The sensitivity of amperometric sensors is typically set by the rate diffusion of the analyte to the electrode surface, so determining diffusion coefficients in various electrolyte solutions is of fundamental interest. It has been theoretically shown and verified that diffusion coefficients of electrochemically generated analytes can be determined using electrochemical time of flight (ETOF), a method that uses an electrochemical array in which one electrode generates a Red/Ox species, and measures the analyte diffusion times to collecting electrodes of differing distances from a stationary generator. ETOF has the potential to greatly simplify the determination of diffusion coefficients as the analyte concentration, the electroactive area, the solution viscosity, and the electron transfer kinetics can remain unknown. Here we demonstrate an alternative data treatment for ETOF in which the electrochemical flight time is measured for a series of different Red/Ox species of known diffusion coefficients at a single distance. We show this a valid application of a method that has existed for almost 30 years, by determining diffusion coefficients for ruthenium (II) hexamine, and diffusion coefficients in solutions of increased viscosity. Diffusion coefficients are important because they set the sensitivity of amperometric sensors and they are a fundamental property both in membrane permeability and in electrochemical measurements. The most common method of determining diffusion coefficients for analytes in bulk solutions or through gels and membranes relies on the rotating disk electrode (RDEs) 1-7 or the rotating ring disk electrode (RRDE).8 This method determines the diffusion coefficients, D, from the slope of a Levich plot constructed by measuring limiting currents, I L , as a function of square root of the rotation rate, w, according to the Levich equation (Equation 1).Accurate values for the area of the electrode, A, the number of electrons transferred, n, the concentration of the molecule, C, and the viscosity of the solution, v, must also be known in order to effectively determine the diffusion coefficient from the slope of a Levich Plot. The diffusion coefficients of molecules through bulk solution can also be determined quantitatively by wall-jet chronoamperometry, 9 or qualitatively by comparing the CV's of different compounds because the shape of the CV is related to the diffusion coefficient of the molecule. [10][11][12] The other primary option for determining diffusion coefficients of a molecule through a membrane coated over an electrode is impedance spectroscopy, [13][14][15][16][17][18] where the diffusion of the molecule through a membrane or polymer is related to the impedance of the polymer or membrane to current flow. As such, the diffusion through the polymer is related directly to the resistance of charge transfer (mobility) through the membrane, which is related to its conductivity and directly correlated to the diffusion coefficient by the Nernst-Einstein equation (Equation 2).Conductance, σ, ca...

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Current methods of calibration of biosensors take place before or after use.  In situ calibration methods are either nonexistent or ineffective at dealing with the changes in sensitivity that occur while the sensor is in situ. Electrode-based sensors are usually covered with a protective membrane that helps prevent fouling and adds some degree of selectivity. External calibration calculates a sensitivity based on an external calibration curve that does not reflect the in situ conditions. As soon as the sensor is placed in situ, fouling occurs at the electrode’s surface/membrane, which changes its permeability, a product of the solubility of the analyte in the membrane and the diffusion coefficient of the analyte through the membrane.  The sensitivity of the electrode will change when the permeability of the membrane is affected by the foulants that adsorb to the surface of the electrode/membrane. In order to achieve accurate results, one needs to have a method for tracking the changes in the permeability of the membrane as fouling occurs. Currently the electrochemical methods for determining the diffusion coefficient of an analyte through a membrane are either to use electrical impedance spectroscopy (EIS) or to use a rotating disk electrode (RDE) experiment.  Neither of these methods are particularly fast, nor do they lend themselves to being performed in an in situ environment. Electrochemical time of flight (ETOF) is an experiment performed using an electrode array where a small amount of analyte is generated at an electrode called the generator while monitoring the current at another electrode in the array, called the collector, which are a known distance apart (Figure 1). The relationship between the distance travelled, d, and the time of maximum collection, tms , is as follows: d=K√(Dtms), where D is the diffusion coefficient and K is a constant based on geometric parameters of the electrodes. ETOF has been used to test diffusional models for analytes, such as ferricyanide or other model compounds, in bulk solution through gels and through solid polymer electrolytes, or to enhance signals of an analyte by redox cycling. ETOF has not yet been used to test models of diffusion through membranes, the effects of foulants on that membrane, or to re-determine the sensitivity of a sensor that has been fouled. The instrumental limitations of most bi- and multipotentiostats, including the popular CHI 750a or CHI 760b potentiostats, have limited the development and applications of ETOF.  None of the most popular multistats  are able to apply a potential pulse to the generating electrode.  Here we report on the construction of a device that applies and controls the generator electrode by connecting and disconnecting the lead to the electrode.  Figure 2 illustrates the connections to the CHI potentiostat, an in-house constructed generation-electrode-controller (GEC), and the National Instruments Compact DAQ through the NI 9403 Digital I/O module.  Circuitry in the GEC uses a flip-flop to catch an outgoing trigger pulse from the potentiostat at the start of an experiment, this starts a timer that times the placement of the potential pulse to the generating electrode. Figure 3 shows an ETOF experiment gathered from a CHI 750 potentiostat using the National Instruments controlled GEC.  The red curve is the generation pulse, then the green curve is the transient of a collector 6 microns away, blue at 14 microns away, and purple at 22 microns away.  The time of maximum collection is measured from the center of the generation pulse to the peak of the collector current.  From this data a line is constructed plotting the separation distance, d, as a function of the square root of tms. The slope contains the geometric constant, K, and the square root of the diffusion coefficient, D. Using an analyte of known diffusion coefficient one can determine the geometry constant of the electrode. In a second experiment and knowing the geometry constant, one can determine the diffusion coefficient of an analyte. Currently ETOF-determined diffusion coefficients in our lab have been measured within 10% of diffusion coefficients determined for ferricyanide in bulk solution using RDE experiments, reporting diffusion coefficients of 2.0x10-5 cm2/s and 1.8x10-5 cm2/s respectively.  Using the newly developed instrumentation, our plan is to use electrode arrays and ETOF to determine diffusion coefficients through a membrane and therefore monitor sensitivity changes in membrane coated sensors. Figure 1