Fast Scan Cyclic Voltammetry
Fast Scan Cyclic Voltammetry (FSCV) is one of the many methods for detecting and estimating the concentration of a species but can only be used for identification in relatively non dynamic backgrounds. Fortunately, the regions of our interest in the brain, are relatively chemically stable which makes FSCV a useful tool to detect sub second chemical species concentration changes in the brain. In theory, it can also be used to study dynamics and reactions provided that there is an exhaustive library of I-V waveforms that we can decompose the signal into (along the lines of the Fourier transform) however, like everything else in life, we're limited by practical considerations.
A species requires a threshold amount of energy to give up an electron and it dissipates a threshold amount of energy to gain an electron. the loss of electrons is termed oxidation and the gain of electrons is termed reduction. It is not necessary that the energy required and dissipated is the same, which means that the process is not symmetric. it might need more energy to lose an electron than it would release to accept one or vice-versa. Also, the amount of energy required to lose the second electron might not be the same amount that was needed to lose the first electron, which means that the process is non linear as well. The same applies to the reduction case.
These two properties are of use to us and are the reasons why FSCV works.
suppose we provide a stepwise amount of energy at 1 J increments every 1 sec to a system . The system contains 1 mole of 1 chemical species: A. A requires 5 J to oxidize and the maximum amount of energy that we provide on the left is 10 J and the starting amount of energy we provide is 0 J.
therefore, at t = 0sec , we are providing 0 J at t = 1sec , we are providing 1 J at t = 2sec , we are providing 2 J at t = 3sec , we are providing 3 J . . . . at t = 10sec , we are providing 10 J
since oxidation requires a threshold amount of energy (nothing or everything) A will not oxidize for t<5 sec or energy <5J. Thus the concentration of A+ will be 0. assume that all of A will oxidize to A+ by t=7sec for 5sec<=t<=10sec or 5J<=energy<=10J. Thus the concentration of A+ reaches its maximum at t=7 and then stays there till t=10.
Now let's add the dimension of space and charge. everything staying the same, lets say that the system is a box and the energy is supplied to the system from the left side of it in the manner shown above while 0J are being supplied on the right side of it. We will also give the right a charge of +1C and the left a charge of-1C. Lets also say that the species is uniformly distributed throughout the box, that is, the amount of species A on the left side is equal to the amount of it on the right side at time =0 or when energy supplied =0. when ramping the energy on the right up to 5J, The amount of A will stay as it is because the energy supplied is not enough to oxidize it and it will not move left or right since its charge is neutral. 5J onwards, the molecules of A nearest to the left will be oxidized first to A+. These A+ ions will now be repelled from the right (positive charge) and attracted to the left (negative charge). thus, they will move all the way to the left until the system reaches equilibrium. This can be seen on a plot of the no. of moles of A+ near the electrode vs energy or time.
- I-V curves
current is defined as the rate of change of positive Charge at a point over time (dQ/dt) (C/sec) while Voltage is defined as an amount of energy per charge (Energy/charge) (J/C). If the charge we are observing has a contribution from a chemical species of interest, then we can build a relationship between the amount of that species and the current flow and essentially realize a signal in the units depicted in the image above. There is a direct proportionality between current and the concentration of the oxidized species. The more the amount of an oxidized species, the more charge there is to be displaced across a point of space in the same time frame, thus, the more current. Hence I-V curves are useful for obtaining a concentration vs energy chart. The specific bumps at a particular energy point are clearly characteristic of the species itself. hence, This allows us to obtain a fingerprint of the species because the whole curve can vary from species to species due to its threshold energy of oxidation and the number of stages of oxidation which creates a large combination of waveform which reduces the chance of overlapping between the waveforms of two species. we can further reduce the chance of this happening by studying its reduction peaks as well.
The need for CFM electrodes (high impedance electrodes ~100's of Mohms) is so that we can detect these chemicals at very low currents (nA) (V=IR) so that the potentiostat system doesn't excite or change the background (the brain) in any significant way.
Steps for making a CFM electrode
- clamp a glass tube horizontally under a microscope
- fill it with isopropyl alcohol using a syringe
- take a few strands of T650 carbon fiber and insert them into the glass tube
- drain out the alcohol
- set it in the glass puller and pull with program 7 (P=500, Heat =555, vel=78, time=250, pull=0)
- cut the carbon fiber off with either a wire cutter (for a lot of strands) or a tweezer(for just a few strands)
- remove the two pulled electrodes, remove any remaining carbon fiber from the filament area
- check under the microscope to see if the carbon fiber reaches sufficiently into the electrode to make any contact, discard if not
- place the electrode tip in water to check if it isn't drawing any of it inside to make sure that the tip is sealed. discard if it is drawing in water.
- solder a male pin to a tungsten rod.
- under a hood (wearing gloves), smear silver print II on the tungsten rod(shake the bottle well first) and insert into the electrode clamped vertically
- let it sit for 15 minutes for the print to dry off and settle near the tip
- dab some super glue at the contact end of the electrode to seal it. let it sit for another 10 minutes till it dries.
- the impedance of the electrode can be manipulated by the length of the CF tip or the number of strands. the working electrode's impedance should always be larger than the counter's.
Steps for making an Ag/AgCl reference electrode
- denude a piece of silver wire at both ends with a flame switch. have the tip denuded over a longer length.
- solder the other end of the wire to a male pin, check for contact with a multimeter
- place the tip of the wire into a vial of bleach. let it sit for 15 minutes
- should be able to see a light brown tip
to gain 5x sensitivity for adenosine, dip the working electrode tip in nafion and let it sit to dry overnight
Circuitry and setup
tape the reference electrode to the side of the beaker. use a micro-manipulator to set the electrodes in the solution. place the beaker on top of a stir plate and always stir to keep the solution as homogeneous as possible. load fscv_summer_2016_pratik.set workspace which uses the holding current module and the FSCVStim module for stepping the species input and applying the voltage pulse respectively.
- add desired volume of ACSF in a beaker
- first tape the reference electrode to the beaker, then set the working and counter electrodes in it
- use RTXI to measure the open circuit voltage of working vs reference and counter vs reference. A DC voltage should be observed when in ACSF. if not, the CFM electrodes should be discarded.
- remove the CFMs from the beaker, connect the electrodes to the circuit, then connect the batteries while still in the air, then lower into the beaker.
- make the desired stock solution of the species in ACSF
- start stimulating for about 20 minutes to allow the electrodes to build a stable double layer
- set holdingcurrent to 0. start recording. give it a minute to record the background. increment holding current, and then immediately add an aliquot. repeat for the desired number of aliquots. after which stop recording.
- carefully remove CFM electrodes out of the solution, and disconnect batteries. store electrodes by lowering them into a beaker of water.
- enter the aliquot size, stock concentration and other variables in the matlab .m file FSCV_rtxi_analysis_summer_2016.m and run the code