nanoHUB-U Principles of Nanobiosensors/Lecture 3.7: Amperometric Sensors - Glucose Sensors I
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[Slide 1] Welcome back. This is, we are in the second set of lectures. We're discussing biosensors. Different types of biosensors and corresponding sensitivity. 

[Slide 2] Now, today we'll be discussing the second set of nano-biosensors which are amperometric. Now you may recall that there are these three types of biosensors we promised that we'll discuss. One is this potentiometric biosensors where the biosensor is like a camera for the charge. And so therefore when the yellow molecule lands on the sensor, which is often a MOSFET in this particular case, it changes the potential of the channel and that changes the corresponding, corresponding current. Now of course that would be still very good because it can be miniaturized, integrated with other components. So that's a very good thing except that we saw that this salt was the trouble maker. Salt was necessary so that the molecules can stay together like the DNA. They are both negative and therefore in that case, unless the salt was present they would simply fly apart. And same was the case for protein and antibody binding. Without the salt present around it, it would be difficult to manage the interaction. So in that case, of course, the trouble was that a significant fraction of this yellow charge was sort of being taken away as stacks for helping the molecule sort of, by the salt itself. And a very little amount was left. And that gave rise to this logarithmic dependence. So this is sort of a screening problem  of potentiometric sensors that we saw there's a way to get around. Like using high with high frequency. But in general, this continues to be a concern. The second sensor that we'll be talking about today, and the third one, the cantilever base sensors, neither have this screening limit as, sort of as being detrimental as the potentiometric case. So let's get started with the amperometric sensors. The basic idea is that in amperometric sensors we'll see that it is the electron affinity of the yellow molecules and its ability to catalyze the, or change the property of the electrode itself. That will give rise to a current. And so when the molecule arrives on the sensor's surface, this current will tell us, the current flowing through this circuit will tell us that a molecule has landed and that will allow us the detection. That's the sensing. So, let's see how it works. 

[Slide 3] Now before I get there I just want to remind you the historic context. That these amperometric sensors are very important. I told you about the potentiometric sensors and how eventually, towards the end of this course, we'll be talking about how potentiometric sensors are used in genome sequencing. But, one of the most important sensors that you will see, most people routinely use. Like, my mother has diabetes and she uses this type of glucose sensors all the time. So, when you're talking about amperometric sensors, we are essentially talking about sensors such as the glucose sensors. So it's very important in terms of the biosensors that we routinely use. 

[Slide 4] Now, if you really took a closer look at the glucose sensor, then you would find the following. That this has multiple layers. This strip. It has multiple layers and the most important layers are the electrodes. So one will be the counter, or the reference electrode. One will be the working electrode. So when the blood comes in, it will first react with this enzyme coating. Then something will happen that will be picked up, the reaction products will be picked up by the electrode and the current will be flowing between the working electrode and the counter electrode. And so, therefore, what we will see, that the glucose count in the blood will be deflected as the current flow. Now, I'd like to break it down and explain each one of these tapes very clearly so that eventually when you talk about DNA sensing using a very similar amperometric sensors, then things are crystal clear to you. 

[Slide 5] The way to view a glucose sensors, I find it most convenient if I divide it into two parts. One part is a normal sensor, so I have two electrodes. Platinum would be the working electrode. And Ag/AgCl, silver silver chloride electrode, will be the counter electrode, or the auxiliary electrode. And we will see that we have to apply a voltage here. I will explain why in a little bit. And what will happen, that this is sort of the key sensors. So, it will sense the main anilites that we are interested in. and the second part of this sensor, which are actually together, but for conceptual reason I have just pushed it apart. So we'll see that the glucose coming in from the blood, glucose coming in and it reacts with oxygen. A gluconic acid will get out. And a hydrogen peroxide will be produced as a reaction product. Now this can only occur in the presence of an enzyme called glucose oxidase. An enzyme allows for specificity. That is, only when the glucose comes, is it catalyzed by this oxidase so that this reaction by-product forms. Now this sensor cannot detect glucose directly. You know, that's what we're after. Sensing glucose. But instead, what it does, is that when the hydrogen peroxide comes in here, once gated by this enzyme itself, then it will react on the surface in the platinum surface, two electrons will get out and two protons will get out. I'll explain all of them, why such electron comes out and why protons come out. But for the time being, just the outline of the problem, the electrons will come out and the two protons will go through the fluid. This is the two electrodes separated by a fluid. And the protons will essentially be combined to electrons that came from the other side. And in the process there will be a current flow which will be proportional to the amount of hydrogen peroxide, which in turn will be proportional to the amount of glucose present. So simply by looking at the current flow through this sensor, I'll be able to say how much glucose has been around. Whether the glucose blood sugar is too much or too little, I can be able to find out by simply looking at the current. So that is the essence of an amperometric sensor. The physics will come in a second. This is very important to realize that this enzyme allows the specificity that only when glucose comes can the reaction occur. And so therefore, this hydrogen peroxide, the platinum itself, doesn't have any specificity with respect to this. Only when hydrogen peroxide comes, assisted by this catalyst process will the current flow. So I want to explain to you how the sensor works. 

[Slide 6] And so, we'll go one step at a time. Now the reaction that happens on the right hand block, remember while we had glucose and oxygen and the reaction by-product. That's the top equation. You see the glucose molecule reacting with oxygen. This is the enzyme and the reaction by-products are hydrogen peroxide. And then there are this hydrogen peroxide goes to the platinum working electrode, two protons are generated, two electrons are generated, which flows through the wire, remember. And the two proton, goes through the fluid and therefore it calculates completely. So, I will start by this second set first. I will start by this second set first. And then in the next lecture we'll go back and look at this. And then we'll put everything together in order to see how amperometric sensors can be used for DNA sequencing. So let's get started.  00:09:17,356 --> 00:09:22,786 

[Slide 7] So before I do so, let me just briefly highlight the important points. The important points are that the reason why we are using amperometric sensors that it obviates the problem of screening. The salt screening associated with it will no longer be as important. Remember, these are electron hole flow. Electron holes, these things are all charged. So, eventually the effect of potential will still be there, close to the electrode will still be there. It is just that it will not be as sensitive to screening as the potentiometric sensor was. Because in this case we are working on electron affinity as I'll explain, not the potential itself. Now, this specificity comes from this oxidase because that allowed specific molecular recognition. You see, just like the DNA binding allowed a specificity, are the specific antibody when the protein molecule came around and exactly sort of matched with this. That also gives specificity. So in this case, the enzyme gives the specificity for the reaction. A random product will not simply be recognized. And as I said, the broad range of sensors are actually used. Now, the important point is that these sensors have a different type of sensitivity for sure. But still, the molecule have to first come in. Remember about the example of the bar I said that the bar, unless it comes to the focus of your camera, then it doesn't matter how many mega pixels of camera you have. So, the diffusion limit is still there. And the other thing you saw that there are multiple electrodes. Here are already three electrodes which are often difficult to miniaturize. And that is a concern. And so therefore, putting all these things together, and is still a big technological challenge. That's why the glucose sensors that you see in the market are so amazing. Think about the millions of people who are using it who know nothing about bio-chemistry, to sort of control their food intake every day and unless this is quite reliable, this would be a very difficult problem. So this remarkable advance in technology has already occured this despite these problems that are associated with it. 

[Slide 8] So let me explain now the physics of-- or the chemistry, of how things go. 

[Slide 9] Now, we will begin with the left side. And we'll begin with this basic sensor and in the next lecture we'll come and discuss the component on the right. 

[Slide 10] Let's go back to college. That would be many years from now for me. But I still remember this experiment in college where you put copper sulfate, that's the solution, the greenish solution, and if you put zinc in it, it sort of a small electrode of zinc, a small piece of zinc in it, then what happens, you will see, gradually this copper sulfate becomes zinc sulfate, slightly pinkish color and zinc begins to dissolve and there's heat produced here. Now, the question is, why does it occur? Why does it occur? And the reason it occurs is because we have zinc when it relates to copper, when it reacts with the copper. Then zinc becomes positively charged, goes in the solution. And copper accepts the two electrons from the zinc. And as a result, zinc with copper becomes neutralized. So therefore we have zinc sulfate and copper. Now this is good. You could say that this reaction is a spontaneous reaction and this is often you are given the analogies as if you are going downstream. Just like waterfalls under the force of gravity from the top energy to the bottom. The zinc goes to copper. The electrons go from zinc to copper in an analogous manner. 

[Slide 11] Now, it is relatively easy to see why that happens. If you call-- recall the oxidation potential, oxidation potential is essentially just desire to give away the electron, if you think about the oxidation potential for zinc, it's quite high. It doesn't like its electron, it just wants to give it away. On the other hand, copper is very happy to accept electrons. Its oxidation potential is negative, minus .337, so what happens is that when you put the two things together, electrons will go from zinc to copper and the reaction will proceed spontaneously and this excess energy will be liberated as heat, something that you can see. Now let's say you don't want this to be wasted as heat, but you want to also go inside and look at how would this current flow across itself. How would you do that? Turns out that it's not too difficult. 

[Slide 12] What needs to be done is that this reaction which is happening in the same chamber, you split it into two. So we have zinc on one side and copper electrode on the other side and you hook it up with the wire. And here is your amp meter. You can measure the current. And so, now what will happen, that if you put zinc and zinc sulfate, let's say a solution, zinc is very happy to go back and forth. It can ionize and de-ionize, going back and forth, but let's say it goes from zinc to zinc sulfate. The zinc positive ions. And the corresponding electrons that have been left behind will now flow to copper. Remember copper is happy to accept electrons. And these electrons will then be, this copper is in the copper sulfate solution. The electron will essentially neutralize the copper ions. Now that's very good. But that's not the end of the story. Because as soon as the copper has been neutralized, and deposited  on this electrode, this nitrate will essentially be alone because its partner has been neutralized. This will go and react with sodium, which is part of a salt bridge, allowing only passage of ions. No electron. Just ions. So nitrate will work with sodium, removing the sodium in the process, liberating NO3. The NO3 will go and talk to zinc. They recombine and zinc nitrate is formed and everything has been back to normal. The extra zinc that came in, the solution now has been reduced to zinc nitrite which has become a part of the solution itself. So gradually, these two processes will continue. And so therefore, you'll see that between zinc and zinc plus, there is a easy transfer between the electrode and the solution, going back and forth, and then there's also an easy transfer between copper and copper plus. And at the end of the day, and once these two reactions are complete, then the process can continue. So in this case instead  the heat, what we have is the electrode itself, the current can be, can be directly measured and you can get it by jewel heating, corresponding jewel heating. 

[Slide 13] OK. That's very good. When the electrons wants to go somewhere, from the high energy level to the low energy level and there is no barrier, that's not a problem because now this process can occur spontaneously. However, however, think about putting zinc in water. Now in this case, going back and forth, unlike zinc sulfate, will be very difficult. And so, therefore, the electron, which is sitting in one of the electrode now have to go up first. Before it can go to the other electrode. And if this level is sufficiently high, then there's nothing that's going to happen because these electrons will find it inconvenient. Although there is a net desire to go from this point to this point. But net driving force-- but the electron itself will not be able to initiate the reaction in its own chamber and therefore no current flow. It's like putting an insulator between two, two metals. Though the electron may want to go from one metal to the other, but the insulator is sort of preventing it. Now assume that you have just introduced, in the solution, a material, a set of molecules which has a slightly lower oxidation potential. Now it is still difficult for the electrons to go here and then to come back. But at least it is easier. Than going to the top level, energy level. In the original solution lets say zinc in water, or platinum in water. And then to come back. Now, this new molecule, of course once it comes in, will facilitate the process and there'll be a little bit of current flow because the electrons will be able to go. And then it will be able to go to the other electrode. However, this current will be exponentially increased if we do the following. That instead of this level, we apply a positive bias so that the level moves up. Now of course it's much easier for the molecule, for the electrons to hop back and forth and, as a result, this process of the oxidation and deduction of this molecule, by the way the word oxidation in this partic-- yeah, the reduction is simply accepting electrons and oxidation means giving away electrons. So this process of giving away electrons and accepting electrons would be the reduction, oxidation and reduction process going back and forth. And as a result, quite a bit of current will flow. Although, the original solution had no current flow. So this is a way one can then recognize a molecule. Through this force oxidation reduction while you have applied a small amount of voltage to facilitate that process. And we'll be using it quite a bit as you will see. 

[Slide 14] So now, let's look at the solution. This particular configuration. We have platinum electrode. Remember I'm going to go back to the amperometric sensor, the glucose sensor. So we have the platinum on one end and we have silver, silver chloride on the other end. You may remember. And when you have the system on its own, no current flows because platinum cannot come here like zinc did because in this water there's essentially no way to initiate the reaction, although you have put a small amount of voltage, no current is flowing essentially, very small let's say. Now what you can do, is bring in this hydrogen peroxide. Hydrogen peroxide will have lower oxidation potential and so as soon as you put 350 mili volts, this reaction will begin. And what will happen that the proton will come this way and the electron will go and this process will continue. So although its not a  spontaneous process. However, with this voltage, this applied voltage, it becomes much easier for this reaction to proceed. And so, therefore, this current becomes directly proportional to the hydrogen peroxide concentration, which is also directly proportional to the glucose concentration. So this is how the sensor would work. Once again, I'll show you the same picture, that this is the platinum level, let's say. This is not exact level. Just the cartoon. This is my hydrogen peroxide level. This reaction is initiated when I have pushed it up by 350 mili volts, say. 

[Slide 15]And therefore the whole process has, can continue. 

[Slide 16] Now very quickly let me tell you a little bit about, so we had been talking about two electrodes, platinum and silver, silver chloride. No problem. But let me very quickly tell you that quite often, two electrodes are not enough. Remember in the case of the glucose sensor we had three electrodes. There is very important to have a reference electrode. Why is this? Because even when you have platinum silver, and you can measure a voltage or measure a current, the problem is that if you wish to control this voltage, remember we want to do it 350, add the platinum electrode so that the reaction can occur. If you simply apply a voltage of 350 directly between the two electrodes, then it may not really work. 

[Slide 17] The reason is, that when you put, let's say in this particle, let's say I apply .8 volts, rather than 350 millivolts, then I can not apply what fraction will get unto the working electrode and what fraction will get to the other electrode. Let's say the counter electrode. I cannot control the partition of the voltages. And if I cannot control it, then of course I may not have a controlled reaction. For every voltage, they'll be slightly different reactions. So I have to do something about it. 

[Slide 18] And the way to do it is to insert a third electrode so that it maintains its potential at a given reference value. And then, with respect to it, then you move things back and forth. And so the working electrode then will always have a fixed potential with respect to the reference electrode. And the reactions will be controlled reactions. Rather than arbitrary reactions depending on the exact configuration of the sensor itself. 

[Slide 19] So, therefore, often you'll have a three cell. There'll be a fixed difference between the reference electrode and the working electrode and the reference electrode and the counter electrode so that you can rely on the reaction that is happening between the working electrode and the analyte that we wish to, 

[Slide 20]that we wish to detect. 

[Slide 21] So let me then, very quickly, show you how this whole process works. So we are thinking about hydrogen peroxide coming in and giving away its electron. Which is getting oxidized in the process. And this reduced place is getting oxidized. And of course, and leaving the electron. And leaving the electron here, remember when the electron goes out, the current flows in the opposite direction. But of course there are lots of electrons here so that reverse reaction is also possible. That HO2 which has just been liberated, the proton, which is hanging around, and the electrons, together they can also make H2O2, also hydrogen peroxide. So both forward reaction and reverse reaction, both are possible for oxidation and reduction. Of course, depends on the exact voltage that you have applied. And so in this case, the more current that flows is proportional to the forward reaction, giving electrons, and the reverse reaction, taking electron back. And so giving and taking it back the difference of it multiplied by the area of the electrode gives you the current. Of course it depends on the voltage also. The voltage is, there's the 350 millivolts here determine the kf and forward and the backward reaction rates. And so specifically, in this case we'll have the hydrogen peroxide and then the oxygen, these two are R and O in our case, and that will determine the current, provided the 350 millivolts has the predictors kf and kb. Now this equation is extremely important. This is called a Butler-Volmer equation. Any time you have an electrode, and there are electrode catalytic processes going on, something is getting reduced and something is getting oxidized, the forward and the reverse reaction is always given by this very simple formula. There are more complicated versions of it but for this course the only thing we need to remember, that if the forward reaction is sort of strong by applying a voltage, for example. So in that case, what will happen, this you can almost neglect with respect to this and then you can see, the current will become proportional to the hydrogen peroxide concentration and which will then indicate how many glucose one has in the blood. 

[Slide 22] And so here is an example, for specific examples. For example, here you have a set of carbon nanotube, let's not worry about exactly how they were fabricated. Here is a electrode. You see this palladium electrode and correspondingly there are the sensing regions, small sensing regions which looks like a nano-cube. So this is really the working electrode that we're looking into. And if you go ahead and then start different-- bringing different amounts of hydrogen peroxide into the solution. Then what we'll see, that the current would essentially jump in steps. Because every time you have a different concentration, remember the current in the amperometric electrode is directly proportional to the hydrogen peroxide and so therefore for every concentration there'll be a response of current. It will stay flat for a little bit, approximately. Then if we increase the response, it will go up. There'll be more current. And so this is almost linearly proportional to the amount of analyte. So simply by looking at how much current you have in the sensor, you could say what is the concentration of the bio-molecules, what is the concentration of hydrogen peroxide present and therefore indirectly, what is the concentration of glucose present in the solution. 

[Slide 23] So let me conclude this first lecture on the amperometric sensors. By summarizing a few things. As I said, the amperometric sensors, essentially responds linearly to the reactants to be measured. Remember that for potentiometric sensors, it became a log because of the screening. Fortunately here, if you increase the concentration by a factor of hundred, you do get a factor of hundred increase in the current. That's pretty good compared to what happened in the potentiometric sensors. And another thing is, the kf and kr, the reaction rates, that depends on the voltage you apply to the electrode itself. You want-- the higher the voltage you apply, better it is because then you'll have more current. The two levels will become more and more aligned. However, if you apply too high a voltage, then yes, this current will be large but there may be parasitic reactions reaching other levels sitting close by which will essentially contaminate your signal. These parasitic channels you don't want to activate because that will reduce your selectivity. Now I mentioned the three electrode configuration is very important in order to get specific ref voltages for each one of the electrodes. Because, otherwise what will happen, the partitioning between the voltages, between the working electrode and the reference electrode may not be well defined. And if it is not well defined then it will not be, it will not be a very robust sensor, a predictive level sensor. But in many cases, putting three electrodes are difficult and so in that case working with two electrodes before extensive calibration before you should take the results for granted. And the important thing is, yesterday I just told you about glucose sensors. No problem. If you wanted to do nitric oxide sensors, remember the NO, remember the three types of molecule we talked about. Small molecules, then we talked about DNA and protein, virus and bacteria, the three classes. Many small molecules you could use the amperometric method to actually sense it. Because very selective and essentially the chemistry is well defined and the response is linear and not logarithmic and so therefore it is really used in wide variety of sensing applications, especially for small molecules. So I'll stop here. In the next class we will talk about, in the next lecture we will talk about the second part of the amperometric sensors. Today we just focused on sensors itself. So, the enzyme, how it reacts with the glucose and all those things. We really didn't think about much. In the second lecture we'll talk about that.