 As we have been discussing about different type of biosensors, we discussed about SPR and now we are discussing about BLI or Bihelier Interferometry. In the last lecture, you got a glimpse of how BLI can be used to perform the kinetic analysis of protein-protein interaction. Today we will be focusing on another application of biosensor that is biomolecular concentration analysis. The task of measuring the concentration of a specific protein in a complex solution is not simple. Therefore, a reliable analysis method for the same is desired which enables the estimation of biomolecular concentration in the complex sample matrix. In this concentration assay, different concentration of the analytes are injected and the response from these different concentrations is plotted against the concentration of each sample. From this, a calibration curve is calculated which is then used to determine the concentration of the same biomolecule in the sample matrix. Today we will be studying the concentration analysis of a protein, a polyproprotein E by plotting the standard curve through bio-layer interferometry based experiment. Let us welcome Mr. Sushil Vaidya for today's lecture and demonstration session. Good morning one and all, myself Sushil Indra Vaidya from Paul Corporation. I am going to give a demonstration on how this BLI technology we can use in the quantitation. Now I am going to demonstrate the experiment here is like that quantitation of the apo-lipoprotein using a antibody agonistic apo-lipoprotein. The biotinulated antibody I am going to use immobilization of the antibody onto a striptavidin sensor. This is the striptavidin sensor I am using for the immobilization system. So here in this experiment we are using a striptavidin sensor. The striptavidin sensor has to be hydrate clear for the immobilizations. Now I am going to do this hydration of the striptavidin sensors. What I am doing is I am I am adding the 1xPBS into the 96-calculator 200 microliter of the 1xPBS. Now what I am doing is next step I am taking the biosensors which is the striptavidin sensors these are the sensors looks like this I am going to put onto the sensor tray this is the sensor holder tray I am putting this sensors like this. Now this sensor tray I am keeping for the hydration for the 10 minutes. So this is I am going to keep like this so now I will set aside this sensors for the 10 minutes. Now I am going to demonstrate you that how we are going to immobilize the apo-lipoprotein anti-Apo-lipoprotein onto the striptavidin sensor 200 microliter of the 1xPBS buffer this is prepared I am going to add 200 microliter each to a well matrix well putting the this is the biotinylated anti-Apo-lipoprotein so now I am going to add 200 microliter of each biotinylated anti-Apo-lipoprotein 96 val plate now this now the sensors are hydrated I am going to place in the instrument the sensor holder I am placing here like this it should be firmly fixed in the groove and now I am going to put the sample plate which contains the biotinylated antibody I am going to place like this in the sample this is we call it as this this particular part we call it as a sample compartment this part we call it as a sensor compartment now I am going to click on to the data acquisition mode so when you click on the data acquisition the instrument now you can see this how the movement of the optical head can be considered of the spectrophotometer itself is initializing in the instrument in the monitor during the initialization we can observe this initialization status once it comes the signal as a ready then we can use for the immobilization step so now you can able to see here the instrument status it is ready when you click on the experimental wizard setup you can see that there is a new quantitation experiment as well as the kinetics experiment here I am doing the immobilization step so that's why I am choosing the basic kinetics with the experiment I am choosing this blank experiment then I will say this go now you can see there is a page in this page it's on your left hand side it's showing that 96 well plate what things you had put it in the 96 well plate here I had put in the in the A1 and the B1 here I had put a buffer that we called as a buffer right click it and say it has a buffer it's indicated by a B then we had to select these two now we had to mention this step as a load load is nothing but the immobilization of the biotinylated antibody against the apo-reco-propane that we had placed in the second A2 and the B2 so we had to mention here in the sample ID just it is a buffer okay then what it is biotinylated a biotinylated apo and I can copy it and paste it here in the next step in the assay definition we have to mention what is the steps the instrument has to be performed the first step is baseline and the second step on clicking on to the add here we had to load the next step is loading step that is nothing but your immobilization step I have to say okay the first step is baseline I had to take a hour mark here right click here in the next step is the immobilization we have to use it loading this is the time here it's mentioned is 30 seconds I'm mentioning around 1000 seconds for the immobilization of the antibody I'm using a strict iodine sensor by default it's coming as a strict iodine this is we had assigned the assay definition step the sensor as I mean where you had kept the sensor in the sensor compartment we had kept a sensor CZ A1 and the B1 it is by default it is showing somewhere it is in the tray we have to assign in the review experiment whether my methods are fine or not we can test it the first step is the sensor it has to be picked from the A1 and the B1 it is going to the baseline that is nothing but your buffer it is highlighting by the black color on the circuit so next step is loading step so it's moving in the run experiments where you want to save the data I will select this in the experiment I can give as a low okay then I can uncheck this these two say go here it's now the sensor is picking from the sensor tray here if you look at there's a shaker here we are working at 1000 rpm speed it's going to the baseline it is acquiring for the 30 seconds the baseline now we can see the immobilization step the biotinylated antibody is going to immobilize on to the step iodine sensors generally we will load it has to reach to the equilibrium roughly around it should be a more than 0.7 nanometer is good enough for the immobilization level we can go for the quantitations now you can see that desired level of the biotinylated antibody immobilized on to the step iodine sensors now we can able to see there is a 1.4 nanometer loaded on to the sensor now I'm going to take back this sample plate and also sensor tray now the sensor actually which is hydrated in the pbs buffer and also our biotinylated antibody also it is in the pbs buffer now I'm going to equilibrate these sensors with the pbst buffer the pbs buffer having the twin in that then this is the pbst buffer because as a sample delin buffer here I'm going to take a 200 micro drop this pbst buffer I will add a 2 a 2 in the b 2 I will now the sensor position is I'm changing from a 1 a 1 b 1 like that to a a 2 b 2 so now I will start the equilibration of the sensor between this this particular buffer we are going to do a quantitation of the apolipoprotein so the in the in the pre-estep what we had done is we immobilize the biotinylated antibody on to the step iodine sensor now we can do the quantitation using that sensors it's pre immobilized so what I'm going to do is I'm making a apolipoprotein standard with the concentration is 50 microgram per ml so I'm here I'm having apolipoprotein standard I'm going to dilute 50 microgram per ml using a sample dilute so from this I'm going to do a the standard I'm going to do a serially dilution like two-fold dilution I'm going to do with the buffer here to that what I'm doing is I'm taking this 96mm plate I'm adding the the buffer into the wells here I will add I'm going to use around the six concentrations two-fold dilutions so I will add here buffers I'm going to do a dilution in the plate itself so 200 microliter each of the sample diluent buffer I'm placing in the six wells of the 96mm plate I'm going to use here three as references this plain buffer uses as a reference here this is the 50 microgram per ml protein I'm serially diluting doing a two-fold dilution in the plate so now we had made a six concentrations of the standard apolipoprotein so starting from concentration range 1.56 to 50 microgram per ml I had put these standards the 1.56 in from A3 to A till A8 that is the last 50 microgram per ml as well as I had put a reference buffer the the buffer which we are using for the reference abstraction here so that I had put it at the B6, B7 and the B8 now I'm going to add a unknown samples we have three unknown samples here so that I'm going to put it in the B3, B4 and the B5 so these three unknown proteins we are going to determine the concentration of the unknown apolipoprotein in the in the samples using the standard now I'm going to add the three unknown samples here I'm going to place at the B3 this is the sample number one which is sample number at B4 the sample number three unknown samples so I'm using the glycine buffer that is a ph2 10 millimolar glycine buffer this is used for the regeneration of the the sensor so this the regeneration buffer I'm going to place at the A11 and the B11 so this will the the bound apolipoprotein will be washed when the the sensor dip into this acidic buffer so also we have to place one more which we we call it as a neutral buffer that is what this pvst buffer I'm going to place this one at the A12 and the B12 now I'm placing this sensor tree into the instrument at the sensor compartment and also we had prepared the sample tree here we had put the unknowns as well as the standards apolipoprotein in the sample plate now our aim is like here we are performing a quantitation experiment so now we have to choose the quantitation mode here new quantitation experiment here in this we are using a basic quantitation with a regeneration because two sensors we have immobilized and the same sensor will be regenerates and can re-acquire the different concentration data so we had to choose the blank experiment and say basic quantitation with a regeneration blank experiments then we had to say grow here in the experiment when you open this experiment you will see the plate definition in the plate definitions what what samples we had put it here so here from A3 we had put around standards A3 to A8 6 concentrations we have chosen this 6 concentration we have chosen this as a standard okay now we can see that this is alerted as in the green color and also here one more we had chosen these three era unknown samples and these three are your reference you can choose the reference or later also we can we can we can take it at the reference into a like even unknowns later in the data also we can that that can be modified as a reference here in the table on your right hand side we have to put the information here in the A3 what you had to and A8 what you had to in the method modify section here single analyte we have to choose 120 seconds we have to use a 400 400 the the regeneration is five seconds it will dip in the acidic buffer five seconds it will dip in the neutral buffer that that cycle has to be performed at least for the three three cycles so then precondition means sensor first will regenerate and then goes to sample post condition is nothing but once the sensor is put it into the sample after that it will go for the regeneration so we have to choose both precondition and the post condition sensors 120 seconds is the acquisition time i can still extend to a 150 seconds here then say okay 400 is the shaker speed i will say okay here then the sensor assignment it is showing so many sensors here but we are using a two set of sensors for the acquiring the all the samples so now we had kept a sensor at the position one so i will say remove automatically your sensor is placed at the second we had kept sensor for the equilibration using the VST buffer in the review we can we can see how exactly the instrument will perform sensor is picking from the second position it is going to a regeneration then if you click on this arrow it is a neutralization regeneration neutralization regeneration neutralization in the method we had said that it has to in the play technician we had mentioned that it has to perform for a three cycles so that's what it is indicated by in the experiment it is acquiring for a three cycles then later it goes to a sample then once again regeneration neutralization regeneration neutralization regeneration neutralization then goes to here second set and regeneration neutralization regeneration neutralization regeneration then the third same same the process will continue till the last in the running experiment where you want to save the data i will mention this file as a The sensor is already highlighted I will uncheck this then I can say go here the instrument is picking the sensor from the sensor back and it is going to a regeneration see the pink one is the one which is unknown sample here and the the green the sensor it is indicating the lowest concentration which is around 1.5 microgram once the data is acquired the green indicates this is the standard and as well as the pink indicates this as an unknown sample now the acquisition is done we will go for the data analysis this is the file this is the acquisition file indicated by a green pillar here so now we can see this the graph it is indicating the all all it was standard this is your standard graph the one which is the 50 microgram is the highest the binding curve is showing in the in the second then the third then the fourth and the fifth concentration and the sixth so highest will be the 50 and the lowest will be the 1.56 microgram for here there are the three unknown samples also we had kept in the in the v3 v4 and the v5 and the three reference samples these reference samples are nothing but your buffers buffers also sometimes gives the artifacts to subtract the artifacts from the from the signal we are we had to use a method called as a reference subtraction method here we are going to click this reference subtraction average because these three are well saw the buffers so it will get subtract from the all the all the all the acquisitions for the different concentrations as there will be unknown from that. So now we have to go here in the result section in the result sections we have a different equations here in the standard curve equations which one you want to choose like linear point to point we have 4 p l methods 5 p l methods as well as linear y is equal to a mx plus c kind of equation a into x plus b so we will go with this equation linear equation here and also we had put standard in the plate itself as well as the the unknowns so we can easily do a data analysis we can quantify the unknown samples then we say calculate binding rate when you say calculate binding rate we will see the strike line the blue dots indicates the different concentrations of your standards and the the red ones which indicates your unknown sample there are the three unknown samples here so the unknown sample we can see standards if you look at the standards to see microgram the the back calculated instrument back calculated it recovered as a 49.2 percent if you see the percent residuals that is what we are we are saying it should be the sub minus 10 percent for the except in the criteria so this is we are getting around 49.2 25 we are getting as a 27 12.5 we are getting as a 12 6.25 we are getting as a 5.73 3.13 we are getting around 3.27 1.56 we are getting 1.3 but somewhat this value should be a more than 10 even we can exclude also from the binding rate calculations not required I think here in the unknown concentration I should look at all the three samples which are the the well concentration is calculated as a 38.7 37.9 it is nothing but 38 microgram and 51 it is somewhat 51 is something like a very high showing but but our standard curve is 50 but still the the instrument extrapolates and calculates the concentrations so now we can save this data we can save this as a save report if you save report it will convert this into excel sheet like you can give a opposed standard curve then say ok it will create as a excel sheet the report will be created as a excel sheet in the summary you will you will you will see the what are all the data where it is and all that and the standard curve if you look at this is the standard curve we had the standard curve also chi square is point 997 r square and our chi square is a 0 this is a very good actually to look at all data points almost on the very near the line and in the plate map where you had put your the samples and the unknown samples in the result section this is your standard along with your percent residuals and also for the unknown samples here it is also an unknowns that that indicated to your calculated concentration if it is any like if your sample is dilution dilution diluted you have to put a dilution factor automatically calculates its the calculated concentration with respect to a dilution factor if you look at the r square values of the all the unknown samples as well as the standards it is a more than quantity so the data is acceptable so with this I am going to finish the quantitation experiment thank you. In today's lecture and demonstration session you have observed another application of bilayer interferometry I hope you made observation that is standard curve obtained for apolipoprotein E was linear and had good dynamic range with high response level these standard curves can be extrapolated to find the concentration of apolipoprotein in the complex solution of course you can choose any other protein of interest and the same experiment can be performed to measure the concentration of your protein of interest from the complex solution. Bilayer interferometry could be used to measure the concentration of a specific protein even in complex solutions without need to verify the proteins. In the next lecture we will see more of these applications of using label free biosensors to study biomolecular interactions thank you.