Vibrating tip conducting probe microscope

A scanning probe microscope is provided for measuring at least one characteristic of a surface, the microscope including a force sensing probe which is responsive to the at least one characteristic of the surface, an oscillator which moves the position of the probe relative to the surface, a voltage source for establishing an electrical potential between the force sensing probe and the surface, and a detector which detects the oscillating component of the electrical current flow into or out of the probe as a measure of the at least one characteristic of the surface. The microscope can be operated to simultaneously acquire both electrical and topographical information from a surface of a substrate.

BACKGROUND OF THE INVENTION
 This invention relates to scanning probe microscopy, and, more
 particularly, to an instrument for the simultaneous acquisition of
 electrical and topographical information about a surface under
 electrochemical potential control.
 This invention was made with government support under Contract No.
 BIR-9513233 awarded by the National Science Foundation. The government has
 certain rights in the invention.
 The scanning electrochemical microscope (SECM) is a device for measuring
 the currents owing to electrochemically active species at, or near, a
 surface, and for mapping their distribution with a spatial resolution on
 the order of micrometers. FIG. 1 shows the schematic layout of a SECM as
 taught by Kwak et al, U.S. Pat. No. 5,202,004 and Bard et al, "Scanning
 Electrochemical Microscopy," Electroanalytical Chemistry, vol. 18:243-373
 (1993). The microscope includes a small metal electrode 1, made from a
 wire 2 covered by insulation 3 which is cut away at one end to expose the
 inner conductor. The electrode is placed in a solvent 4 containing
 dissolved ions 9, 10. The tip of the electrode is held in place some
 distance above a sample to be examined 5 which may, or may not be a
 conductor. The electrode 1, is connected to a potentiostat 8, to which is
 also connected an auxiliary electrode 7 and a reference electrode 6.
 Sample 5, if conducting, may also be connected to the potentiostat. The
 potentiostat is used to control the potential of the electrode 1 and the
 sample 5 (if conductive) with respect to the reference electrode 6 by
 means of a potential applied to the auxiliary electrode, as is well known
 to those skilled in the art.
 The dissolved ions 9, 10 can exist in one of several charge states, for
 example Fe.sup.++ or Fe.sup.+++. Referring to the less positively charged
 state as R and the more positively charged state as O, these ions,
 together with their associated dissolved anions, form a mediator, so
 called because they mediate the currents that flow between the electrodes.
 Suitable salts for forming mediator solutions are described in Bard et al.
 The two charged species exist in equilibrium at an electrode held at the
 formal potential, E.sub.0 for the process O{character pullout}R.
 If the electrode 1 is held negative of the formal potential, species O
 become reduced to R, giving rise to a current flow through the electrode
 1. As a result, the concentration of species O falls in the vicinity of
 the electrode 1, so that the current also falls. Eventually, the current
 falls to an equilibrium value determined by the geometry of the electrode
 and the speed with which replacement ions O can diffuse to the electrode
 1. For a disk electrode of radius a, this limiting current is given by the
 equation:
EQU I.sub.L =4nFDc (1)
 where n is the number of electrons transferred at each reduction, F is the
 Faraday constant (9.6.times.10.sup.4 Coulombs per mole of charge), D is
 the diffusion constant of the ions (often assumed to be the same for O as
 R and on the order of 5.times.10.sup.-6 cm.sup.2 /s) and c is the
 concentration (in moles per cm.sup.3, if a is in cm and D is in cm.sup.2
 /s). The time for the equilibrium current to be reached is small in the
 case of a small electrode, being on the order of a.sup.2 /D, or only a few
 milliseconds where a is on the order of a micron and D=5.times.10.sup.-6
 cm.sup.2 /s.
 The SECM profiles a surface by utilizing the manner in which the surface
 affects the diffusion of ions to the electrode. If, for example, the
 sample surface 5 is an insulator, it blocks the flow of ions to the
 electrode if the electrode is placed within a distance on the order of its
 diameter (d=2a) of the surface, as illustrated in FIG. 2. The ion species
 O, 9, is now constrained to flow in from the sides only, flow from below
 being blocked by the surface 5. If the electrode tip 1 is now scanned over
 a surface of varying height, then the flow of current will increase as the
 surface retreats from the tip, and increase if the surface approaches the
 tip. This current signal may be used to control the position of the tip
 and to form a map of the surface, as described by Kwak et al, above.
 The SECM may also be used to profile conducting surfaces as illustrated in
 FIG. 3. In this case, the flow of current is enhanced as the surface of
 sample 5 is approached. This is because ions that are reduced at the
 electrode 1 may be rapidly re-oxidized at the sample surface, thereby
 increasing the supply of ions O in the vicinity of the electrode 1.
 This scheme suffers two drawbacks: First, the resolution is limited by the
 exposed electrode area (being about an electrode diameter, d, under
 optimal circumstances). Second, it is difficult to profile heterogeneous
 surfaces which consist of both insulating and conducting portions. This is
 because a conducting surface which recedes from the probe gives a falling
 current, just as an insulating surface which approaches the tip.
 One solution to this problem has been proposed by Bard and Wipf, U.S. Pat.
 No. 5,382,336, which solution is illustrated in FIG. 4. In this scheme,
 the electrode 1 is oscillated up and down by an amount .delta. (16) so
 that the gap d (17) changes from d+.delta. to d-.delta. at the extreme of
 each oscillation as shown in the FIG. 5 plot of distance versus time. The
 corresponding oscillating component of the current (i.sub.cond) a
 conducting surface versus time is shown in FIG. 6. A similar plot of the
 current (i.sub.ins) for an insulating surface versus time is shown in FIG.
 7. The signal for the case of an insulating surface is in phase with the
 applied modulation, and, consequently, the output of a lock-in detector
 fed with this signal would be a positive voltage proportional to the
 amplitude of the oscillating current. The signal for a conducting surface
 is out of phase with the modulation, and so the output of a lock-in
 detector fed with this signal would be a negative voltage proportional to
 the amplitude of the oscillating current signal. In this way, the output
 of the lock-in detector can be used to generate a feedback signal which
 has the correct sign in all cases. However, this scheme suffers from the
 limited resolution inherent in SECM probes with micrometer dimensions.
 Attaching the SECM electrode to the force sensing cantilever of an atomic
 force microscope (AFM) would improve resolution because the high
 topographical resolution of the AFM could be combined with the chemical
 sensitivity of the SECM. Macpherson et al, 118 J. Am. Chem. Soc. 6445-52
 (1996) have attempted to do this by insulating a conducting AFM probe as
 illustrated in FIG. 8. An AFM probe 31 is coated on one side with a
 platinum film 32 contacted by a conducting clip 34. The clip is in turn
 connected to a conducting wire 36. The entire assembly is coated in a
 polystyrene film 33 to render it insulating. Operation of the cantilever
 in an AFM is assumed to have abraded away the insulating film in a small
 region near the tip 35, leaving an otherwise insulating film on the
 cantilever. The cantilever is inserted into an electrolyte 4 above a
 sample 5. Reference 37 and auxiliary electrodes 38 were also inserted into
 the electrolyte. In this case, the AFM was used for high resolution
 imaging, and the AFM cantilever coating was used as an electrode to
 generate a high concentration of the desired ions in the vicinity of the
 sample 5. SECM imaging was not attempted. This scheme has the drawback
 that the desired level of insulation is very hard to achieve. The currents
 through the cantilever are on the order of ten microamperes for an
 electrolyte concentration of 0.05 mole/liter. Using D=1.3.times.10.sup.-5
 cm.sup.2 /sec and I=10.sup.-5 A gives, from equation 1 above, a=0.08 cm,
 or d on the order of 1 mm. This is a very large exposed electrode area.
 None of the existing SECM or AFM prior art techniques can detect the very
 small currents associated with electrochemical processes in single
 molecules. Such small currents have been detected by using a well
 insulated scanning tunneling microscope tip (Fan and Bard, 267 Science
 871-74 (1995)) or by working in an insulating fluid (Han, Durantini et al,
 101 J. Phys. Chem. 10719-725 (1997)) (where the quantitative advantage of
 potential control is lost). These experiments show that detection of
 electrochemical signals from single molecules requires a sensitivity in
 the picoampere (pA) range, six orders of magnitude smaller than the
 leakage signal from poorly insulated AFM tips.
 Accordingly, there remains a need in the art for a technique and system to
 produce an SECM signal that is highly localized to a region close to an
 atomic force microscope tip. There also remains a need for a technique
 which is able to detect very small currents associated with
 electrochemical processes which avoids the problems of current leakage
 from AFM tips. Finally, there also remains a need for a technique and
 system which is able to acquire simultaneously AFM topographical images
 and SECM current data.
 SUMMARY OF THE INVENTION
 The present invention meets those needs by providing a scanning probe
 microscope for measuring at least one characteristic of a surface, the
 microscope including a force sensing probe which is responsive to the at
 least one characteristic of the surface, an oscillator which moves the
 position of the probe relative to the surface, a voltage source for
 establishing an electrical potential between the force sensing probe and
 the surface, and a detector which detects the oscillating component of the
 electrical current flow into or out of the probe as a measure of the at
 least one characteristic of the surface. In a preferred embodiment of the
 invention, The at least one characteristic of the surface is the
 electrochemical potential associated with the surface, such
 electrochemical potential resulting from molecules or ions on the
 substrate surface.
 In a preferred form, the force sensing probe comprises an atomic force
 microscope cantilever including a tip. Preferably, the cantilever and the
 tip include at least one surface which has been coated with an
 electrically conductive material such as, for example, platinum. The
 cantilever is substantially completely covered with an electrically
 non-conductive material to render it non-conductive except for a portion
 of the tip. In a preferred form the non-conductive material comprises a
 polymer such as, for example, polystyrene.
 The force sensing probe is oscillated in one of two preferred manners. In
 one embodiment, the oscillator comprises an acoustic transducer in
 communication with the probe. In another embodiment, the probe includes on
 a surface thereof a magnetic or magnetostrictive material, and the
 oscillator, preferably a solenoid, creates an oscillating magnetic field.
 The detector the detector comprises a lockin circuit which provides a
 measure of the oscillatory component of the current. Preferably, the
 reference signal for the lockin is generated by the same signal used by
 the oscillator.
 The present invention also provides a process for measuring the
 electrochemical properties of a surface of a substrate which includes the
 steps of providing an oscillated force sensing probe, establishing an
 electrical potential between the force sensing probe and the surface of
 the substrate in an aqueous electrolyte, the potential being insufficient
 to cause ions in the electrolyte to undergo oxidation or reduction, moving
 the oscillated force sensing probe across the surface, and measuring the
 oscillatory component of the electric current resulting from contact
 between the probe and electro-active species on the surface of the
 substrate. In a preferred embodiment of the invention, the electro-active
 species comprise molecules or ions. The process is particularly useful in
 the measurement of the electrochemical properties of proteins such as, for
 example, beta-carotene.
 In yet another embodiment of the invention, a scanning probe microscope for
 the simultaneous acquisition of electrical and topographical information
 from a surface of a substrate is provided and includes a force sensing
 probe, an oscillator which moves the position of the probe relative to the
 surface, a voltage source for establishing an electrical potential between
 the force sensing probe and the surface, a first detector which detects
 the oscillating component of the electrical current flow into or out of
 the probe as a measure of the electrochemical potential associated with
 the surface, and a second detector which detects the deflection of the
 force sensing probe as a measure of the topology of the surface.
 In operation, the microscope includes a conducting AFM probe (cantilever
 and tip) which is electrically insulated so as to expose only a small
 region in the vicinity of the tip. The probe is oscillated, for example,
 either by acoustic excitation or by applying a magnetic force to a
 magnetic coating on the cantilever. A lock-in is used to detect the
 oscillating component of current that arises from the oscillation of the
 tip with respect to a sample surface containing electrochemically active
 molecules. The electrolyte is chosen so as to provide a current path to
 the tip and substrate but with a formal potential far enough removed from
 the surface molecules that any additional electrochemical current on
 approaching the surface comes only from electrochemically active molecules
 on the surface under study. Because the leakage current is not
 oscillating, detection of the oscillating signal in a sufficiently narrow
 bandwidth (such as, for example, 500 Hz) permits extraction of signals
 from the surface which are orders of magnitude smaller than the leakage
 signal owing to bulk electrolyte. As the oscillating signal is confined to
 the region of the tip closest to the surface, high resolution
 electrochemical imaging is possible even if the insulation of the tip is
 exposed over a much larger area. Conventional AFM topographical scans may
 also be acquired at the same time.
 Accordingly, it is a feature of the present invention to provide a SECM
 signal that is highly localized to a region close to an atomic force
 microscope tip. It is a further feature of the invention to reduce the
 noise from unwanted leakage currents not associated with the surface under
 study. It is a further feature of the invention to permit simultaneous
 acquisition of AFM topographical images and SECM current data. These and
 other features and advantages of the present invention will become
 apparent from the following detailed description, the accompanying
 drawings, and the appended claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
 The microscope of the present invention is shown in its preferred
 embodiment in FIG. 9. The microscope comprises an atomic force microscope
 (AFM) cantilever assembly 41 which has coated on one side thereof an
 electrically conductive material 42 such as a metal. In a preferred form,
 electrically conductive material 42 comprises platinum. A wire 43 is
 attached to this conducting layer and the cantilever assembly is made to
 be electrically insulative. This may be accomplished, for example, by
 encapsulation of the assembly in an electrically insulating film 44 such
 as, for example a polymeric material such as polystyrene. The connecting
 wire 43 is insulated with a dielectric sheath 52 so that no part of it is
 in contact with the electrolyte 45. Electrolyte 45 typically comprises an
 aqueous solution containing dissolved ions which can be chemically reduced
 or oxidized. The dissolved ions render the electrolyte electrically
 conductive.
 In this preferred embodiment, the microscope is designed to detect and
 measure the electrochemical properties of molecules or ions on the surface
 of a sample. Thus, as shown in FIG. 9, sample 47 contains
 electrochemically active species 48 anchored to its surface. This species
 has different charge states designated as O.sub.1 and R.sub.1. These
 species 48, in the form of ions or molecular ions, may be chemically
 tethered to the conducting substrate 47 or packed within a molecular
 monolayer self-assembled on the sample surface. The species could be
 naturally assembled, as, for example, in the case of electroactive surface
 proteins embedded in a biological membrane which has been spread onto the
 electrode 47. The sample is covered by and immersed in a supporting
 electrolyte 45. The dissolved ions in this electrolyte O.sub.2 (54) and
 R.sub.2 (55) have the property that they are much less easily reduced
 and/or oxidized than the ions or molecules on the sample surface.
 For example, the molecule on the surface (48, O.sub.1 R.sub.1) could be
 beta-carotene, an organic molecule that is oxidized at the formal
 potential of +0.53V on the saturated calomel electrode (SCE) scale. The
 source of dissolved ions 54, 55 could be sodium perchlorate which produces
 the sodium ion, Na.sup.+, and the perchlorate ion HCLO.sub.3.sup.- in
 aqueous solution. These ions do not undergo bulk-solution oxidation or
 reduction in aqueous electrolyte. However, the electrolyte serves the
 purpose of rendering the solution electrically conductive, so that tip 58
 and sample 47 may be maintained under electrochemical potential control by
 auxiliary electrode 50 with respect to the reference electrode 49. A small
 additional bias voltage V from source 46, may be applied between the
 conducting AFM tip 58 and the sample 47as shown in FIG. 9.
 The AFM tip is oscillated up and down by a small amount .delta., 51 by an
 oscillating voltage 64 at a frequency f. In preferred embodiments, such
 oscillation is accomplished by the use of either an acoustic signal or a
 magnetic signal as will be described in greater detail below.
 Because the species of interest 48 is attached to the sample surface, then,
 in the absence of a mediator which is reduced and oxidized within the
 potential difference between tip and substrate, electrons from voltage
 source 46 can only be transferred into or out of the molecule in question
 by the tip 58 if it comes sufficiently close for direct electron transfer
 to occur. For direct electron transfer to occur requires that the tip 58
 touch the species of interest 48. Thus, the current from the tip 58 will
 not vary in an oscillatory fashion unless the tip is directly over the
 species of interest 48, at which time a current on the order of picoamps
 to hundreds of picoamps will flow (see, Han, Durantini et al., J. Phys.
 Chem. 101:10719-10725 (1997)), depending upon the potential of the surface
 and the voltage applied between the tip and substrate 46.
 This current is detected by resistor 61, amplified by amplifier 62, and fed
 to the lockin circuit 63. As is known in this art, the lockin circuit 63
 has the capability of measuring the magnitude of a signal that is in phase
 with a reference oscillation, and the magnitude of the component that is
 out of phase with a reference component, averaged over a selected
 integration time. The lockin circuit reference oscillation signal is
 generated by the same signal used to oscillate the tip 64. The output
 signal 65 is proportional to the amplitude of the oscillating current from
 the oscillator.
 With care, an exposed area of electrically conductive material of only a
 few microns by a few microns at the tip can be achieved by covering the
 tip with an insulating material and then scanning the tip over a hard
 electrically conductive surface and abrading away the insulating material
 until an electrical current is detected. Operation of the tip in a
 supporting electrolyte concentration of 0.01 M/liter gives rise to a
 leakage current of only a few nanoamperes (nA), as can be derived from
 equation 1 with D=5.times.10.sup.-6 cm.sup.2 /sec. The noise from this
 background signal in a bandwidth of B Hz is dominated by shot noise and is
 given by the equation:
EQU I.sub.noise =2+L qI.sub.laek +L B (2)
 where I.sub.noise is the RMS value, I.sub.laek is the leakage current, and
 q is the charge on an electron. Taking B=500 Hz and I.sub.laek =1 nA, a
 value is obtained for I.sub.noise of 0.4 pA. Thus, oscillating currents of
 sub-picoampere (pA) magnitude may be detected in the presence of DC
 leakage currents of nanoampere magnitude with an instrument bandwidth of
 500 Hz. If each pixel of image information is acquired in 1 millisecond,
 this results in acquisition of a 256.times.256 pixel image in just over a
 minute.
 In one mode of operation, a small bias voltage 46 (ca. 0.1V) is applied
 between the tip 58 and substrate 47, and side-by-side images are acquired
 from the normal AFM topographic signal and the current signal output 65
 from the lockin circuit 63 and amplifier 62 as the potential of the
 substrate is varied. It has been shown that the electron transfer through
 the surface species is a maximum close to the formal potential for
 oxidation/reduction of the surface species 48. See, Tao, Phys. Rev.
 Lefters, 76:4066-4069 (1996). Thus, the current image will show a maximum
 brightness when the surface potential is set near the formal potential of
 the species on the surface.
 In a preferred embodiment, the tip 58 is oscillated by an oscillating
 magnetic field as disclosed in Lindsay, U.S. Pat. Nos. 5,513,518, and
 5,612,491, and 5,753,814, and pending U.S. patent application Ser. No.
 08/905,815, now U.S. Pat. No. 5,983,712, the disclosures of which are
 hereby incorporated by reference. This arrangement is shown in FIG. 10.
 There cantilever 71 is coated on one side thereof with an electrically
 conductive material such as, for example, platinum film 72. The cantilever
 is coated on its opposite surface with a magnetic or magnetostrictive film
 74 such that the direction of magnetic moment M is along the soft axis 75
 of the cantilever 71. The entire cantilever assembly is then made
 electrically insulating by encapsulating the assembly in an insulating
 film 73 of an electrically nonconductuve material such as polystyrene,
 except for a small area around the tip 58 which is removed by the abrasion
 that occurs when the tip is scanned across a hard surface as explained
 previously. The motion of the tip is detected in a manner conventional in
 this art using a laser source 80 to produce a laser beam 76, 77 which is
 reflected off of the surface of the cantilever 71 and detected by a
 detector 82. A solenoid 78 is placed in close proximity to the magnetic or
 magnetostrictive film 74 to generate motion by tip 58 when it is driven by
 an oscillating voltage 79.
 In another embodiment of the invention which is shown in FIG. 11, a
 cantilever 71 having a conductive coating 72 and an insulating
 encapsulating layer 73 is securely attached to a piezoelectric transducer
 90 which is driven by an oscillating voltage 79. The corresponding
 mechanical excitation of tip 58 leads directly to motion of the end of the
 tip if a suitable resonance is driven.
 While certain representative embodiments and details have been shown for
 purposes of illustrating the invention, it will be apparent to those
 skilled in the art that various changes in the methods and apparatus
 disclosed herein may be made without departing from the scope of the
 invention, which is defined in the appended claims.