 In the next lecture, I will focus upon the label-free detection platforms and their applications. In today's lecture, I will walk you through various label-free detection techniques available and their advantages over the label-based detection platforms to study the molecular interactions and their kinetics. This is always the debate whether we want very accurate quantification using labels or labels are going to affect the properties of biomolecule and we should use the innate properties and have the label-free quantification. So, we will have these discussions in this lecture. Further, we will discuss about a few label-free platforms in detail and their interpretation of these insurograms obtained. So, let us continue with my lecture which I delivered in this workshop. Let me start with the outline what I am going to talk, a couple of things which you have been hearing already the field of intractomics, I will not take much time on that. But then I will move to comparing the detection platforms, which is because you are hearing lot of lectures from Josh on protein microarrays, you have heard many of their speakers on reverse field arrays and peptide arrays, all of those require some level of label-based detection system and study you also started getting exposed to some of the label-free platforms. So, brief comparison of those two type of detection platforms, some of the overview of the label-free technologies, there are many in fact, you just got some exposure of SPR study surface plasmon resonance, but there are many emerging technologies which are already in have started making some impact, but many of them are still in the testing phase. Some of the latest popular label-free technology platforms as we go along in the course and then some of the brief overview of applications, I will not have too many cases studies, but I will at least give you the flavor of what all things can be done because our intention with this course is that many of you can start planning your experiment and think about you know how broadly these technologies could be used for everybody because they are not biased for any kind of sample, they are not biased for any specific project depending on your objectives, I think you can very much modulate these things for your own research. So, I am sure by now you are convinced that you know these kind of technologies are very useful to study the entire intractome which is involved in many of the important activities whether you talk about signal transaction to splicing morphology growth, metabolism, translation, DNA replication, these are all pretty relevant for any kind of physiological processes which we want to study. So, therefore many type of protein interaction methods have come forward, the traditional approaches includes yeast to hybrid and different type of IP methods and the latest technologies you are now happy and convinced that you know protein microarrays are emerging as one of the solid platforms including cell free expression based arrays which includes naparrays and then we have couple of label free platforms, the biosensites which are equally crucial as well. So, let us talk about detection platforms and their comparisons, so you want to detect the signal after doing the assay right and that is where whatever you have hypothesized at the end only looking at the dots or looking at the curve you know that your experiment has worked or not. So, therefore you need to have some robust detection platforms and to do that either you are using some sort of label to follow your experiment and then you are trying to measure those labels either with the fluorescence based methods or chemiluminescence based methods some some even radioactivity basis can be very powerful. So, you require some sort of labels which could be used as a marker to follow your experiments or you can also think about label free approach because whenever you are labeling a protein irrespective of what chemistry you use you are adding something from outside on a given molecule and that is going to definitely in some way affect the overall structure overall binding and it may result into certain artifacts. So, for many kind of you know critical drug discovery platforms it is you know because more apparent that you probably do not want to change the native structure of the molecules and you want to study them in their own you know natural environment as much as possible without adding any sort of extra label on top of them. As it is that many type of label free platforms have emerged which essentially aims to look for the properties of the molecule itself. If two molecules are binding what kind of mass is getting changed what kind of dielectric properties are getting changed do we see some sort of percentage reflectivity change because of the binding intensity can we see some sort of interference change many of the physical principles are being used to look at the molecules and molecular interactions and can we measure those properties. So, as I mentioned the label based the readouts could be many of these fluorescent radioisotope HRP based systems for label free you got little exposure of SPR even many of the nanotechnology based platforms like carbon nanotubes etcetera also equally powerful and they have been used for doing these kind of measurement as well. Label based one of the major advantage here is that these technologies are available the kind of you know every lab every center will have those scanners. So, that you have easy readouts you can definitely do the scanning I am sure in any given building you will have some of these instruments available. Regents are easily available even if you do not have HRP you do not have Psi 3 or any of the dies I am sure you can borrow from somebody in the neighboring lab and you can do the experiments, but you cannot do these kind of experiments with the label free kind of approaches when the technology they are very specific. However, the label free platforms are avoiding the tag related issues which you observe in case of label based and most importantly if you are actually monitoring the biomolecular interactions in the real time manner and that is pretty powerful because all the protein micro technologies are great, but it is like you know takes whole day time to do the experiment sometime when you are longer if you are doing a blocking and all and at the end of the day then only you are relying on the scanner to show you that you know your spots are lighting up or not or you see some sort of you know huge background you see you know your controls did not work out and you are actually you know hugely disappointed, but you had literally no control on as the experiment progressed whole day. In this case here your binding is not working you will you know after you will watch just for 20 minutes time stop it and now you will bring a solution change the concentration change the temperature do different pH scouting. So, you have many ways of planning to modulate the experiment you are not going to just wait for something to happen 10 hours time and then you say that is happen or not happen more importantly that you know in case of micro array or other kind of label based platforms you are only measuring the signal at the end which gives you an idea it happened or did not happen. So, you have a positive signal or a negative signal. So, an interaction happened or a biomarker is present you can just say qualitatively it is showing you signal and of course you know that you have many different measures of having different controls which could be used to do the quantization as well. But in this case when you are talking about some of the label free platforms you have even ability to say that you know yes binding is happening, but what can be on rate what can be off rate what can be overall decision constant for these and how my kinetics is actually getting changed. So, these are the added information on top of what you would only obtain in the micro array based or any kind of label based platform. So, that is added advantage of the label free technologies. So, label based because of the tax you know they are might interfere with the function you may have certain kind of issues which can give you artifacts and this is end point measurement because you are not having control on how the assay progresses. Label free technology having lot of advantages, but there is still various specialized many of them are not available in many labs you will not find the those biosenseats available everywhere. They are sometime more costly and again you still have to ensure that all the signal which you measure is coming because of the actual binding and not because of the bulk effect not because of something else which is happening and you are seeing an artifactual response. So, many of the things are still under the kind of you know more infancy more early development stage which needs to be tested out well. So, now just to give you feel that you know we are covering two or three label free technologies in this course, but there are many which are actually under development and there are lot of physics people who are actually you know contributing towards this way of making many type of biosensors, where they are involving different type of physical principles how they could be used to measure molecular interactions. So, you know so many I am sure you know you can find out SPR just one among them one of the label free platform which can give rise to multiple type of even technology platforms including surface plasma resonance which we talk briefly surface plasma resonance imaging based platform SPRI or nano holer is all these three depends on one of the principle which is SPR. Then there are many platforms which are emerging based on the ellipsometry, we have interference based methods, we have electrochemical impedance spectroscopy, atomic force microscopy, enthalpy arrays, scanning Kelvin nano probes micro cantilever. So, I am sure you appreciate there are many technologies which are under development right now they are all looking at different type of physical principles which could be used for looking at the biomolecular interactions in the label free manner. So, while we cannot talk everything right now, but at least some of them I will I will talk I will give you some examples of those, but idea here is that you know many times when a physical scientist work on these kind of biosensors their objective is to only show that two molecules are interacting and by looking at interference or conducting change or you know reflectance change you can measure binding. And then they stop over there because they would have taken very standard protein sets you know they will take BSA and IBSA or they will take you know if you tell there are five more interesting proteins to test out they will take those highly abundant protein and the robust antibodies to test out those assays. And these things are usually not good because when you want to try out your actual experiment which is depending on many time low abundant proteins and antibodies are not so great for them, then I think you know your assays do not work as robust as you would have seen the you know proof of concept level. So many of these technologies are still under proof of concept level which needs to show that what could be done at the actual biological sample level. Therefore what all I have shown here not everything has reached to biological lab and not everything is actually holding up to the biological experiment because biological samples they have lot of low abundant proteins you will not have that is strong and lies to test out. Alright so all the label free measurements irrespective of which platform we talk about they are all measuring the biomolecular properties and it is pretty much kind of without changing anything on top of them and it is adding the information which is providing you either kinetics information or you are looking at the affinities these are added information what one could obtain along with you know just yes no answer which you can obtain from other binding experiments. You know more importantly the new area which are emerging which is essentially system biology area the intention is can we generate lot of these values which is on rate off rate for different type of binding interactions which are happening and now develop those as a model and wiring diagrams which could be used to predict many unknowns because how many actual experiment can you do for so many you know proteins. So let us say you know for a given protein wild type condition and this many mutant forms which you want to test their affinities certain you know with a drug or with other molecule now you can build let us say from 50 of their mutants you can develop some of the values which can give you the some models and now can those models be used to predict the behavior of another unknown mutant. So that kind of stuff is going to really happen if we have the available information or at each level of on rate of binding off rate of binding what is the KD value and then you build the models and that is a lot of system biologists have started working this area and are going to contribute much more heavily towards these directions. So different type of label free techniques as I mentioned are in various developmental phases of course Bayekor is pretty leading in that way because you have already over now 20 plus years of the technologies in field available but many technologies you know which are still just emerging including some of the biolayer interferometry based methods have really come up very forward fast even MST technologies are emerging so those could be good platforms to be used. I am just showing you very briefly some of the principles involved of various type of label free technologies one of them is showing you here you know these kind of nanowires nanowires sensors on which antibodies are immobilized if a protein binds specifically to this antibody then you will see a change in the conductance happening and this is recorded here over the time period. Simple technologies simple concept can have so much utility clinical wise imagine that you know when lot of SARS issues were happening different type of viruses people are getting affected that time you know immediately on lot of you know metal detectors on the airports these type of platforms were used to detect that whether patients or the individuals who are coming are they carrying these kind of antigens possible and then they were quarantined and all kind of these things are happening on the airports. So just you know immediately these papers were published lot of you know applications started emerging out of them simple concept you are measuring antibody antigens interactions looking at the conductance change and if that can give you some idea that you know some individuals are carrying those antigens which are you know going to give rise to certain viral disease then probably you can do much more specific test on those. So another platform which is you know which has shown some promise because of the low cost and that is ellipsometry based platforms where you are measuring how much polarization change you can see as a result of binding and you know again it's very a simple setup with involves a microscopy and CCD camera and it ideally measures the protein contents which is not unlike the other platforms which are requiring lot more costly setup this platform is very cost effective method although it has not shown as many biological application as we would have seen from other platforms but this was a robust thing to show that in cost effective manner you can do label free technologies. One more thing which you know we have seen some good promise of this paper based on a spectral reflectance imaging biosensor or SRIB where you have you know different layers of silica and silicon dioxide and by varying the you know concentration of this if you have now the protein printed in different amount you can see their change in the binding happening and this particular concept was again you know published in PNAS which shows how beautifully just looking at the change in the interference you can see that how much material is printed in different quantity and can be measured in the label free manner. Then comes the SPR technology which is surface plasma resonance which you are exposed already on a prism and on a gold side you have certain antibodies printed for example some protein molecules are flowing here and as you see the binding is happening by changing the percentage of reflectivity you can measure those particular things in the real time manner and what you are actually measuring these kind of plasmons which are on the interface of the gold surface when these you know electrons are being generated and those you are measuring throughout your you know reaction and by changing the solution then you are seeing what is the SPR angle being changed from one to other condition and though the SPR angle is being used to monitor your entire binding reactions and that you measure in the technology which is used as SPR sensorogram. These sensorograms could be used to monitor your entire binding activities for the reactions for example initially you when you are just you know having the molecules coming in and start to bind with the antibody for example you can start seeing there is an initially baseline there which is stable as the reactions are happening there is some binding happening you can see an on rate happening association of these molecules and then after sometime they will you know get saturated you are still injecting your buffer and then ideally they should start washing off and then you will see you know off rate going in and now the you know you are doing these experiments on the noble metals which are very costly essentially gold you are using so you want to regenerate you want to reuse the same slide again and again at this couple of times so then you are trying to do regeneration and you are using some mild acids to chop off your this binding of molecules without disturbing your you know printed molecules and then this chip can be used after you know again you have to wash off with the buffer couple of rounds and the same chip could be used again for next round of interactions so in this way you know you can reduce the cost of the experiment otherwise the gold slide and all these chips which we are buying are way too costly so but what in it's offering you of course it's offering you a much more powerful information as we talked you are getting the kinetics or the rate of reaction how you know you can monitor the label free and the real-time manner you are looking at both on-rate and off-rate of these reactions to happen unlike the label-based methods where you are just talking a binding is happening or not happening here you are saying that okay binding is happening but probably the on-rate was very fast or off-rate was you know very slow so those kind of information you can obtain and many of these informations having lot of utility in the pharma sector and that's where you know even we see that you know more than academic labs the you know Bayekor and SPR and many of the label free technologies are much heavily used by the pharma sector who discovery and drug designing and testing kind of you know experiments they have lot more utility for you know using these platforms because it's just having binding information is not sufficient how the binding is happening on-rate and off-rate differences are much more crucial to know over there so advantage of SPR I sure you are all convinced that you know you have lot of promise here you are not doing any sort of extra thing on your molecules which you want to test for interaction so there is no labeling which you are applying you are directly measuring the biomolecular interactions using the physical properties and then you have full control on how the reactions are progressing and as a result you can actually modulate the conditions to make it work many times we are trying out you know different type of concentrations of different antibodies and we are doing the first initial screening or you know you can use different type of flow rates to even try out so many things you can play with it in that in the initial round of experiments to find out what can be the best condition for your binding to be observed and now once you finalize those conditions then you can apply the same on your full experiment with many of the molecules of interest alright so I think we have talked that you know there is a lot of merit for using these things but of course there are limitations as well because everything what you are relying looking at the mass changes and sometime this could also come from the bulk effect that is may not be the actual binding happening from the molecules so where you are immobilizing the molecules depending on the size of those detection also you know going to get less or more sensitive because exponentially as you know the distance is further from the chip surface you are going to see the loss in the signal and the detection limit estimated around 200 nanometers in this cases limited to the choice of you know metal which you are using because that is a gold metal which is useful for doing this kind of experiments your samples has to be really clean it has to be homogeneous you have to do the you know proper removal of all the gas etc just so that you know bubble is blocking your columns is there is nothing is getting clogged as your you know flow rate is changing for measuring the binding. So sample preparation and these you know probe attachments could be difficult but I am sure it is either some of the generic thing which is applied for any kind of you know the chip based as soon which you have to follow you have to ensure that you know your washing and your all kind of cleanup steps are well performed otherwise you will have many non-specific interactions which may result to ensure that you are getting a specific signal it is good idea to have a lot of positive controls and you can vary the concentrations of those and see you know till what is smallest lowest concentration you can still measure those signals and those can be used to follow your various you know not everything you can do on one chip if you have to test many of your molecules. So then those kind of you know one flow cell let say I am sure you are aware of the different flow cells involved in the Bayer core experiment for example. So one flow cell you can just block it which can be used for you know your QC checks for many experiments and then those could be and you can vary the other flow cell for testing your unknowns. So therefore you will have a still full control even as you are going along the chip has not gone bad and your you know the molecules are for positive controls are behaving same way. So of course you know many of these things in the field of label free technologies you have to avoid ensure that the binding what you are seeing is actually real binding it is not artifacts it is not and sometime you will see a reaction is happening on the temperature dependent manner. So you have to also you know play with those I think fortunately not some of these instrument and technologies have not to give you the range from 4 degrees to you know you can vary till 37 to 50 degrees even to try your different conditions. Of course you know we are talking in this course about high throughput platforms intentionally I am sure you know with Josh talk you are pretty much you know convince that if you can have the chip which can screen 10000 proteins of course you know you are going to talk very quality high quality data you are reducing your time you are reducing the cost everything and so much variation which comes by doing you know two experiments on if you had 10000 protein on one chip versus 10000 on one chip right. Same thing is applicable here as well on the SPR based platforms where if you are doing only four maximum interactions or four maximum testing of the you know binding on the via core range of platform what is it if you can do thousands on a different platform and that is where the concept of SPR imaging became very powerful with hope that can we build the platform which can do the high throughput interaction studies using the same principle what we have used for SPR. So I hope after attending my today's lecture you are now able to appreciate the utility of using label free detection techniques to define the biomolecular interactions and analyzing their binding and kinetics. Although there are many label free detection platforms currently available but most of them are still under infancy and they have shown the utility only at the proof of concept level it means most of these new technologies and biosensor platforms they have shown the experimental evidences that these principles were only with the known pair of robust antigen and antibody based detection. However when you have to test out the actual biological samples and the biomolecules which are in the physiological context they are very very in subtle amount very low concentration then one need to think about how to use technology platforms robustly. In this slide one of the technology based on the SPR and the Bayer Coat technology have over the period from long time shown its utility also a new advancement in the area biolayer interferometry technology has also shown a lot of promise. Nevertheless SPR is one of the most acceptable platform to study the biomolecular interactions whose principles and data analysis was explained in this lecture. I hope now by looking at a sensorogram looking at these binding curves you can now make sense of what has happened in this particular experiment especially for the interaction kinetics. We will continue our discussions on latest advancement in this area and also how to integrate different technology platforms with existing label free biosensors in the next lecture. See you then thank you.