 In this course we are emphasizing the need for high throughput approaches for studying proteins and proteome. For such kind of studies protein microarrays have become a very robust platform. You have seen there are different ways of making protein arrays starting from printing antibodies or purified proteins or even tissue lysates or cell lysate or even just simply printing the cDNA and make the proteins on the chip using NAPA technology or nucleic acid programmable protein arrays talked about with Dr. Josh LeBeurre. So, there are many ways of printing the features on the arrays and you can have different type of contents which could be printed on the chips. However, finally what actually makes huge difference is how good your printing is, how reproducible your chips are from one to other batch there is no variability and the spot features are really defined really circular and you are not seeing any diffusion from each of the features. So, printing technology plays a very important role in whole of the microarray experiments and especially in the case of protein microarrays when we have different type of components to be printed on the chip it becomes much more crucial. So, we have invited Dr. Saloni Sonawala from Arrajet who is going to talk about non-contact inkjet bio printing which is one of the fastest printing technologies. At Arrajet her prime contributions have been in designing and optimizing projects performing assay transfer studies and leading advanced technical training sessions for microarray users worldwide. In today's talk Dr. Saloni is going to talk mainly about what are the key considerations for doing good printing for microarray slides especially the bio printing versus microarraying. I hope you will enjoy this lecture. Good afternoon welcoming all the new people coming in I will take some time for you to settle down today I am going to talk about Arrajet solutions you probably all have worked or done some work with microarraying designing an array experiment printing arrays can anybody tell me how many maximum features have you been able to print on a slide anybody who's worked on arrays. Okay so they are used to the HuPro 20,000 features that's good to know because we spent about it was myself working with the team at CDI who spent two or three years developing the HuPro array and finally I'm so pleased to see that it's in India and it was developed with our technologies we do know that there are issues in our technologies today and we are lacking in some of the critical highly sensitive methodologies where thousands of interactions can be processed in one simultaneous manner but it has to be cost-effective it has to use less of your precious sample because that's the most important thing you're trying to conserve save samples and get as many accurate runs out of it as possible and that is why we've got inkjet bio printing we are from Scotland Edinburgh that's where I live but I was was born in Mumbai so I still love the Scotland in terms of the collaborations we have done with the institutes in India and a key goal for me to try and see what requirements proteomics in IIT has or any other academic institutions to try and fill that gap. So it's a complete bio printing solution which means that there are R&D systems that you can have in your lab and then there are once you've developed that assay once you have a larger library to screen then there are same technology can be scalable so it goes to a higher level of a system so not necessary that you have to start with a high throughput platform you can start with the same technology with an R&D scale and then go upwards one of the key things that we are doing is the Arrayjet Advanced Services these are collaborative approaches with yourself as your scientist and our company scientists to develop the assay on the platform so we've done a lot of Eliza text transfers so right as you probably have all done Eliza's and you know that Eliza is quite time-consuming it requires a lot of sample you can hardly do few Eliza's before you get few errors etc so what we do is we are doing assay transfer projects from Eliza to inkjet and then we obviously provide consultation and other gaskets consumables printed slides so we sell the Hubro slides because we developed it simple why Arrayjet it's a complete solutions provider it's the fastest printing technology in the world so if you are to compare this with any other method of screening or printing or a ring it wouldn't give you the kind of efficiency that you would get in just 20 minutes of finishing your assay and spending the rest of the day actually doing analysis part which is crucial for your for your project rather than sitting three days and just by petting things like I said we have global pleasant but especially in India we've got lab India instruments I'm not sure if how many I think you some some of you might know the company it's a large distributor in India and they are helping us with a lot of academics institutions to try and get projects together to make sure the students are able to get the samples and analyze and print them and a particular facility these are some of the key institutions and companies that we've worked with as you can see there is a nice spread of academic institutions like the Sanger Institute United States Medical Research Institute Rosalind Institute where the Dolly was developed Griffith University Monash University so reproductive health science so these are we do work with a lot of academics because there are so many different assays and projects that different applications but instead of investing in five six different platforms the key idea is to have one platform every department chemical engineering proteomics genome makes glycans they all can come and use it and it is using a piezoelectric technology so the printing is as quick as this to be very honest with you it's a point two meters per second this is something that I like to circulate across you guys this is a liquid sample handler and it is able to handle biological samples in terms of 12 multiples of 12 or multiples of 36 so depending on how many samples you have in a 384 well source plate it aspirates the sample upwards and it attaches itself to the printhead this way so it makes a nice little attachment and what happens is afterwards you don't need these pins at all this is the biggest difference most of the technologies use spins they take your sample they pin it they take the sample and they pin it whereas for us we don't need these pins because they are brittle they break they get clogged and there is a lot of replacing maintenance all that is involved so what we've done is we bypass that so half of the printing or the hundred percent of the printing happens with this printhead so imagine it's like an HP color printer in your house now imagine the color printer is printing all your biological samples on the fly without touching the slides or contaminating with the slides so you are reducing the error rate you're reducing the samples that actually go and get picked up and get deposited because everything is happening with the printhead I'll just show this across to you try not to try not to touch the pins because they're a little sensitive to breaking so you can see that the jet spider is something that is in-house patented and developed it can simultaneously aspirate a set of 12 samples together and print them simultaneously so you imagine it's not just one one two two it goes 12 12 in 20 I think it's 20 meters per second and goes back picks up another 12 goes back prints it again 20 meters per second so the way we calculate the the fastness of the efficiency of it is 640 features per second so it's quick 640 features per second is super quick so sometimes you don't even know whether your sample is printed or it's it's ongoing because it's that quick when it moves so this is just to show you this is your 384 well plate this is your jet spider attached to the printhead and it it just dips itself and it picks up as little as 1.3 microliters enough to print 75 slides so imagine people struggling with 30 microliters sample 20 microliters for the whole year we only need 1.3 microliters as the minimum to be able to screen an array of 75 slides which is enough to give you more than enough results so how much sample are you saving so let's think about it that way again this is the printhead this is the jet spider and this is the sauce plate what happens is the sample gets aspirated upwards goes inside the printhead and it just prints that's the printing so this is the connection between the printhead and the jet spider and it prints this is a bit too technical it shows you how much volume of a sample you can get in your capillaries of the printhead because it's an industrial printhead it's extremely robust and anti corrosive so you can even get to the level of understanding how much volume you of sample you need for the whole year to be able to print let's say 100 slides or process 96 alizahs in less than a week so you can we can help you calculate this again I'll move through the video because I'll be able to show you offline this is taken from one of the studies we did very similar customer to Hebrew CDI very similar customer but we helped him to do 65,000 features in one slide so this was high throughput printing style but you see the morphology and you see the assay results that you get is highly reproducible so your one slide will be able to do the same job as your slide number thousand and this is what people have obviously this is coming from appendorf and we all know appendorf this is the results they got with their contacts pins for to where it took them maybe a week to do this or maybe two weeks to do this this is the work we did with Array jet not only for Hebrew not only for this customer but for many of them and we do this work it is highly precise for so you don't see any merging you don't see any dirt or missing data or anything of that sort again sensitive versatile reproducible multiplex because we support a lot of alizah tech transfers this technology is highly efficient to transfer any immuno assay into inkjet because any immuno assay that you're doing has certain limitations that all get transferred into positives so that most of your research is focused on getting the actual analysis the actual data high throughput screening this is something that we support in terms of whether it's antibody discovery host pathogen interaction biomarker discovery epitope mapping hybridoma screening there's a lot of discussion on Napa arrays hybridomas so we do that we've helped a lot of people today that you can see in this symposium as well to develop projects around hybridoma screening where you have your lysates they get printed off on one layer so different lysates get printed off on different slides then you have your target antibody of interest that gets printed on top of each other so you can imagine there is a spot and then there is another spot on top and because of that binding of one spot to another spot it's called a spot-on-spot assay so it's a spot-on-spot type of printing where you can make sure the entire interaction or the screening is done while it's getting printed antibody validation which we've all done small molecule library skinning again this is for drug targeting therapeutic antibody screening and gene expression profiling now you are going to think what samples can a rigid handle all samples can be printed so we go from nucleic acids so genomics lab can be used you've got cell lysates you've got serum or plasma that can be spotted small molecules aptomers hybridoma supernatons carbohydrates nanoparticles and polymers also and of course we do cell tissue micro-ring as well so the more things you can imagine outside the box what can this platform support the more answers you will get yes we can do it so it's quite flexible in terms of what your project is and what samples you have and then how how can we transfer those samples on to inkjet style of printing obviously again I'm saying this is not restricted to slides so again I'll let you pass this on I can pass this on myself but yeah so this is this is the plates and the slides that we can do so imagine doing one entire Eliza in one well and doing 96 Eliza's in one plate at one time and doing hundred such plates so 96 my maths is very bad that's why my biologic person but if you count this if you calculate this yourself you will be able to understand how many Eliza's you can do and how much time you can save to actually analyze the data points to get your data right because it's going to be highly accurate so I'll show you this is the plate that I'm circulating across in this plate there is one well your entire one Eliza can happen in one well instead of one 96 well Eliza plate for one reaction that one plate that one plate can all get concise into just one small tiny well so you can see that we can not only print on two wells but on two plates on two bio chips majority of the work I'll tell you is on slides to be very honest but I don't know how many of you have had any experience with SPR imaging SPRI technology but this is something that again is used for drug targeting and we are able to print on to the SPR prisms as well so there are companies that require SPRI as one of the key methods to get your drug target but we can we can reduce the process by helping them with the SPR prism printing it's very simple it's speed it's precision and its consistency there is a reason why yesterday you all could do work on new pro-arrays because there is a reason every array is accurate because it's printed with an ink jet technology so bio printing versus micro ring there are very key differences why people say oh but micro ring is outdated it's it's now everyone's move to next generation techniques whereas here you are we are giving you a complete understanding on traditional micro ring and what bio printing with Arreja do we have an inline optical quality control camera where we do the QC for you you don't have to have a separate QC step we'll do the QC for you and if we see that your important antibody is missing the coolest thing about the software is it remembers which slide your antibody is missing you go back and print it so at the end of the run you've not wasted your antibody you're able to get a full set of data from that one printing because it remembered that it is if it is missed somewhere because maybe you missed putting the sample or it was a bit sticky and it couldn't get aspirated in many of these techniques the software which is called the iris the iris as the eye can remember recognize which ok slide number thousand has my antibody five missing so what am I gonna do I'm not gonna have thousand slides with all the antibodies missing it's a waste of my experiment so what it does it is remembers that one antibody 12 is missing it'll go back and print antibody 12 to all the thousand slides in case you've forgotten to put the antibody or it has missed so it will make sure that all the data you get is a complete set of data and not just missing contents which sometimes we do see with other a ring technologies that you see missing missing content so here we are again bypassing that missing content again I mean this is this is a very easy table I would say using the pins not using the pins slow printing sample concentration is very critical here we have we can print on four degrees so we actually convert the whole machine goes inside a big fridge so the fridge is like this tall probably yeah it's probably this tall and it is this wide so what happens is the machine goes inside a printer and this is how the Hebrew arrays are actually made in Baltimore so when I went there for the setup the whole lab is converted into a four-degree fridge so what happens is the entire arrays that are spotted on the slide they are extremely sensitive and functional so they can be used and sold and a lot of people can make sure that the technique is quite standard again higher setup and maintenance that is again something which is bypassed because we don't require any extra fancy readers or fancy equipment or hidden consumables it's very simple the whole system works on liquid hydraulics all you need to do is prepare a glycerol buffer in your lab which you get a recipe you prepare a glycerol buffer that buffer goes into the system and that's really it that's all you need to make this work trust me that's all you need so people say oh you need this scanner to go with it you need a reader to go we don't need that it is compatible with lots of scanners which today you have in many labs so it reduces a lot of hidden work that goes into making an assay so for me it is what do I need to make this system work I need a glycerol buffer which I can prepare in my lab in five liters four liters that goes inside the system and that makes the system run and then slides which we all can buy from a lot of suppliers here so what really you need is just the running time again this is something that people have asked me in the past is so are you the only ones or are there other people so I thought I'll show it to you to see to make you see the difference and what is the edging effect here they're all non-contact so nobody uses pins we're all using this printhead mechanism but there are there are large differences in how we can handle each and every sample I'm gonna go back so you can see that the number of plates we can do the number of samples that can get handled are quite high and the deposition rate is the fastest which is why we are the fastest in the world so if there are assays that need to be done in a timeline and you have to report results in a week and you're not getting success with pipetted Eliza's what are you going to do you're gonna quickly run to an arrager printer print your samples as many slides as you want and process those assays instruments so this is again like I was saying it's not just it's not just for companies it's not just for research or high throughput scientists it is for R&D work as well I would say 50% of the people have worked with personally to develop assays including the Hubro guys there they're all institutions we work with Johns Hopkins we work with Sanger we worked with Monash University we worked with Griffith so there are lots of institutes that require these platforms more than the companies I would say so this is our entry-level system which is called the Marathon Argus it does not have that camera features that will remember and reprint this all these are the systems that are there have the camera feature which will remember and recognize and reprint the spot this is how the machine looks it's it's got glass panels so it's quite easy to see what you're doing you can actually see the spots getting printed this is the space here these are the two bottles I'm showing you where you can prepare your own glycerol buffer and you can and that's all is needed so you have your glycerol buffer in the system you put your slides you have your 384 sample plate here you have your slides printing here and that's it so it's quite easy it's really quite easy I started doing this technology when I was I think this is ages ago but to be very honest I was 22 when I started this and it was easy for me to grasp it it was easy for me to understand what the platform it's not really high high level high tech it's not that bad so for students who are using these platforms it needs to be quite easy for you guys to do things it shouldn't be that advanced it has to be easy it has to be user-friendly and it has to be fast this is something that we've developed it's an in-house servicing where we work in partnership with you you tell us about your projects we assign a scientist who can understand what kind of projects you're working on whether it's an Eliza based methods it's an immuno assay whether it's something else whether you're developing a chemical product whether you have some chemical samples anything you'd speak to us we will develop a protocol for you with our experience and the knowledge that we have shared with lots of industries to have a very easy cost effective method to transfer this into inkjet so we will print the samples for you it takes a week because it's very quick so it comes let's say on Monday we'll do the printing on Tuesday we'll give you the report analysis so it'll take a week by the time it gets shipped which is shorter time than many people here locally in India can also give you back as custom printed arrays and correct me if I'm wrong but I have been told that this sort of time lead to get the arrays back after printing is about two to three weeks depending on how busy they are or you know depending on how many projects they have so because it's fast we are able to do a lot of printing for a lot of students quickly so in a day we can finish off a lot of projects so you don't have to wait for your results or your reports so manufacturing services custom array printing again custom panels are available custom antibody panels are available that we we can pick and choose for you you must have from yesterday's you pro you have you know the Hebrew content it's got custom arrays you've got custom panels so what we can do is we can pick and choose different antibody panels that you want for your assay and we can print them however in whichever fashion you like that is with a rigid advance so different ways that we collaborate with students and researchers especially is we have a basic proof of concept like a pilot study where you understand what are the requirements of an assay transfer and then you outsource them the other few options are these so there are institutions where funding is extremely critical and this is why these are the approaches we can take so we've supported Indian Institute so IISC Bangalore for the UK IR and Geeta projects for their grant for the system where they feel that all the departments can make use of this we've supported working with Biocon in India in Bangalore sorry to get their immuno assays developed on the platform there are few other institutes in Pune as well as in Bangalore where the grant funding has been done so we are providing complete grant support so if you do need or you have something which you feel will require the system or will require the services and then there is a scope for a grant then we can give you all the grant support for justifying why the technology is required very simple workflow so you'll have an initial discussion with somebody like myself in India and we'll go to our head scientist or the team of scientists in UK we'll have a collaborative discussion on what you want to develop actually what is your assay what is your criteria what do you want to achieve out of it and then we will develop a printing protocol a printing support mechanism which at the end of the day will give you guarantee that yes I can come to them six months down the line for a next project and they'll be able to do the similar job for me so I think this is something that is a crux of the Arreger technology it's a software that is able to make you design the entire array so this is your command center this works in conjunction with the with the technology so when you're trying to print samples first you need to design your arrays you need to design your assays and this is done with the help of a command center software so let me just take a second here and show you a video so this is how the technology really works how the printing is done you can see this is the platform you've got a tray of 25 slides and that's how it moves each time it moves it prints 12 samples on one slide second slide third slide fourth slide till 25 slides and then on the fly motion this is how it's picking up its sample this is getting into the source plate where your biological sample is it aspirates once the aspiration is done it transfers the sample into your industry printhead which is like your color printhead so now there is no need of these pins there is no need to use these pins now it's all in the part of the printhead so your sample is here and then wait for a minute this comes forward and off it goes and each time it is doing 12 samples at a time across 25 slides that's your first redone moves again 12 samples at a time secondary done moves again 12 samples third redone it's gonna be less than two minutes for me to finish my 12 samples across 100 slides so this is the quick motion this is how quickly it moves as a printer once it's finished doing it it obviously has a standby mode where it is able to wash itself so many people must be concerned how do you do the contamination is it contaminated if you're picking up another sample does it know it has its own individual wash cycles so it does washing automated so whole thing is automated so it not only does the washing after every time it picks up a new set of 12 it washes internally and once the washing is complete it makes sure that the samples are getting back and your new set of samples is going back in let me show you another video this is how we have used a variety of different platforms or surfaces this is the one that times you so this video will give you a little bit of a timing this is in fast motion but within a minute you can have your entire panel of antibodies quickly printed and you see how this is moving this is how the aspiration takes place your sample goes inside the printhead then this is where it ejects out this is your software that I'm going to tell you about these are your plates where you can design what plate you want this is the washing step this is a test slide this will show you how many slides or how many spots are actually there look at how tiny the spots are look how many spots we can fit on a slide again temperature like I told you we can print from 4 degrees to 35 degrees and humidity is again really high from 80 percent we work with customers with 80 percent humidity or 40 percent humidity this is the washing cycle this is where the washing happens and once the washing happens which is the bit which takes some time because it's very critical to wash your one set of 12 samples to avoid any contamination we want to make sure these are some of the spots we've printed with label-free technique and I think I believe they are not printed on a slide they're printed on a completely different surface so you have a slide you have a multiplex slide you can even do go as big as a plate so each Eliza can happen on a plate you've got chips if your anybody is doing microfluidics work or any of the lateral techniques they can all be transferred so as I mentioned in the beginning the success of microarray based experiment also lies in how reproducible our printing technologies are how good our arrays have been made and there is no variability or very little variability from one batch of printing to the other batch of printing imagine when you are preparing the slides for doing micro experiments you are printing in hundreds of slides you know large number of slides at the same time and if there are a lot of variation to start with from slide number one to slide number hundred then your entire biological experiments and reproducibility will be compromised so it's very important to pay good attention to the quality control checks which are required to make good arrays and of course you know as you proceed to perform the experiments there has to be various QC checks to ensure that the quality is good for your printing and what slides you are going to use I hope in this lecture you have learned about inkjet printing and its benefits you're also taught about different kind of substrates which are used for this printing and advantage of this technology over other technologies in the next lecture Dr Saloni will continue and talk to you about how exactly this technology works and how it can be used for many microarray based applications thank you