 Welcome to the CIS Distinguished Lecture Series. Today we are very fortunate to have Dr. Renu here to give a really exciting talk about the precision in medicine, point of care systems by medical sensing. And Dr. Renu has three affiliations. He's Harvard Medical School, the Children's Hospital in Boston, as well as Northeastern University. So he has a very long resume, so I have to just read most of the highlights here. He received his bachelor's degree from Magistrate University and then did master's program at Columbia University with PhD from State University of New York. And he studied with the Nobel laureate, Professor Harold Urey, and which is a very distinguished scientist. And he also published more than 250 publications and two monographs. He's currently working on the textbook level monograph on the bio-nanoscience. And he's a member of several academies. He was the editor-in-chief for Journal of Bio-Nanoscience and associate editor for Journal of Nanoscience and Nanotechnology, and currently serves on six editorial boards for various journals. He has been interacting with a number of leading researchers in the world from universities like MIT, Harvard, Stanford, Berkeley, et cetera. So he's very well known for the biosensors at the interface of engineering, materials, and biomedical applications. So without further ado, I would like to invite you to join me in welcoming Dr. Renu for this wonderful talk. Thank you. Good afternoon. I'm very pleased to be at the University of Wisconsin in Milwaukee campus. I had been to the Madison campus many years ago. It's quite a surprise waiting for me here that it's expanding. I have been very fortunate in knowing Professor Zhenhong Shen, with whom we share many common scientific interests. And I think our partnership in census will be a tour of the force. Having said that, what I'm going to talk today is on personalized precision, personalized medicine and the various tools that are emerging from nanoscience and nanotechnology, protein engineering, and a number of other areas, and medical areas like nephrology, endocrinology, cardiology, urology goes on and on. If you look at the way this country is operating, we have a health care industry that is escalating. And there's been a lot of discussion in the last 10 years how to reduce the cost. If you look into the breakdown of the cost of the major illnesses, lifestyle diseases, one thing that comes to the attention, redundant, repetitive diagnostic tests, and a somewhat reluctant medical community to share the information. Because of several complications, doctors are not talking to themselves. It is a collaborative effort. It's not a single physician alone can solve the problem. So the repetitive redundant cause or a very large component of the total health care cause upwards, spiraling upwards, past a trillion dollar mark. In fact, a trillion dollar mark would be approximately one-third the GDP of France. Therefore, there is an urgent need to avoid repetitive tests and to make it more accurate. That is where the recent developments in a number of areas, material science, nanoscience, nanofabrication, protein engineering, molecular biology, biotechnology, and engineering all converge. If you do that, the tests become more accurate. Second, why is that health cost increasing? For example, glucose medias have been around for a long time. Measuring cholesterol at home is not easy. Measuring triglycerides is not easy. Now, there are so many analytes, the liver profile, cardiac profile, the nephrology profile. Some of them can be done by an educated patient at home. That will reduce the cost and the information will be automatically relayed either from home or from an ER, from an ambulance or an emergency room. And there is a lot of miscommunication between the two and we are making these tests again and again and again. So, taking advantage of the great developments in material science, nanoscience, it is possible to make these point of care devices home-based physician office, emergency room, or in a continuous dynamic mode where we can measure from a smallest amount of blood, the entire biochemical profile of a person, and from there the diagnosis begins in a collaborative collective effort. In the case of children, this poses neonase, this poses a problem. In the animal science area, this poses a problem because we don't understand so much of, although we are using animal models in biology, there's a lot of gaps in the veterinary science which came to my attention the last four weeks, four months. And I've taken that as a major mission to see that all this, we know in human medicine is transplanted to the animal world as we become more and more civilized towards the end of the century. Now, in the case of neonase, there are special complications. Life is just beginning. At the beginning of the life, there is the biochemical reactions that are taking place. The entire process of developmental biology is not known to us. Now, with all the advances, infant mortality is not insignificant, it's quite high. Now, our groups are working on a fusion of different disciplines of science and technologies. While we say something, say certain types of research we do may seem out of place in a medical school, yet you can see a common thread running through the entire mosaic. Now, we work on energy, photovoltaics, that's mostly and fuel cells at the Northeastern University, sensors, picomolar sensitivity, graphene-based microfluidics. That is done in a collaborative fashion between the Boston Children's Hospital which is teaching affiliate at Harvard Med School and University of California Berkeley, Stanford Medical School and Rice University in Houston, also with MIT and Columbia's partners. So, you can see we've got some of the elite institution, now the University of Wisconsin joins us. Now, we also been known for high density memory. Everything we do is bio-based. We believe in a bio world dominating, entirely replacing the presence inorganics and organic materials and making it more green and energy-friendly. Now, the high density memory storage had been a project that has been kind of hush-hush due to the commercial interest involved in that. All of this, some of it started at MIT and Harvard, then went to the Florida International University in Miami, is a collaboration between NEC, Sony and ourselves. The idea is to create a thin film, protein-based film, which is a photochromic material that can be used for writing and reading and densities can go upwards of 50 terabytes. All idea is to have a thin film embedded in a smartphone or in a tablet. That is quite exciting, but it is taken more than 15 years. Some of the initial ideas were taken from Soviet Union. Naturally, they are very upset with the whole thing and I've not seen a single Russian-made appliance or a consumer product in any department store, anywhere in the world, including Moscow. Now, we were also working and imaging fluorescence tomography, but that project is not moving anywhere. Then we have the bio-nanorabotics, which is funded by the Gates Foundation. The whole idea is to import more human-like characteristics to robots by using biosensors. Then we also have a last area, smell receptor arrays. It started with the founder of the Bose Electronics. The whole idea is to create sensors, smell sensors. How do we digitize smell? That is science fiction at this moment. So, we are supported by many laboratories, protein structure, dynamics, protein engineering, protein-based device fabrication, which is challenging. Many, many device fabrication technologies cannot be used when biomaterials come in. They are very sensitive. Carbon nanotube, which is based out. Graphene, which is mainly with Rice University, Professor P. M. Ajayan. And now with Professor Jan Hong Chen here. And University of North Texas was also a collaborator and the Vanderbilt material characterization center for nanoscale systems, solar cell assembly and testing at the National Renewable Energy Laboratory. All the clinical part, the medical part is all done at the Boston Children's Hospital, Brigham and Omen's Hospital and some at NIH and UCSF. Microfluidics is in the laboratory of Professor Dorian Leapman at University of California, Berkeley in California. There are many numerous collaborations and network of collaborations across Europe, Japan, some in China, Australia and Canada too. So, point of care system for ultra-sensitive quantitative detection of blood analyze relevant to diabetes and coronary diseases. If you look at the chronic illness in the USA, there will be a percent of the US healthcare dollars spent in chronic illnesses. Now, if you look at the two lifestyles, one of them is a lifestyle, this is diabetes. Diabetes is a silent killer, where the diabetes type one, type two and there must be some way of measuring the glucose. Glucometas have been around since 1975. We can also measure the HVA1C, that's fairly constant over a period of time. There is no need to measure them continuously because the dynamics of glucose is very different from the glycosylated hemoglobulins. Then we have the heart disease, the deadliest disease, the cholesterol, high density lipoprotein, low density lipoprotein and triglyceride. So, we need a point of care device that in a single platform can measure glucose, cholesterol, total, break it into HDL and HDL and look at the ratio as well as the triglyceride and an HVA1C channel two. If you look at the glucose excursions in type two diabetes, if you look at the variation into the day, it kind of fluctuates depending of what time you take the meal, it goes up and then down and up and down. Now, a stage has come and the glucose measures we are using are not sensitive. 15% or even 10% or 9% accuracy is not acceptable to the FDA because more original cases of diabetes will be treated as diabetes and you start giving the medications, whatever it is, glucophage, glucomet and genuvia or many other and metformin as well. So, the side effects of some of these is enormous. So, we got to really pin down who is really diabetic or pre-diabetic. That means we need an accurate measurement of glucose. Pancreas is the most sensitive sensor for monitoring glycemic dynamics. The pancreas detects the change in blood glucose concentration and releases the appropriate hormone by a complicated interconnected signaling pathway. So, we have a project organ and a chip, pancreas and a chip. That project is still in its infancy. There are quite a few groups working on it. We were in collaboration with a Wies Institute at the Boston Children's Hospital and also with the School of Engineering and Applied Sciences in the main campus. There are many biosensors, sensors and biosensors. You have an analyte and a response. There's an analysis, signal detection, sample handing and preparation. There are many ways by which there are many different techniques for biosensors. Fluorescence, DNA microarray, surface plasmond resonance, impedance spectroscopy, scanning probe microscopy, atomic force microscopy as quartz crystal microbalance, surface enhanced Raman, electrochemical, amperometer. The last one field effect transistor is the mode we have chosen because it's very sensitive and very accurate. Amperometric lab and a chip consists of at least four microchannels. Flow to micro chambers, one consists of glucose for the second one is for cholesterol, third for triglyceride and the last one can be turned on and off. That is for the glycofumoglobin. Now, this is a flow chart of the device. We work with the tiniest amount of blood that you can think of. There is no point in getting large amounts of blood in certain cases. In the emergency room, it's got to be the smallest, tiniest of tiniest. Then it undergoes a separation into four channels, as I mentioned. One glucose, HBA once it can be turned on and off, LDL, total cholesterol and triglyceride. The basic processes involved in using for example, glucose oxidase, cholesterol oxidase, cholesterol esterase or lipases for the detections, ultra sensitive detections depends not only on a platform, that platform I will come to is a graphene, single layer. It can, the basic processes are electrochemical in nature. Redox reactions, transfer of electrons, occurs at working electrode, produces current, electrons flow to the counter electrode and the current is purported to the concentration. They are very simple things. So, there is an oxidation. The loss of electron and the reduction again of electrons is the redox reaction. So, there is a movement of electrons all across. In the case of glucose determination, we use glucose oxidase as a probe. Glucose oxidase undergoes a chemical reaction, releases hydrogen peroxide and the electrons flow from the glucose oxidase from the center of it, called the flavin adenine dinucleotide on to the electrode. Either carbon nanotubes or graphene, now graphene, they are completely abandoned carbon nanotubes and direct transfer is inevitable. Now, the fact which is the electrochemical hot of this enzyme is a cofactor, very important cofactor. Without it, glucose oxidase is not active. It's oxidized, electron acceptor, FAD cofactor is involved in several important reactions. Not only in glucose oxidase, it can exist in two different redox states and its biochemical role usually involves changing between these two states. The complete oxidation reduction cycle involves two protons and two electrons. Cholesterol determination, LDL and HDL concentrations are the two different enzymes. One is cholestrolox esterase, the other one is cholestroloxidase. Both of them contain the same cofactor, flavin adenine dinucleotide. LDL determination will block the LDL. We use the anti-human beta-lifer protein to block it. So, we are looking only at LDL and the total. Convert to cholesterol using cholesterol esterase, oxidize the LDL cholesterol, we use cholesterol oxidase and finally, it can go into an amperometric detection or interfere with the FET sensor. Total cholesterol determination extracts the total cholesterol ester, convert to cholesterol, oxidize and then it goes into FET or amperometric. Amperometric, we are not doing anymore. Triglyceride, we use a different enzyme. That enzyme has been cloned in our laboratory. We make all of our proteins. They are protein kitchens. Lyserol-3-posphate oxidase called GPO. It also has a fat cofactor. Conversion of triglyceride oxidize the glycerol-3-posphate and eventually goes into FET. Now, in the case of the glycosylated hemoglobin HVA1C, it undergoes the glycosylation to fructosyl peptide, then proteolysis, then fructosyl valine is actually fructosyl valine. Aminoxidase is the enzyme, releases hydrogen peroxide and the electron. Now, we were earlier using carbon nanotubes. We are decorating the carbon nanotube by covalential linking various types of proteins and lately microRNAs, too. Immobilize the protein. Three-dimensional electrode improves sensitivity. Immobilization is done by introducing carboxylic acid groups by acid oxidation. Activate using EDAC, which is 1-ethyl-3-diamethyl amino-prothyl-carbodiamide. Stable active ester, N-hydroxy succinamide. There is amide bond formation, nucleophilic substitution reaction. Now, all the proteins are expressed in yeast system. We are using E. coli. Problem with E. coli, highly glycosylated, phosphorylated, proteins cannot be expressed. So, they will not fold correctly. They are folding in the protein. When you express it, it is very important. So, we have switched to the yeast system very successfully. Now, expression and purification in pro-proteins transform into p-care-pastore stream. Play transformments. There are several steps involved here. Now, the general strategy is whether you look at a solar cell, photovoltaic cell, or a biosensor to design a point of care, or you look at any other area of topic that you are working on, like memory project, for example, photovoltaic. There is an organic, inorganic material, a synthesis cloning by molecular biology in the case of proteins. It forms nanostructures and there is a nanointerface. That nanointerface is a gateway. So, then there is an organic-organic interface, inorganic-organic interface, inorganic-biointerface, metal oxide, organic-metal oxide, bio-nanointerface. So, if somebody were to ask me why in the children's hospital we are not shining light on the patients to generate electricity, but there is a common element here. Unless we understand how the electrons flow, there is no way we can design the optimal, efficient biosensor or point of care device. Now, graphene functionalization has been problematic. We use a linker molecule. We work on single layers. All that is done in the laboratory of Professor P. M. I. J. M. at the Rice University in Houston. And then we functionalize it using a linker, organic-linker molecule. So, very simply in organic compound, we should synthesize in large amounts and that links to the enzyme or the protein. Now, the functionalization protocol, you incubate the glucose oxidase with a fly millimolar linker molecule, one pyrin butanoic acid succeeding in the middle ester in dimethyl formamide for two hours at room temperature. Then it is washed with pure DMF and deionized water. The linker modified graphene was then incubated with 10 ml. Now, glucose oxidase is again originally obtained from the fungus. We can commercially buy them but you got to purify them extensively. Protein purification is an odd. There is nothing called a 100% pure protein. We can call it 99% plus. Now, therefore, we have cloned the glucose oxidase and so we use the cloned recombinant glucose oxidase. We also have developed by protein engineering method very large number of mutants that can interface with the single layer graphene very effectively. Then we use the Fourier transform in Roman in the signature region, amide one and amide two in infrared and amide one and amide three. They call Boxer stretching region which show perturbations that will indicate that the enzyme has been successfully bound. It is retained in that state. Otherwise, it is useless. If you are evidence of corboxyl functionality. Then doping leads to the discovery of new layered materials. One of the common doping that we found very effective is the fluorine doping. There is lightly fluorinated graphene, moderately fluorinated graphene and highly fluorinated graphene. Size specific fluorination or doping is still a black box. So, we are trying to use some enzymatic methods by which the fluorine atom can be placed in the center of the graphene or the size of the graphene or the edges. We were using carbon nanotubes up until about five, six years ago. The discovery of graphene completely overshadowed the carbon nanotube. It's a very simple discovery. Theoretically, it was possible to take a carbon nanotube, rip it apart, lay it on a table, on a flat table. You basically end up with a graphene. When I visited the laboratory of Sir Konstantin Novosilov and Henry Guine at the University of Manchester, about approximately 10 years ago, I came back not very impressed. Much earlier to that, a postdoc of mine came to my laboratory, to my office, at about one in the night. He was a little unusual person and he said, I did something bad. I said, what happened? I broke, broke what? I took a carbon nanotube and made that into a flat shape. My next question is, is it stable? Theoretically. He came back and said, it's low and behold, it's stable. And I asked him, where are we going with it? He said, I don't know. There must be some experimentalists who can create this. The meanwhile, graphene was born in different laboratories. It's simply not one laboratory alone. There was a lot of controversy on the water of 2010 Nobel to Henry Guine and Konstantinovacero. They were taken by surprise too. Scientific community was taken by surprise. Today, we've gotten to a stage. There's no need to go back to carbon nanotube. We never go back in science and technology. Sometimes we revisit or quote some classical papers in physics, for example, in mathematics or even in chemistry. So the question is not what we can do with graphene, the question is what we cannot do with graphene. Therefore, we are looking beyond graphene. We are looking into silicene, organosilicene. We are looking into molybdenum sulfide, dope graphene, fluorine dope graphene, boron dope graphene, which is suddenly assumed. Tremendous importance in the last two weeks. The rate at which you see news items and graphene is coming is astounding. Nothing like this has happened before. What's the because we have the internet? Any information from any place goes to another place very quickly and people are hungry for information. Now, having said that, we were using the carbon nanotubes as a platform long time ago. No matter where you use graphene or carbon nanotube, the first step is immobilizing the protein. When proteins are coupled to carbon nanotubes or graphene, there's a charge transfer. I talked about charge transfer before. From the protein to CNT or graphene, which is the fundamental principle, foundation for the construction of any biomolecular sensing device. The nano dimensions of single wall carbon nanotubes and graphene, of course, their electronic properties make them an ideal substrate candidate for anchoring the proteins for biochemical sensing. Integration of carbon nanotubes proteins by covalent attachment influences the conductance of CNT. The three steps, carboxylic acid groups are introduced by acid oxidation. Activation is done by EDAC, 1-ethyl-3-diamethyl amino-proxyl-carbodiamide, then stable active ester using N hydroxyl succinamide. Then there is an amide bond formation between biological macromolecules, protein for example, or an enzyme and the single layer gases. All these proteins are expressed in our laboratory. Expression of glucose oxidase was being done from fungus. Isolation was done. Now, we have cloned it. They created a library of mutants. What is subjective in using protein engineering here? Number one, to be able to produce them in large amounts. Number two, to be able to site specifically mutate them to make the interaction between the enzyme and surface, one side of it and the single layer graphene. So, they switched to yeast system, picaplastores, which is an ideal system for those proteins that are highly post-translational remodified. Now, the expression and purification consists of several steps. The project strategy is basically either CBD, I mean graphene is produced with CBD, a dope graphene or it could be an organic molecule or it could be an inorganic surface. Making a nanostructure, there is a nano interface through which electrons have to go through the gateway. No matter whether you look at biosimpsons or point of cat devices, photovoltaics, memory devices, there is an organic, organic or inorganic, organic or inorganic biointerface. We have got to understand this interface very clearly. Now, graphene discovery goes back to Manchester according to the Nobel. Today, there are probably about 18 or 20,000 papers that are flowing in. The rate at which we are reviewing papers, all of us, on graphene is astounding. I wonder that I am sure there is a lot of repetition, a lot of incremental things being reported. So, the CVs are bloating. When you see numbers 200, 300, 500 papers, I think if you finally it looks like a kale in the supermarket, huge. When you bring it home and boil it, it shrinks. So, there is a lot of redundant, repetitive papers that are coming up too. Now, graphene synthesis is straightforward. That is a by CVD chemical vapor deposition. I will not go into it. Transfer the graphene for a flexible electrode. Then, the functionalization to create corboxal functionality. The same thing we did with the carbon nanostructures. Now, the functionalization protocol, GOX was incubated with a 5 mm linker molecule, a simple organic molecule in dimetal form of mine for 2 hours at room temperature and washed with pure DMF and deionized water. The linker modified graphene was then incubated with 10 milliliters of the rest of the buffers and we used the Fourier transform and infrared. You can see the infrared here. You can see a shift in the carbon illustration region, indicating that the attached, covalently attached protein or enzyme is really bound to the graphene single layer surface. You also use Raman. Raman is more conclusive. You take the Raman and infrared. Basically, your evidence that the functionalization is gone through and the proteins are attached are quite intact. You have been looking at graphene forms. This was in collaboration with Vanderbilt University. We are not doing it at this moment. Now, the doping leads to the discovery of new layered materials. Fluorine dope, hydrogen dope, nitrogen dope. There are several dope. We are an European dope graphene. Fluorographene, graphene, the problem is a zero bandwidth. So, when you create a device out of graphene, switching on and off becomes a problem. If you open the bandgap by means of doping, slight specific doping is still stopped. You need some enzymatic methods by taking the fluorine, putting it in selective spots that is still much more a conjecture at this point. Now, boron doping has been looked at experimentally and theoretically. We use a density functional theory. We do a lot of things. Physical chemistry is the same time. So, to do this all under one roof is impossible. So, we collaborate with many institutions and laboratories on the campus and out of the campus and out of the country too. Fluorographene comes from Professor Ajay's laboratory and in future it will be coming from the University of Wisconsin-Milwaukee from the laboratory of our GenFoN channel. Now, we also use the fluorine 19 and the carbon 13 magic angle spinning to characterize these graphene and right now we have done a lot of atomic force from studies at the highest resolution possible. We also do small angle neutron scattering as well as inelastic neutron scattering. Now, cryo electron microscopy is done in Oxford an instrument that can go up to 2.7, 2.8 angstrom resolution. Cryo electron microscopy is getting to age, it is catching up. But ideally if you can crystallize the material with very poor long range order, it is very tough. If we can do it, then we can use X-ray crystallography. So, X-ray crystallography will not work. We have got to look at small angle neutron scattering, small angle X-ray scattering, cryo electron microscopy as well as molecular dynamics which use X-ray crystallography. Now, the protein engineering at glucose oxidase, these are some snapshots at 200 nanosecond simulations. Now, we have gone into microseconds. Microsecond simulation of a graphene complex for protein sitting and graphene surface requires petaflop computer. We have gone to the highest level possible sphere. This is a 100 nanosecond snapshot from molecular dynamics simulation and glucose oxidase interaction with graphene. What are we trying to do here? We are trying to make the interface between single layer graphene and the protein as efficient for optimal transfer of electrons. Now, there is a cross talk between the enzyme, the probe and the graphene surface. Electrons get quite lost, although the electrons can travel up to 30, 40 angstroms quite easily. So, the current that you observe is directly proportional. That is where the FET sensor becomes very sensitive. Optimizing the electrical communication between enzymes and the electrode is critical in the development of biosensors. That is, in fact, a major, major challenge. Enzymatic biofuel cell and other bio-electro-catalytic apparatus. One approach to address this limitation is the attachment of the redox mediators or relays to the enzyme. Here, we report a simple genetic modification of a glucose oxidase that is being published in materials today to display a free thiol group near its active site. This facility is the size-specific attachment of a nullumized modified gold nanoparticles enzyme which enables direct electrical communication between the conjugated enzyme and say graphene surface. Glucose oxidase, cholesterol oxidase, cholesterol estuaries, lipases, in the case of HbA1c, fructosylvalene, amino oxidase, and you are going to hear the ammonia sensing in plasma, which is one of the most complex projects coming up in a short while. So, glucose oxidase is a particular interest in biofuel cell and biosensor application and the approach of pre-wiring enzyme conjugates in a size-specific manner will be valuable in the continued development of these systems. Now, if you look at this, these are some amperometric results. They are not very satisfactory before we switch to FET. We switched to FET approximately 2012, about five years ago, four years ago. Before that, we were using amperometric acrochemical methods as sluggish. They are not very sensitive and you want to reduce them to a handheld device to measure glucose, cholesterol triglyceride, HbA1c and several others. You need to have only FET type of sensors. This is a cycling ultramograph for cholesterol bio probe with different cholesterol concentrations. It is sensitive, but not as sensitive as you would like it to be. This is a graphene-based sensor response to glucose concentration, the resistance on this axis and the glucose concentration on the x-axis as a function of glucose for a single sensor is shown for three sequential experimental measurements. Now, if we reduce all the data that we obtain on the glucose FET sensors, cholesterol, triglyceride, HbA1c and now with the liver enzymes, with the nephrology, for example, kidney injury molecule, creatinine, blood urea nitrogen, the whole variety of things. In fact, the other day somebody was telling me we can have a couple of blocks of people. We can line them up in a cube. Neurologists, obstetrician, gynecologists, nephrologists, urologists, cardiologists, it goes into several areas. All of them are interested in the sensitive point-of-cat devices. Now, microfluidics has been around for a long time. The flow of a viscous fluid like blood requires understanding and manipulating the microfluidics to ensure the efficient functioning of the design biosensor. It provides an understanding of the behavior, the size, control and manipulation of fluids that are geometrically constrained to a small, typically sub-millimeter cave. Microfluidics goes back to 1950s. What is happening now? I hope this trend becomes solid. The discovery of something in the laboratory, a prototype sitting in the laboratory, going into some kind of a pre-commercial prototype and then various phases of it before it ends up in the market. It's a long chain. It's kind of getting reduced now. That trend is good. If you look at the history of science, everything that was discovered in the last century, some of them took a long, long time. There was no communication at the time between scientists. The left image shows a microfluidic test bed made from polycarbonate using hot embossing. The fluid channels are 100 micrometers wide, 75 micrometers deep. In the middle of the device is a graphene FET sensor with gold contacts that are accessed through the holes in the plastic cover. The right image shows a magnetic sensor chip with micro-fabricated electrodes integrated into the polycarbonate. All this is done at the University of California, Berkeley in the laboratory across the Dorian River. Now, initial experiments with the device, large range of glucose concentration is got to work in low glucose, mid-level glucose and high glucose. High sensitivity at low concentration work repeatedly with somewhat reduced amplitude. This is the figure at the bottom. It shows a microfluidic channel in the plastic polycarbonate shielding, gold pads opening for electrical connection. Sample volume in a microfluidic channel is approximately two microliters. The device fabrication, I am not an expert in microfluidics. All of them is done in Berkeley in the laboratory across the Dorian River. As I said earlier, it is in thermoplastic devices, polycarbonate or acrylic. Hot emboss devices. There are several steps involved in this. In the sensor fabrication, a gasket is created in a plastic sheet via hot embossing. A functionalised graphene sensor is placed inside the gasket. Using a mask, two gold pads are spreaded for electrical connection. Now, the IGN ships the entire device with a gold contact to us. Then we make sure they are alright. Functionalization is done in Boston. Microfluidics are there. And MIT comes in in a variety of stochroscopic techniques like surface environment in Norman. With a group in Columbia, we collaborate with also. Microfluidic channels are printed on a double sided tape and placed on top of the graphene sensor. The device is covered with a plastic sheet leaving openings for the electrical connection and inlet and outlet of the microfluidic channel. This is a schematic of the biosensors chip. There is the inlet for the blood here. The plasma separation is also a pump a micro pump and there are several channels that you see that in a little better. The prototype micro scale device that we have created single platform again I repeat is a multiplex sensor. One of the very few multiplex sensors that may become available in the market. So you don't have to be running around with different point of care devices. One for color store, one for glucose you know that will act to the cost. And the cost of this device is relatively it's affordable. If the glucose meter runs in about 18-20 dollars somewhat subsidized with glucose and blue shield this may come to maybe another 10 dollars more. Provided the patient is educated and he doesn't scared looking at the values. Another problem, a fear complex for example in micro RNA detection let us say a patient wants to test his blood sample for micro RNA for any type of carcinoma and he sees a value that is abnormal. What should we say? He gets scared so we want to avoid that shock and so some of these devices may still may have to be used in a physician's office or in an emergency room or an ambulance by trained paramedics but others can be done at home. Now since there are several field effect transistors the channel of which a pre-functionalized graphene and operated back gated mode. Each covalently anchored protein and graphene would be sensitive only to a particular targeted species this is a very important point. Emerging two-dimensional nanomaterials alone cannot are not expected to show any specificity. Specificity is something that is germane to the world of biology. A single enzyme can detect only one probe at a time sometimes multiple probes. So the specificity you want to call it two-dimensional nanomaterials comes only when you bring in the world of biology and life sciences into two-dimensional nanomaterials. Now one of the most complex projects we have is the high-sensitivity multifunctional biosensor for microRNA quantitation this entire project except for some parts of it in Chilin's hospital MIT, Berkeley is located at the Stanford Medical School of Radiology and the PI of this project is the person with a long name not as long as mine. Gene expression regulation by microRNAs in cell mechanism. MicroRNAs interact with the 3 prime UTR of messenger RNAs. Low microRNA, mRNA-based specificity can block translation each microRNA can potentially interact with several hundreds of mRNAs. So gene expression by mRNA degradation of translation the role of microRNA and as a biomarker we are all after biomarkers has been emerging in the last five years. The whole thing started with a crisis in the year 2013 2012 when a patient had a rare type of cancer bile duct cancer called Cholanguocosinoma then the oncologist in Danufarba Mayo Clinic also suggested let us measure the microRNA see we cannot be making experiments with a patient who is sick finally he died at that point there were only two or three laboratories Mayo Clinic University of Tokyo Danufarba no one else can measure, quantitate the microRNA now when we lost a few line species at home presumably due to carcinometrosis there is we try to measure the microRNA even the veterinarians will not agree that microRNA is biomarker for a few line stations but we did send the samples to University of Tokyo Mayo Clinic Stanford as well as Danufarba all of them confirmed the absence of microRNA later unfortunately after the passing of this line we found by an autopsis there was carcinometrosis which caused peritoneal fluid to accumulate and histona was bulging some of the veterinarians were so comical they thought giving him a four course dinner and making him eat because he has been starving for a week was an absolutely preposterous every crisis, every challenge is utilized whether human or non-human towards some new scientific discovery and this goes on endlessly all the 24 hours so the point here is the microRNAs are biomarkers before the the present surgeon general of the United States who is in Washington at this moment I have no idea at all he was proposing let us have a screening at the neonatal stage for microRNA can we determine this is also catastrophic if a child for example can be abused is diagnosed as a proclivity to its carcinoma in the later life what do we do it can be abused medical abuses are quite dangerous and they are going to be there for a long time to come so the idea is to have a microRNA screening in the life what do you do with it that is another question that is where the cancer therapeutics comes the role of microRNAs has been very well established in breast cancer, certain types of cancer and they are all 22 male polynucleotides so we make a sense and they are conjugated hybridized on the surface it is a very complicated concept the multifunctional PLGA nanoparticles for antisense microRNA corgidine peptide there are several steps involved in that strategy is currently used for microRNA quantitation quantitative RT-PCR persists the copy numbers from microRNAs TACMAN probes the quantitatively measured microRNAs molecular beacons microRNA hybrids next generation sequencing microarrays the scheme of graphene based sensing so microRNA consists of a synthetic polynucleotide 22 residue long synths and antisense are hybridized we look at the fluorescence properties for example graphene concentration dependent quenching for the 165 antisense microRNA as you increase the concentration so we use the fluorescence here fluorescence is a very sensitive type you also use impedance spectroscopy surface enhanced roman now to get the microRNA to a POC a point of care device it is very complicated there are many microRNAs they are in a sea of microRNAs I am not sure how we are going to solve the problem but the role of microRNA in carcinoma is getting firmer and firmer and firmer day by day like breast cancer lymphoma I think it is pretty well established we still have the pancreatic cancer liver, hepatococcinoma it is very tough we are working with transplant surgeons to get biopsy samples from outside the country I had to be able to extract the microRNA and look at them so here comes the transplant surgeons if you are complaining about one brand that was left out see this multi-channel the schematic illustration I propose microfluidic sensor functionalized with multiple microRNAs targets for evaluating a panel of microRNAs from blood samples now comes the most challenging project they were taken up ammonia gas sensors have been there for a long time they are quite sensitive but to be able to sense ammonia NH4 plus not NH3 in plasma it generates at the bedside in a static mode and a continuous mode we use a family of proteins called ammonia transport protein AMTD ammonia transporter from AMT or orange family structurally and biochemically well characterized under lab as extensive experience in purifying and handling these proteins XA crystallography and ammonia transport protein has been done fortunately in order to visualize the conformational rearrangements there is a channel there so this is a channel protein that is embedded in a liposome NH4 goes plus 4 goes in finally there is a release of electron and on to the graphene suffer but the problem of getting a protein a proteolyposome to bind graphene is not yes where we are still experimenting with it now in order to test the AMTD activity we reconstitute the protein into liposomes and the activity is measured using surface supported membrane electrocosiology so there is a channel protein this protein is so big there are 11 trans membrane channels in it this is the early molecular dynamic simulation of the ammonia transport protein inside the bilay this is average at 10 dummy atom models of the ammonia transport protein from cryo electron microscopy not from our laboratory from our collaborators at the university statclad in Glasgow in a partnership with Oxford University and Grenoble in France so it is a multi national effort that is required here the future perspectives we are trying to use a mediated transfer a mediator is usually a ferrocenium the ferrocy nanoplasmonics for the detection of glucose now the challenge comes in continuous glucose monitoring what do we need that we all saw the variation of the glucose at the time of the day therefore you need to have a bedside glucose continuous glucose monitoring I think this is still a far cry I was tossing into some of the drug companies none of them are sure that they can come up with a continuous monitoring we need one for cholesterol so it is a lot of work here but when we do research we are concerned about something very important here simply we do not want to make a device we want to understand the science behind it would that science open up new avenues for example a merger between material science and enzyme catalytic as I mentioned for site specific doping, fluorination enzymatic processes these are some areas which will bring in new science the science as it is developing now we are more going into application science but while forgetting fundamental seminal advances in chemistry and physics is what prevails the whole that is what I would like to see any project this lab and a chip has been around for a long time but a multiplex sensor is a technological engineering challenge there are emerging platforms boron nanotubes molybdenum single layer is very promising our team should have my name at the very end Dorian Leapman at the University of California Berkeley P. M. H. M Ph. T. Rice University of Houston, Texas another long name from Stanford Medical School J. R. T. Berry M. D. is an expert in periodic diseases Michael August is a medical intensive care unit Joseph Bonaventure renal division Harvard Medical School urology Harvard Medical School or not J. Well at the University of Stathclades Flavon-Mathfilipec at the University of Warsaw and Dr. Saunia Bishwanathan at the Newton University Hospital she is an internist those three people inspiring us are not humans at the bottom of the list so this is an overview of the Harvard Medical School the quadrangle of course this place had been very historic children's hospital itself had two or three Nobel laureates three Nobel laureates the way you count on Harvard is a bit funny because people are everywhere every hospital tries to put their name so totally it is generated maybe a couple of dozen Nobel Prizes in Physiology and Medicine one way or another and quite a few of them still are around you can see them in the cafeteria sometimes colliding with them but I don't think there are any any special facts about them they're just like that the Children's Hospital in Boston this is my lab, this is the 14th floor my lab has two neighborhoods Harvard Yard great inspiration as I said this is a partnership not only between humans as well as non-humans right now as I told you we're extending this to an important problem renal collapse right now creatinine and blood urea nitrogen are two biomarkers we have a kidney injury molecule CHIM-1 and CHIM-2 came out of the beginning of the hospital we are doing a lot of structural biology too at one time I was only doing structural drugs but nobody buys it now unless it is tied to a disease or to a device or something that the consumer best buys otherwise you don't get it even with all that we don't get money scientist and money are two folds apart they are not being good entrepreneurs only a fraction of the intellectual property generated right in this building probably finds his way to the master's place unfortunately so working in all kinds of directions I want to thank all of you for listening to me, thank you Thank you very much Dr. Manu for the wonderful talk we have time for questions so many questions for Dr. Manu actually I have a question I know we have many students here so what they would like to know probably is what are the most opportunities for the biosensor research in particular development for engineering and real applications which we are focusing here on so maybe you could give some advice to them some advice Sensors are there to stay with us no matter where you look all kinds of sensors have been around for a long time biosensors that is the procedure in your laboratory for example here and bios and several others have become very important in medical diagnostics right you may also find applications in security for example in homeland security right when a hidden explosive or some thing is in the suitcase you got to go to parts per billion some of them may emanate or nitrogen oxides may come out of it ammonia may come out of it or it might be some other substances abuse so it has applications in those areas as well and these are getting so small that we do not know we may not be even aware that a sensor is around us therefore in terms of job opportunities first of all training in this area as I say it we got to create a new workforce that workforce will be very different from the workforce we need the training at one time maybe 10-20 years ago we talk of biosensors we need a strong background not only in biology but in biochemistry as well right and the ability to express these proteins is a biotechnology concept so the type of courses that a student should take to prepare himself for the future job market is quite complex we are not sure we got the faculty in all the universities to be able to teach this we may have to create a new generation of faculties we will not have any mind sets or open mind like all of you the younger generation here so job opportunities are growing if you look at the number of medical companies that are working in diagnostics take Minnesota now we have got the electronics we have got the Boston scientific and Maple Grove Decton Dickinson then you have got the Thermo Fisher right there are several companies that are coming up worldwide market for this is increasing I have seen some of these figures being put out when you ever start the company like you the statistics I wonder they are true they are probably true and also there are other companies competing with us this is a politically contentious issue right now companies in China in India in Taiwan and just about everywhere and therefore job opportunities should be opening up I am also intrigued how much of this workforce will remain here you know the eastward migration may occur too that is already happening in areas like information technology and software and so on and so forth therefore I think the future is very bright the question how we train them and also the background of the student assistance 4 year colleges are not doing a good job students end up in the 4 year college from a school that is lackluster has not paid any attention to training all these things worry me so there must be a very robust teaching program jointly for example the medical college of Wisconsin should join Heinz medicine campus your has been well established I know whenever you have a huge towering institution like Harvard and MIT Boston University and others feel a little not so happy and they try to compete I don't think competition is there the collaboration is the way to do it even the magnetization I start talking about the material modification you mentioned to functionalize the big protein or enzymes on the carbon nanotubule but there are also records saying easy to peer all those big functional groups do you have any suggestions for utilizing the boundary between the protein and two ways of looking native enzymes what we call as wild type can do the function but certain residues in say for example an ammonia transfer protein might be binding to the graphene so if you mutate those residues electro statics you may be charged there lysine, oxygenine mutamic acid you can modify them so that you can make the attachment much stronger that is where protein engineering comes but I have not seen we have done a lot of glucose oxidase mutation ammonia transfer protein is a beast if anything can happen to mess up a protein in nature ammonia transfer protein would be the most notorious member of that so that is hard so you can genetically modify them using biotechnology that is what I have studied I do not think it is any rule of thumb question related to the glucose sensor so we know for the home testing now the glucose has stretched across about one dollar or less a piece so I consider that as pretty affordable so what do you think about detection at home are you trying to make it cheaper or are you trying to make it more robust cheaper see there was a time when we were working on the memory project a questioner has supposing we need one ton for making let us say protein based memory devices thin film to be put into the tablets and smart phones at that time the price was about 600-700 dollars per gram today thanks to China the prices come to something like 10 or 20 or 30 dollars per gram so the cost of the one component of the material is coming down now the question of graphene mass production is being addressed again there are Chinese companies that seems to be ahead of just about everyone in the world so the material cost is less fabrication cost and testing and other things will be there so what will be the cost of the glucometer using the new technology using graphene microfluidics where not made any accurate calculation I don't think it's going to be bad but cost again is dependent on the demand so if the cost is comparable or in two times the cost of the glucometer as the volume of the sales increase it is going to come down and the lifetime is another question there is a time when people are saying I will only buy devices that will last lifetime I don't think it makes sense people who want to throw away they throw away a culture I think that this country has been singularly blamed will continue for some time many of us go back to an old device and take it to repair it who will repair it for what telephone breaks it breaks what we do with the components inside it or the green or the benign that's another question that we have to begin to work if the cost will come down eventually it's comparable it's not too folk maybe too folk now the question is now how many different analyzed in the blood you want to measure continue glucose is one now what happens in the continuous monitoring wirelessly the information is sent to a central desk in a critical carry on it there are the human elements to the concern she has to pass it on to the physician right wherever he is and if you are going to for example I tell you this outsourcing business in healthcare one block number one sensitive information is going on by the time I get a number to ring in India and pick up I want to pick up are we going to a lunatic asylum nobody picks up the phone send me a text message what's that somethings don't work by this so there is a human element involved so how many analyzed we want to measure continuously glucose maybe ammonia plasma and if you are talking of really very large scale multiplex system that can measure everything all you need is let us say 5 microliters of blood that information integration of all that information requires a physician's intervention that's where I think we believe in the medical community believes artificial intelligence for pain and fucking growth it will take a large amount of data collate and say these are the problem 3 4 5 6 7 8 9 even before the patient arrives in the emergency room so it has got to happen with such great speeds right go back to artificial intelligence will become very common algorithms are getting all the time better there is a number of interesting challenges and questions what will be the healthcare like in 2050 2100 we can make some educated guess about what will happen or at the same time the human life span is going up too not noticed by many people are living longer posing a major problem for the social security administration, Medicare, Medicaid and whatever that plan that we had being revamped into whatever that it may be revamped we do not know collaborating not one single person can do it thank you thank you