Double barrel tip

Several of the procedures described in the previous sections can be advantageously carried out with double barrel tips. Such a probe consists of two capillaries (see Sec. V.B), one of which acts as the potentiometric sensor, while the other is used to determine the tip-substrate distance. For example (79), a gallium microdisk was combined with an ion-selective (K+) potentiometric probe to image K+ activity near the aperture of a capillary (see Fig. 7). Similarly (77), a double barrel tip with one channel as an open Ag/ AgCl micropipette for solution resistance measurement and the other channel as an ion-selective neutral carrier-based microelectrode for potentiometric measurements was successfully used to image concentration distributions for NH4 (Fig. 8) and Zn2+ (Fig. 9). While dual-channel tips facilitate the approach of the substrate and permit a direct determination of the absolute tip-substrate distance, their difficult fabrication severely limits their use. Reference 80 compares the above methods. 

The tips discussed so far have been amperometric probes, typically of Pt-Ir, that produce faradaic currents reflecting redox processes at an exposed surface. However, it is also possible to use potentiometric tips, such as ion selective electrodes based on micropipets, in an SECM (28, 29). These produce potentials (with respect to a reference electrode) that depend logarithmically on the solution activity of a specific ion. Tips of this type that can detect H, Zn, NH4, and with a resolution of a few pirn have been described. Tips of this type are especially useful for detection of nonelectroactive ions, like many of those of interest in biological systems. However, such tips are passive probes, in that they detect the local activity of a given species but do not sense the presence of the substrate. They cannot be used to determine d, so they must be positioned with respect to a substrate, such as, in studying a concentration gradient at an electrode, by visual observation with a microscope, by resistance measurements, or by using a double-barrel tip that contains both an amperometric element and a potentiometric one. 

SECM associated with scanning ion conductance microscopy (SICM) requires a double tip, on one side of which is a conventional microdisc electrode and on the other side is a narrow pipette filled with electrolyte and an electrode that measures ionic conductance through the mouth of the pipette with respect to another electrode in the bulk solution. When the pipette mouth is within one pipette tip radius away from the sample surface, the conductance varies sufficiently to be used as a control signal to maintain the z-position of the tip during the scans, thereby affording constant-distance SECM operations [133,134]. This methodology is fast and apparently less-challenging to implement than shear force SECM, but it requires the fabrication of double-barrel tips in which one channel is left empty and the other is filled with a conventional microdisc. 

Analogous to SECM amperometric methods, SECM potentiometric methods are based on the measurement of electrode potentials (tip and substrate, AE-j- and AEg, respectively) as a function of various parameters, including tip-substrate distance (d), XY location, and time. Again, a potentiometric tip responsive to the desired solution component is necessary to perform any SECM potentiometric experiment. To use potentiometric probes in SECM, it must be possible to exactly evaluate the tip-to-substrate distance. Different electrochemical methods to evaluate the potential-distance dependence have been investigated, for example, utilization of metal/metal oxide electrodes as both amperometric and potentiometric tips for pH measurement," double barrel tips having one... 

Several of the procedures described in the previous sections can be advantageously carried out with double barrel tips. These probes basically consist of two capillaries, see Section 10.1.2.4, where one... 

In case of passive tip, when feedback effect is not available other distance measuring method has to be selected. Double barrel tip, one barrel for imaging, the other one for distance measuring, has been successfully applied [51,52]. In case of pH imaging double function antimony tip [62] could be used for both distance measurement (amperometric mode) and potentiometric mode (pH imaging). The constant height imaging mode is appropriate in case of thin film samples. It requires simpler apparatus. 

The electrodes used in the above studies were double-barreled glass pH sensitive microelectrodes, and the spatial retinal pH profile was recorded by withdrawing the microelectrode tip at a rate of 1 //m/s or lOOpm/step across the retina in vivo or in vitro. In a typical retina pH profile (Fig. 10.9), measured in cat retina by the microelectrode, started from the choroids (Ph = 7.41, at distance Ojum). The pH steadily decreased to a minimum value (a maximum [H+] concentration) in the proximal portion of the outer nuclear layer (pH = 7.14 at —140 jum), then increased to —7.28 (at —310 pm) at the vitreous retinal border. The peak [H+] concentration in this layer indicated that a net production of proton occurred across the avascular outer retina [76],... 

When making actual intracellular measurements, the microelectrodes are mounted in micromanipulators for cell penetration. It has been found that beveling the tip of the micropipet ISE aids in the ability to enter the cell and also enables the fabrication of electrodes with smaller tip diameters of O.I (im. Once inside the cell, single-barrel liquid membrane micro-ISEs (as described above) allow for the measurement of only steady-state ion activities. For excitable cells, where ion levels change rapidly, one cannot differentiate the potential changes resulting from variations in the intracellular activity of a specific ion and the living cell membrane potential. For such situations, double-barrel-type liquid membrane micro-ISEs have been developed (K2). 

When performing potentiometric measurements with a traveling probe, one needs to take into account the effect of ohmic drop. Whereas this effect is the basis of SRET measurements, it becomes a nuisance in potentiometric SECM applications. If the substrate is an electrode involved in a Faradaic process, the current flowing between the substrate and the counterelectrode leads to potential gradients in solution. The tip will be sensitive to the potential distribution, and this may overcome the signal due to the concentration change for the ion of interest. This is particularly pronounced if the reference electrode associated to the tip is located far away in the bulk and of course if the solution conductivity is low. To remedy this situation some researchers have used double barrel electrodes where one channel acts as the ion-sensitive element and the other acts as a reference electrode (81,82). In the life sciences intracellular measurements are usually carried out in this way. Alternatively, it is possible to subtract the ohmic drop from the tip... 

Figure 2 The principle of measurement by means of an ion-selective double-barreled microelectrode inside a cell. The cell is in a bath the solution of which is grounded via a reference electrode. Each barrel is connected via a chlorided silver wire (shown coiled) to amplifiers (triangles). The reference barrel of the double-barreled electrode directly records the intracellular electrical potential, the membrane potential (Em). The ion-selective barrel, indicated by the plug of ion exchanger in the tip, records the sum of the membrane potential and a potential , related to the chemical potential of the ion in question (of activity a) (see eqn [1]). / is obtained by electronic subtraction. The influence on from other ions (indicated by index j and the valencies Zy) can be obtained from calibration.

Fig. 13 a Scanning Electrochemical Cell Microscopy (SECCM). b SEM image of the tip of a double barrel capillary. Reproduced from [37] with the permission of Wiley... 

A major concern in this study was localization of the tip of the double-barreled microelectrode (Fig. 1) in the intracellular compartment of proximal tubular cells. The reference barrel is the electrical sensor the other barrel being the chemical (potassium or chloride) sensor. The leads of the two barrels were connected to one electrometer and this gave the ionic potential. The leads of the reference barrel and an external 3 M NaCl single pipette were connected to another electrometer and the PD between them represented the membrane potential. The ionic potential (K or Cl") and the... 

From this equation it can be seen that it is necessary to make two measurements in order to determine the intracellular potassium activity. You have to measure AE with the ion selective electrode and then you have to measure % with a conventional saturated KCl filled pipette. Ideally this would be done with a double barrel pipette, one barrel being the K" " electrode and the other one the saturated KCl filled for measuring the membrane potential. To date we have not been able to construct a double barrel electrode that is satisfactory. The K barrel usually works very well but the reference side always has a tip potential that is too large for it to be useful for intracellular recording. What we have done is to make 5-10 measurements with each of the two kinds of electrodes in every preparation, then used the average values from these two sets of measurements to calulate the intracellular potassium activity. 

FIGURE 15.13 (a) Schematic representation of the 0-pipette, (b) an SEM image of the pipette with/ =65 nm and (/= 36 nm, and (c) the G/C IT process at two liquid/liquid interfaces supported at the pipette tip. (Reprinted with permission from Hu, S., Xie, X., and Meng, P. et al.. Fabrication and characterization of submicrometer-and nanometer-sized double-barrel pipettes. Anal. Chem., 2006, 78, 7034-7039. Copyright 2006 American Chemical Society.)... 

SECCM probes are fabricated from either borosilicate or quartz double-barrel pipettes, pulled to a sharp point in a laser puller (typically a P-2000 from Sutter Instruments). The size of the probe can be controlled by adjusting the pulling parameters, with probes between 100 nm and tens of microns across at the tip end fabricated easily and quickly. A field emission scanning electron microscopy (FE-SEM) image of a typical (relatively large) SECCM probe is shown in Figure 19.2a. The pulling parameters differ between laser pullers, but for a typical 500 nm borosilicate probe on a P-2000 laser puller, the parameters are as follows line 1 heat=600, filament=4, velocity=30, delay= 150. pull=20 line 2 heat=500, filament=4, velocity=30, delay=150. pull=60 line 3 heat=500, filament=3, velocity=30, delay = 135. pull=60. 

Yamaguchi, H. 1986. Recording of intracellular Ca from smooth muscle cells by sub-micron tip, double-barrelled Ca +-selective microelectrodes. Cell Calcium 1 203-219. 

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