying a potential between said substrate and said counter
electrode to deposit and secure said polymer onto said substrate
for examination by said microscope..Iaddend..Iadd.13. A method
according to claim 12 in which said substrate comprises
gold..Iaddend..Iadd.14. A method according to claim 12 in which
said reference electrode is silver wire..Iaddend..Iadd.15. A method
according to claim 12 in which said
counter electrode is platinum wire..Iaddend..Iadd.16. A method
according to claim 12 in which said buffer solution is NaH.sub.2
PO.sub.4 at a pH of 6. .Iaddend..Iadd.17. A method according to
claim 13 in which said substrate is activated to a voltage in a
range of about -1.2 volts up to about +1.3 volts with respect to
said reference electrode by application of a suitable voltage to
said counter electrode..Iaddend..Iadd.18. A method according to
claim 14 in which said substrate is activated to a voltage in a
range of about -1.2 volts up to about +1.3 volts with respect to
said reference electrode by application of a suitable voltage to
said counter electrode..Iaddend..Iadd.19. A method according to
claim 15 in which said substrate is activated to a voltage in a
range of about -1.2 volts UP to about +1.3 volts with respect to
said reference electrode by application of a suitable voltage to
said counter electrode..Iaddend..Iadd.20. A method according to
claim 16 in which said substrate is activated to a voltage in a
range of about -1.2 volts up to about +1.3 volts with respect to
said reference electrode by application of a suitable voltage to
said counter electrode..Iaddend..Iadd.21. A method according to
claim 13 in which said reference electrode is silver
wire..Iaddend..Iadd.22. A method according to claim 15 in which
said reference electrode is silver wire..Iaddend..Iadd.23. A method
according to claim 13 in which said counter electrode is platinum
wire..Iaddend..Iadd.24. A method according to claim 16 in which
said counter electrode is platinum wire..Iaddend..Iadd.25. A method
according to claim 23 in which said buffer solution is NaH.sub.2
PO.sub.4 at a pH of 6. .Iadd.26. A method according to claim 25 in
which said substrate is activated to a voltage in a range of about
-1.2 volts up to about +1.3 volts with respect to said reference
electrode by application of a suitable voltage to said counter
electrode..Iaddend..Iadd.27. An electrochemical fluid cell and
substrate assembly for use in studying molecular adsorbates with a
scanning probe microscope, said cell comprising:
an electrically conductive substrate;
an electrically insulating cell wall having an inner boundary
defining a fluid container open at its top, said fluid container
having a bottom boundary defined by said substrate;
said substrate having a portion extending under and beyond said
inner boundary;
a reference electrode extending from beyond said fluid container
into said fluid container through said top;
a counter electrode extending from beyond said fluid container into
said fluid container through said top and maintained in spaced
relationship to said reference electrode;
a voltage potential capable of being applied to said substrate by
connecting said voltage potential to said electrically conductive
substrate at said portion extending under and beyond said inner
boundary..Iaddend..Iadd.28. An electrochemical fluid cell and
substrate assembly according to claim 27 wherein said substrate
comprises gold..Iaddend..Iadd.29. An electrochemical fluid cell and
substrate assembly according to claim 27 wherein said reference
electrode is silver wire..Iaddend..Iadd.30. An electrochemical
fluid cell and substrate assembly according to claim 27 wherein
said counter electrode is platinum
wire..Iaddend..Iadd.31. An electrochemical fluid cell and substrate
assembly according to claim 28 wherein said reference electrode is
silver wire..Iaddend..Iadd.32. An electrochemical fluid cell and
substrate assembly according to claim 28 wherein said counter
electrode is platinum wire..Iaddend..Iadd.33. An electrochemical
fluid cell and substrate assembly according to claim 28 wherein
said reference electrode is silver wire and said counter electrode
is platinum wire..Iaddend..Iadd.34. An electrochemical fluid cell
and substrate assembly for use in studying molecular adsorbates
with a scanning probe microscope, said cell comprising:
an electrically conductive substrate;
an electrically insulating cell wall having an inner boundary
defining a fluid container open at its top, said fluid container
having a bottom boundary defined by said substrate;
said substrate in electrical contact with a conductor having a
portion extending under and beyond said inner boundary;
a reference electrode extending from beyond said fluid container
into said fluid container through said top;
a counter electrode extending from beyond said fluid container into
said fluid container through said top and maintained in spaced
relationship to said reference electrode;
a voltage potential capable of being applied to said substrate by
connecting said voltage potential to said electrically conductive
substrate at said portion of said conductor extending under and
beyond said inner boundary..Iaddend..Iadd.35. An electrochemical
fluid cell and substrate assembly according to claim 34 wherein
said substrate comprises gold..Iaddend..Iadd.36. An electrochemical
fluid cell and substrate assembly according to claim 34 wherein
said reference electrode is silver wire..Iaddend..Iadd.37. An
electrochemical fluid cell and substrate assembly according to
claim 34 wherein said counter electrode is platinum
wire..Iaddend..Iadd.38. An electrochemical fluid cell and substrate
assembly according to claim 35 wherein said reference electrode is
silver wire..Iaddend..Iadd.39. An electrochemical fluid cell and
substrate assembly according to claim 35 wherein said counter
electrode is platinum wire..Iaddend..Iadd.40. An electrochemical
fluid cell and substrate assembly according to claim 35 wherein
said reference electrode is silver wire and said counter electrode
is platinum wire. .Iaddend.
Description
BACKGROUND OF THE INVENTION
Various ways have heretofore been proposed for chemically reacting
molecules with a metal substrate in an electrochemistry cell. In
some cases, the prior methodology allows molecules to be bonded
strongly enough so that they can be imaged in a scanning tunnelling
microscope (STM) or in .[.the.]. .Iadd.an .Iaddend.atomic force
microscope (AFM). However, in the case of negatively charged
molecules, such as DNA, it is extremely difficult to get them to
adhere to an electrode because most metal surfaces are
intrinsically negatively charged and, as such, repel the molecule.
Thus a clear need exists for new and improved technology for the
potentiostatic preparation of negatively charged .[.molecules.].
.Iadd.molecular .Iaddend.adsorbates such as DNA to enable them to
be properly bonded to .Iadd.a .Iaddend.suitable substrate so
.Iadd.that .Iaddend.they can be imaged in .Iadd.a .Iaddend.scanning
tunnelling microscope .[.(STM).]. or .Iadd.an .Iaddend.atomic force
.[.microscopes (AFM).]. .Iadd.microscope.Iaddend.. It is
.[.toward.]. .Iadd.to .Iaddend.this end that the present invention
is directed.
BRIEF SUMMARY OF THE INVENTION
The present invention is predicated upon the discovery of a
remarkably simple procedure for getting negatively charged
molecules onto a substrate and holding them there. The new
methodology is based in part on the concept that DNA (or any other
negatively charged molecule) can be attracted to a surface which is
positively charged by virtue of its interaction with an electrolyte
(.[.See: Lindsay et al, 1988.]. .Iadd.See: Lindsay, S. M., and
Barris, B., "Imaging DNA Molecules on a Metal Surface Under Water
by STM", Journal of Vacuum Science and Technology. Vol. A6, Pages
544-547 (1988)).Iaddend.. What is new and unexpected is that the
same forces that attract the molecules to the surface are capable,
in the practice of the present invention, of holding such molecules
in place on that surface for study in an .[.SPM.]. .Iadd.STM
.Iaddend.or an AFM. More particularly, the present invention
relates to the potentiostatic preparation of molecular adsorbates
for scanning probe microscopy in an electrochemical cell having a
gold substrate, a platinum wire counter electrode and a silver wire
reference electrode placed upon the microscope. The polymer to be
observed is dissolved into a buffer solution which is non-reactive
with the gold in the substrate and thereafter quickly deposited
upon the substrate. Once the cell is filled, the reference
electrode and the counter .[.electrodes.]. .Iadd.electrode
.Iaddend.are connected and a stable layer of adsorbate is formed on
the gold electrode where it can be readily scanned with the
microscope probe.
Accordingly, the principle object of the present invention is to
provide new and improved methodology for preparing molecular
adsorbates for scanning probe microscopy.
Another object of the present invention is to provide methodology
especially adapted to potentiostatically prepared negatively
charged molecular adsorbates for scanning probe microscopy.
These and still further objects as shall hereinafter appear are
readily fulfilled by the present invention in a remarkably
unexpected manner as will be readily discerned from the following
detailed description of an exemplary embodiment thereof especially
when read in conjunction with the accompanying drawing .[.in which
like parts bear like numerals throughout the several views.]..
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings:
FIG. 1.[...]. is a schematic drawing of a simple electrochemical
cell .Iadd.according to the present invention.Iaddend.; and
FIG. 2 is a cyclic voltammogram of a solution used for deposition
of DNA taken in situ using a phosphate buffer solution adjusted to
pH=6 with NaOH and containing .[.5 micrograms 5/ml.]. .Iadd.5
micrograms per milliliter .Iaddend.of DNA.
DESCRIPTION OF PREFERRED EMBODIMENT
A small electrochemistry cell is mounted on an STM or AFM as
described in my prior U.S. Pat. No. 4,868,396. A sketch of the
current simplified cell is shown in FIG. 1. The substrate is Au
(111), the counter electrode a platinum wire and the reference
electrode a silver wire. It has been discovered that silver wires
produce results that are identical to Ag/AgCl/KCl reference
electrodes in these particular solutions, but are much easier to
use and do not cause chlorine contamination of the substrate.
One practical arrangement of an electrochemistry cell is shown in
FIG. 1. Referring to FIG. 1, the electrochemistry cell is
designated by the general reference 10 and comprises a gold-on-mica
substrate 11.[.,.]. .Iadd.and .Iaddend.a glass cell 12, having a
polished bottom that forms a seal against the gold substrate. A
platinum (Pt) wire counter electrode 13 and a silver (Ag) wire
reference electrode 14 extend into cell 12. Each of these wires are
longer than needed and a fresh cut surface is introduced into the
cell for each experiment by advancing the .[.electrode.].
.Iadd.electrodes .Iaddend.13, 14 in .[.its.]. .Iadd.their
.Iaddend.respective electrode .[.holder.]. .Iadd.holders
.Iaddend.15, 15. A stainless steel plate 16 is glued to the lower
exterior surface of glass cell 12 to hold down the substrate 11 and
make electrical contact with the gold.
Cleanliness is critical. As will appear, an excess length of wire
is used for each wire electrode. Before starting the next run, the
used portion of the wire is cut away and a portion of the fresh
wire is advanced into the cell. A fresh gold substrate is also used
for each run.
In one practice of the present invention, a substrate is loaded
onto the SPM. Clean reference and counter electrodes are placed
into a clean glass cell on the substrate as shown in FIG. 1. The
polymer is dissolved into a buffer solution that does not react
with the gold over the appropriate range of substrate potentials.
One suitable buffer solution for use with Au (111) between -1.2 and
.[.=.]. .Iadd.+ .Iaddend.1.3 V (vs. the Ag reference) is NaH.sub.2
PO.sub.4 .[...]. (10 mM adjusted to pH6 with NaOH). A cyclic
voltammogram taken in situ is shown in FIG. 2. For sparse coverage
of the electrode, a solution that, at full adsorption, gives less
.[.that.]. .Iadd.than .Iaddend.a monolayer coverage of the
macromolecule is used. For example, in a 50 microliter cell (0.5
cm.sup.2 electrode area) less than 5 micrograms of DNA per mL of
solution are required.
The solution is placed onto the substrate as quickly as possible
(to minimize contamination) and, once the cell is full, the
reference and counter electrodes are connected. Any positive
potential (in case of DNA) between the potentials at which
reactions occur (from -0.2 V vs. Ag to +0.6 V vs. Ag; the DNA bases
oxidize at higher voltage) may be applied to the substrate. There
are some small reversible phosphate .[.absorptions.].
.Iadd.adsorptions .Iaddend.at lower potentials, but in the
double-layer region macromolecules can be seen in stable
arrangements all the way up to about 1 volt. The voltage employed
in any given reading is correlated to the reference electrode. The
voltage required for deposition is dependant upon the salt solution
used. The values reported herein are for the phosphate buffer
solution.
If the solutions are free of contamination and, in the case of the
STM, the tip is well insulated, a very stable layer of
.[.absorbate.]. .Iadd.adsorbate .Iaddend.is formed on the gold
electrode. It may be scanned in situ repeatedly with no sign of
sample movement or degradation. Indeed this is the salient feature
of this invention, namely that .[.the.]. an adsorbate, when under
potentiostatic control, is remarkably stable. Furthermore, the
adsorbate layer may be lifted .[.on and off.]. .Iadd.off and placed
back on .Iaddend.the electrode surface at will simply by cycling
the substrate potential between a positive value and -0.2 V (vs.
Ag). Of course, these conditions represent a much diminished
disruption of the solvated structure of the polymers compared to
methods where the adsorbate is chemically reacted onto the
substrate.
The coverage of the substrate, even with the simple layout shown in
FIG. 1, is remarkably homogeneous. The whole problem of molecular
microscopy is now reduced to scanning an area large enough to
contain a few molecules (as calculated from the expected coverage,
given the cell geometry and sample concentration) and then applying
a suitable potential. Furthermore, reactions and various dynamic
processes may be studied simply by allowing them to proceed in the
cell (using components and a potential that avoid irreversible
reactions) and then applying .[.and.]. .Iadd.an .Iaddend.attractive
charge to the substrate.
Finally it should be noted that the problem of contamination is
greatly reduced. Only those molecules that satisfy conditions for
physical adsorption appear in the image. In contrast a vacuum or
ambient image would show all contaminants.
This method can also be used to hold positively charged molecules
providing the reaction current due to dissolved oxygen is
eliminated. This may be done .Iadd.by .Iaddend.using degassed
solutions and by operating in an inert gas .[.enviroment.].
.Iadd.environment.Iaddend..
Experiments demonstrate that this method yields excellent high
resolution images of macromolecular .[.absorbates.].
.Iadd.adsorbates .Iaddend.in both the STM and the AFM. The
electrodes and buffers herein disclosed are intended as
representative preferred materials and not by way of limitation
thereon.
From the foregoing, it is readily apparent that a useful embodiment
of the present invention has been herein described and illustrated
which fulfills all of the .[.aforestated.]. .Iadd.aforementioned
.Iaddend.objectives in a remarkably unexpected fashion. It is of
course understood that such modifications, alterations and
adaptations as may readily occur to the artisan confronted with
this disclosure are intended within the spirit of this disclosure
which is limited only by the scope of the claims appended
hereto.
* * * * *