Wearable Lab and Biofab on Body : Towards Closed-Loop Bio-Digital Human Augmentation

Wearable Lab and BioFab on Body : Towards Closed-Loop Bio-Digital Human Augmentation

by Pat Pataranutaporn

B.Sc., Biological Sciences, College of Liberal Arts and Sciences & Barrett, the Honors College at Arizona State University (2018)

Submitted to the Program in Media Arts and Sciences, School of Architecture and Planning in partial fulﬁllment of the requirements for the degree of

Master of Science in Media Arts and Sciences

at the Massachusetts Institute of Technology September 2020

Author...... Program in Media Arts and Sciences

September 1, 2020

Certiﬁed by......

Pattie Maes

Professor of Media Arts and Sciences

Thesis Supervisor

Accepted by ......

Tod Machover Academic Head, Program in Media Arts and Sciences Wearable Lab and BioFab on Body : Towards Closed-Loop Bio-Digital Human Augmentation

by Pat Pataranutaporn

This thesis has been reviewed and approved by the following committee members

Professor Pattie Maes………………………………………………………………………………………………….. Professor of Media Arts and Sciences, MIT Thesis Supervisor

Professor George M. Church…………………………………………………………………………………………. Professor of Genetics, Harvard Medical School Thesis Reader

Dr. David S. Kong………………………………………………………………………………………………………… Director, Community Biotechnology Initiative, MIT Thesis Reader

Wearable Lab and BioFab on Body : Towards Closed-Loop Bio-Digital Human Augmentation

Pat Pataranutaporn* Thesis Committee Pattie Maes, MIT Media Lab George Church, Harvard Medical School David S Kong, MIT Media Lab

We explore the vision of closed-loop bio-digital interfaces for human augmentation, where the bio-digital system allows for both sensing and writing biological information to the body. Current-generation wearable devices sense an individual's physiological data such as heart rate, respiration, electrodermal activity, and EEG, but lack in sensing their biological counterparts, which drive the majority of individual's physiological signals. On the other hand, biosensors for detecting biochemical markers are currently limited to one-time use, are non-continuous and don't provide flexibility in choosing which biomarker they sense. We believe that the future for wearable biosensors lies in going beyond speciﬁc sensing capabilities and becoming a wearable "lab" on body, where a small device can offer a fully integrated and re-conﬁgurable system that mimics several processes usually performed in the laboratory for clinical diagnostics and analysis of human health. To illustrate our vision of having a lab on body, we prototyped "Wearable Lab" a bio-digital platform for sensing biochemical and digital data from saliva. Our platform contains digital sensors such as an IMU for activity recognition, as well as an automated system for continuous sampling of biomarkers from saliva by leveraging existing paper-based biochemical sensors. The platform could aid with longitudinal studies of biomarkers and early diagnosis of diseases. We present example data collected from the device, show a preliminary evaluation, and discuss the limitation of our platform. The ability to write information back to the body is the second half of the closed-loop human augmentation cycle. Similar to how a closed-loop glucose-insulin monitoring and delivery platform has been proven to have superior clinical impact for diabetic patients, we imagine that in the future, a closed-loop wearable system might be able to sense multiple biomarkers and physiological data, and deliver multiple types of intervention to keep the person healthy as well as augment their capabilities in time of need. As a second part of this thesis, we present "Wearable BioFab", a programmable bio-digital “organ”, consisting of an on-body digitally-controlled biosynthesis platform for personalized, on-demand production of biological compounds. We prototyped a millifluidic bioreactor to produce biological compounds through the use of digitally controlled multi-wavelength mechanisms leveraging engineered genetic circuits. To interface biological production with a digital system, we deployed an RGB optogenetics circuit, a multi-channels light-activated system that allows for wavelength switching to activate production of different components using digital control. By mounting miniaturized light sources with a microcontroller, liquid circulating, temperature control system, and digital display interfaces, we can fabricate a compact millifluidic wearable device. We present our characterization of this platform, show preliminary evaluation, and discuss the limitation of the platform. Together, "Wearable Lab" and "Wearable BioFab" demonstrate our vision of closed-loop bio-digital interfaces for human augmentation, which we hope will inspire the future integration of biology and computing, and shape the new era of human-computer integration.

To Mom & Dad and Dinosaurs! Wearable Lab and BioFab on Body : Towards Closed-Loop Bio-Digital Human Augmentation

Author ...... Program in Media Arts and Sciences August 09, 2020

Certiﬁed by...... Pattie Maes Professor of Media Arts and Sciences Thesis Supervisor

Accepted by ...... Tod Machover Academic Head Program in Media Arts and Sciences

Acknowledgement

I would like to ﬁrst thanks my thesis readers : Professor Pattie Maes, Dr. David Kong, and Professor George Church.

Professor Maes is my great mentor and advisor, she gave me a lot of insightful advice and the intellectual freedom to explore the many futuristic ideas that I wouldn't ever have thought of otherwise, and most importantly being an inspirational role model that makes me want to be a better person every day. Dr. David Kong for encouraging me to always be positive about research and life, especially during challenging times. Dr. Kong's research has inspired and encouraged me to pursue interdisciplinary research on bio-digital interfaces at the Media Lab, and also reminds me of the power of working with community. Finally, I am always excited to listen to the bold and futuristic ideas and visions of Professor Church. His talks always spark my curiosity. I hope that one day he will decide to create living dinosaurs for all of us. I would like to thank my collaborators M.S. Suryateja Jammalamadaka (the guru), Brandon Dorr, Abhinandan Jain, Nicita Mehta, Valdemar Danbury, Casey Johnson, Orisa Coombs, Mariana Avila, Will Reinkensmeyer, Judith Amores, Oscar Rosello, Angela Vujic, Jack Forman, Sunanda Sharma, Rachel Smith, Valdemar Danry, Felix Mosser, Luis Ruben Soenksen, Peter Q. Nguyen, Anusha Manglik, Shruthi Shekar, Misha Sra, and Glenn Fernandes for working with me on all these crazy ideas, giving me inspiration, and lifting my spirits during a most challenging time.

I also want to extend my gratitude to my fellow members of the Fluid Interfaces Group: Adam, Tomas, Guillermo, Camilo, Jaya, Mina, Aubrey, Jingwen, Sang, Jess, and Holly for our friendship, late night hangouts, and support in research. My friends at the Media Lab, Ravi, Elena, Irmandy, Patrick, Mike, and many more for all the fun and mischievous things that we did together and making my years at the Media Lab super memorable.

I would like to thank Ariel Ekblaw, Maggie Coblentz, Mehak Sarang, Xin Liu, and the team of the Space Exploration Initiative for giving me the opportunity to work in the exciting area of space exploration. Professor James J. Collins, Professor Chris Voigt, Professor Werasak Surareungchai for all the support with the project. The IDEO team for helping run the wearable biotech & growable interfaces workshop, which inspired many of the ideas in this thesis. I would also like to extend thanks Dr. Leland Hartwell for his wisdom and to Professors Neri Oxman, Hiroshi Ishii, Joseph Paradiso, Ed Boyden, Joseph Jacobson and Tod Machover for being my intellectual heroes at the Media Lab!

I also want to thank my friends in Thailand, Prompt, P’ Gear, P’ Golf, P’ Sonny, Nattanon, P’ Bank, P’ Sean, and other freakers who have contributed greatly to my intellectual journey. P’ Kai Nottapon Boonprakob, Nawapol Thamrongrattanarit, Kriangkrai Vachiratamporn for exchanging many great ideas with me and giving me a hope that there would one day be a science ﬁction ﬁlm in Thailand.

Joost Bonsen and Dan Novy for sci-ﬁ inspiration and keeping the futuristic culture at the Media Lab alive.

I am thankful for the support of the MAS team, the MIT Media Lab communication team, the Necsys team, and all the staff at the Media Lab during my ﬁrst 2 years at the Lab.

Formlab

Finally, I would like to thank NASA TRISH (The Translational Research Institute for Space Health) as well as the MIT Media Lab consortium for funding the research presented in this work. Table of Contents

Prelude 01

Chapter 1 : 15 The Rise of Augmented Human

Chapter 2 : 25 Bio-Digital Interfaces

Chapter 3 : 35 Sensing the Body

Chapter 4: 45 Wearable Lab on Body

Chapter5 : 55 Writing Information to the Body

Chapter6: 60 Wearable BioFab

Chapter7: 80 Conclusion

Prelude

15 Bangkok spaceport 2050

People say that every ending is a new beginning. But I always wonder, how can we break free from this cycle?

Maybe going to alpha Centauri light years from earth is not that different from a 20 hours ﬂight from Bangkok to Boston. But how can I know unless I get there?

But one question that never left my mind is “What is the meaning of life? and what is our place in all of this?

We always remember our ﬁrst time excitement, but once something becomes a habit we move onto the next adventure and forget our past. Sometimes, I feel that I am standing still as this always proceeds in cycles. 16 My American friends would argue that freedom is the most important essence of life. We have the free will to create our own meaning of life as we please

And technology is deﬁnitely a big part of this.

Manfred Clynes, father of cyborgs, once said that “becoming one with technology is to be free — Free to explore, create, think, and feel”

But once you taste that technological augmentation can do many things, you will run into another problem…

You will be lost in space! The space of ideas and information... The space of possibility, and the literal space of space…

You can go anywhere, but you don’t know where to go…

17 I don’t know if I will ever have satisfactory answers to any of these questions. But I remember that Einstein once said that we can't solve problems by using the same kind of thinking we used when we created them.

AS my biological brain is working hard, my second brain might be able to inspire me further…

HEY EINSTEIN, what is the most important question to think about?

“I think the most important question facing humanity is whether the universe a friendly place?”

18 “if we decide that the universe is an unfriendly place then we will use our science and technology to achieve safety and power by creating bigger walls and bigger weapons to protect us and destroy all that which is unfriendly...”

“But if we decide that the universe is a friendly place, then we will use our science and technology to create tools and models for understanding that universe. Because power and safety will come through understanding its workings and its motives...”

“I believe that God does not play dice with the universe”

“But If we decide that the universe is neither friendly or unfriendly and that God is essentially ‘playing dice with the universe’, then we are simply victims of the random toss of the dice and our lives have no real purpose or meaning...” 19 It is funny, that in the end, it all Anyway, my Alpha Centauri ﬂight is almost came down to what we choose to ready. I need to start preparing now… believe…

I activate my biofab Interface that programs my cells to produce biomolecules to get myself ready for space travel

My Wearable Lab is doing a last minute check up of my body by sampling biomarkers to see if there are any signs of health issues 20 I hope that all of these devices work well and my space ﬂight will go smoothly…

But before I board my ship, I have one last thing to do…

21 “Stay focused… focus… and focus, and don’t forget to turn in your thesis…”

“Thank you for your wisdom!”

Being focused in this post-digital era is hard, even for myself.

But I am not alone…

Even though, I am a single child, I have an AI twin modeled after me that keeps looking out for me.

I have great friends and mentors that share my passions and curiosity...

22 I am becoming augmented ready for the great unknown, and destined to be among stars…. I wish to ﬁnd the answers that I am seeking somewhere in this universe!

“Life, forever dying to be born afresh forever young and eager, will presently stand upon this Earth as upon a footstool, and stretch out its realm amidst the stars”

- H. G. Wells

23 Introduction About This Work

“We like to invent new disciplines or look at new problems, and invent bandwagons rather than jump on them” — Pattie Maes

If you could augment yourself and download a new capability to your body, would you do so?

Manfred Clynes, father of the concept of "cyborg", once said that becoming one with technology is to be free — Free to explore, create, think, and feel. I believe this is the case because future technology will act like an organ that interfaces intimately with our biological body, safeguarding and augmenting our capabilities to enable us to become the best version of ourselves. As I illustrated in the comic earlier, there are future scenarios in which this human-machine symbiosis will enable humanity to take the next step of human endeavors, such as space exploration and beyond.

In this thesis, I explore the vision of closed-loop bio-digital interfaces for human augmentation, where a wearable digital system coupled with a biological system allows for sensing and writing biological molecular information to and from the body using two prototypes: "Wearable Lab", an integrated bio-digital platform for continuous sensing of biomarkers, and "Wearable BioFab", an on-body, digitally-controlled biosynthesis platform for personalized, on-demand production of biomolecules. This thesis is organized as follows.

24 In Chapter 1, I begin with the introduction of the concept of "Cyborg" and human augmentation, exploring the foundation and history of the idea as well as early works in wearable digital interfaces.

In Chapter 2, I discuss the emergence of bio-digital interfaces, where the computation extends beyond the digital to the biological realm. My colleagues and I have developed a theoretical framework for this vision called "Living Bits", and I will discuss Figure 1 : Wearable Lab, an integrated bio-digital some examples of research that demonstrate this platform for continuous sensing of biomarkers vision.

In Chapter 3, I focus on "sensing the body", and how bio-digital interfaces allow us to read electrophysiological and biochemical information from the body through on-body devices. I focus on emerging research in biosensors, exploring the principles and related work that inspire my own research in this area.

In Chapter 4, I introduce the "Wearable Lab" Figure 2 : Wearable BioFab, an on-body, digitally prototype, an integrated bio-digital platform for -controlled biosynthesis platform for personalized, on-demand production of biomolecules. continuous monitoring of biomarkers using saliva biofluid. I present the vision behind the prototype, the technical implementation, and show example data collected from the device. I also discuss the evaluation and the limitations of our platform.

In Chapter 5, I focus on "writing information back to the body", and how bio-digital interfaces allow us to actuate electrophysiological and biochemical information and enhance the body through interventions.

25 In Chapter 6, I introduce the "Wearable BioFab" prototype, a programmable bio-digital “organ”: an on-body digitally-controlled biosynthesis platform for personalized, on-demand production of biological compounds. I present the vision behind the prototype, the technical implementation, and discuss the evaluation and the limitations of the Figure 3: Wearable Wisdom, an intelligent wearable platform for mediating wisdom and advice from mentors and personal heroes platform.

In Chapter 7, I conclude my research, discussing the implications and ethical considerations of this research in bio-digital human augmentation.

Throughout my two years in the masters program at the MIT Media Lab, I was also able to explore additional forms of wearable augmentation that appeared in the comic above. Speciﬁcally, I Figure 4: Wearable Reasoner, a symbiotic system embedded with an explainable AI system for focused on the use of artiﬁcial intelligence, natural supporting rationale decision making language processing, and generative algorithms to create “wearable wisdom”, an intelligent wearable platform for mediating wisdom and advice from mentors and personal heroes (such as Einstein) [1], as well as “Wearable Reasoner”, a symbiotic system embedded with an explainable AI system for supporting rational decision making [2], and “Machinoia”, an on-body machine that allows the user to interact with his/her digital twins created from the user’s computational model of attitudes. Even though these are very different from my bio-digital research, I see them as existing in the same futuristic ecology, supporting and enhancing Figure 5: Machinoia, machine of multiple me human life. While that work will not be covered in this document, the reader can consult the publications resulting from that research.

Last, I was fortunate to explore this futuristic work at the MIT Media Lab, where there is a long history of "visionary innovation" that exists beyond its time. I was encouraged to dream big, think about an exciting future, and realize my vision in functional prototypes. In the past, these prototypes — while imperfect —were the first versions of the the digital touch screen, the first wearables, the first recommendation systems, the first robots for education, and more. Their goal is to demonstrate a bold vision preceding the world for decades. When the time comes, "many of these pioneering works will make a tremendous impact to society, and may become recognized as the trail blazers of new fields". As Steve Jobs, the founder of Apple once said: "You can't connect the dots looking forward; you can only connect them looking backward. So you have to trust that the dots will somehow connect in your future". I hope that beyond the excitement that I felt while working on this project, one day, 30 years from now, I can look back at these prototypes and see their impact realized around me. Chapter 1 The Rise of the Augmented Human

28 Chapter 1 The Rise of the Augmented Human

“The purpose of the Cyborg, as well as his own homeostatic systems, is to provide an organizational system in which such robot-like problems are taken care of automatically and unconsciously, leaving man free to explore, to create, to think, and to feel” — Clynes and Kline

Our species is characterized by the ability to invent tools and technologies that extend our capabilities and allow us to adapt and thrive in an ever changing world. Needless to say the tools and technologies that we have invented also invent our identity and imagination, shape our interaction with the outside world, and have a tremendous impact on our existence as a species. Thus, when we imagine, design, and invent new technology, we in return are re-inventing ourselves.

Computing technology is arguably one of the most powerful tools invented and one of the most signiﬁcant milestones of humanity. When we look back in history, each era of computing paradigm is not only characterized by its increased performance, but also by the unique relationship between human and machine represented by the ratio of users to computers: “The mainframe era of one-machine-to-many-users shifted to the one-machine-to-one-user era of the personal computer, followed by the one-user-to-many-machines era of mobiles, to ﬁnally the many-machines-to-many-users era of today’s ubiquitous computing [3]”.

29 Figure 6 : The Paradigms of Human-Computer Interaction However, researchers suggest that the next step for computing can no longer be described in the same way as before. The line between human and computer is blurring, and the two entities are becoming one. This concept of human-computer integration was recently proposed in 2020 as a result of a groundbreaking workshop by the leading ﬁgures and experts in computer science, interaction design, and other related areas throughout the globe [3]. The concept is rooted in the pioneering research by Kline and Clynes on “Cyborgs and space” published in 1960. They laid the foundation of human-computer integration by developing the term “Cyborg,” referring to a “Cybernetic Organism”, in which computing technologies that include sensors and actuators serve as extended digital organs [4]. These cybernetic systems understand the physical and mental state of the user, regulate resources, and provide real time assistance/interventions, and augmenting human biological system.

30 Figure 7: The Closed-Loop Human Augmentation Cycle Kline and Clynes argued that a closed-loop symbiosis between human and machine, biological and digital, is necessary for enabling the next advancement in human endeavors including performing complex tasks and living in extreme environments, even becoming an interplanetary species, by taking care of mundane self-regulation tasks and freeing up mental faculty for more important work:

“If man in space, in addition to ﬂying his vehicle, must continuously be checking on things and making adjustments merely in order to keep himself alive, he becomes a slave to the machine. The purpose of the Cyborg, as well as his own homeostatic systems, is to provide an organizational system in which such robot-like problems are taken care of automatically and unconsciously, leaving man free to explore, to create, to think, and to feel ”.

31 Figure 8 : The early wearables developed by MIT Media Lab Around the same time in the 1960s, J. C. R. Licklider [5] and Doug researchers in 1997 Englebart [6] further explored the idea of ”Man-Machine Symbiosis” by focusing on technology for augmenting human cognitive processes as well as intellect. This is achieved by having computers provide rapid access to useful information and increasing users’ comprehension of complex situations. Since then, the vision of “cyborg” has been manifested by researchers in the form of “wearable technology”, which enables on-body monitoring of human physiological and behavioral information via integrated sensors as well as providing just-in-time information, feedback, and assistance to the user. The term wearable technology was coined and pioneered by several research groups at the MIT Media Lab, and the same researchers went on to found the very ﬁrst commercialized wearable computer - Google Glass [7]. Current wearable devices come in various form factors such watches [8], necklaces [9], glasses [10], and beyond, and have been integrated in human daily life alongside cutting edge research.

32 Figure 9 : Examples of Wearable Technologies that The ultimate research goal for wearable technology and human enhance different aspects of human cognition augmentation is to enhance — not replace — human capabilities [11]. These technologies should be aware of our desires, rights, and individuality and use those principles as the basis of augmentation. Further, the application domains of wearables span over physical domain such as: strength enhancement through exoskeleton suits [12], muscle stimulation [13], prosthetics [14], robotic symbionts [15], as well as cognitive enhancement including: enhancement of memory and recall [16-19], attention [10][20], motivation [1], reasoning [2], emotion regulation [21],, creativity [22][23], and more. Some wearable devices for applications such as augmented reasoning require the full mental awareness of the user and interface with the human conscious mind [1], while others take advantage of unconscious or semi-conscious states and interface with the user in intimate moments such as sleep [9] [23].

33 Figure 10 : The Continuum of Augmentation [23] The level of augmentation can vary between uses and scenarios. For example, some people may seek assistive enhancing technology that enables them to carry out a task similar to a healthy individual despite an impairment or disability. Extreme users such as astronauts could seek superhuman- like capabilities and augmentation to allow them to survive and perform in extreme environments beyond the ordinary such as in the microgravity of space [24]. One of the signiﬁcant outcomes of wearable technology is the tremendous data being collected and generated by the on-body sensors. This information could be used to understand and gain insights of the user’s physical and mental conditions, make predictions about future states of the user, as well as for training the model to become more personalized over time [25]. However, in the end the user should have ultimate control and agency over the data thus collected to prevent the abusive use of data, and ensure the safety and privacy of the user.

34 Further, to prevent over-reliance on the technology, in certain scenarios the user might prefer to have the integrated computer assist them in acquiring the skills rather than having the technology perform the task on their behalf.

As research on augmented humans continues to unfold, Stelarc, one of the greatest artists working on human augmentation, has remarked that “what it means to be human in the next era is perhaps not Figure 11 : Stelarc, one of the greatest artist working to remain human at all” [26]. He argues for a future on human augmentation with his “Third arm” project of augmented beings that are unbound from their original organic form, constantly experimenting, re-evaluating their bodies, and re-designing ways to enhance their functionality.

This perspective is controversial and could raise many ethical and societal considerations for the future of humanity, especially the fundamental question about human identity and integrity, as well as practical concerns regarding unequal distribution and access to augmentation technology. Who has the right and privilege to be augmented? How could we prevent the augmentation from becoming an amputation [27]? And how do we ensure that the augmentation is the user’s choice of expression, rather than an option being forced upon them by society?

35 Nevertheless, I agree with Melvin Kranzberg, who came up with Kranzberg's six laws of technology that “technology is neither good nor bad; nor is it neutral” [28]. Researchers and societies must challenge the tendency towards “technological determinism” [29], rejecting the reductionist assumption that technological innovation alone is purely good or evil, while also acknowledging the fact that “unneutral” and “biased” implications may arise from technology in the socio-technical system. Thus, it is more important than ever that researchers in human-computer integration continue to pose critical questions to themselves regarding their works. As a result of this practice, humanity would be able to safely and ethically elevate to the next level, equipped for the next endeavor by merging with technology, and becoming the “new species” that computer scientist Jun Rekimoto called ”homo cyberneticus” [30], emphasizing the signiﬁcant next step of human species as we become augmented by technology.

36 Chapter 2 Bio-Digital Interfaces

37 Chapter 2 Bio-Digital Interfaces

“I think the biggest innovations of the 21st century will be at the intersection of biology and technology. A new era is beginning” — Steve Jobs

As we continue to envision the future of augmented humans in the context of human-computer integration, we must challenge the conventional approach, medium, and means of computation to think beyond digital interfaces. In addition to wearable computers on the body, there are trillions of living biological “computers” on, inside, and around the human body. Half of them are our own human cells, but the rest are other microorganisms that co-inhabit our body [32]. This network of living cells in our body is arguably the most sophisticated form of a complex system. They are constantly communicating, sensing the environment, and producing all kinds of molecules e.g. proteins, enzymes, etc, to keep the body alive.

As we invent the next generation technology for human

This chapter is adapted from our publication augmentation, there is an opportunity for coupling bio-digital with the same title [31]. interfaces together. To achieve this, we must use on-body devices to sense and actuate with a living system to achieve new capabilities that cannot be realized purely with digital systems. Thus, the ﬁrst step towards bio-digital interfaces is to frame our understanding of the fundamental unit of biological system, the living cell as a programmable computational unit. We introduce ”Living Bits”: the integration of living cells in, with and as computing systems for the future of human augmentation.

38 Inspired by how Tangible Bits sought to bridge the gap between the digital and physical environment [33] and how Parkes & Dickie created a biological imperative for interaction design [34], Living Bits rethinks living cell as the endpoint for programmable bio-digital interfaces allowing the digital system counterpart to intimately tap into the biological information and its functionality [31].

Figure 12 : Engineering RGB color vision into Escherichia coli The term ”Living Bits” signiﬁes the emergence of digital ”bit” properties and the abstraction of computational parts in the living system. Similar to digital circuits, synthetic biological circuits offer the potential to “wield computational control over biology” [35] allowing researchers to reprogram living cells to sense, compute, and actuate at the micro-scale [36]. Researchers have also developed methods for the cell to have digital logic gates like computation, performing Boolean functions, as well as designing programming languages and software to create them [37]–[40]. Examples that highlight the convergence of digital circuit and synthetic biological circuit include the engineering of RGB color vision into E. coli using multiplexed sensor array circuits, allowing the cell to distinguish between red, green, and blue (RGB) similar to a digital sensor [41]. Further, researchers Figure 13 : Programming Escherichia coli to function as a digital display also reverse engineered a digital clock display using a biological chip containing engineered E. coli with multiple genetic repressors and activators in the circuit. The chip received molecular inputs representing the digit to be displayed, and output ﬂuorescence molecules in the form of a display [35].

39 Figure 14 : The framework of Living Bits, which considers a Through various kinds of receptors such as immunoreceptors, living cell as a biological computer with various input photoreceptors, mechanoreceptors, and more, distributed on the and output modalities. cell membrane, a living cell could receive different inputs such as chemical cues or physical signals from its surroundings to metabolize and respond to the environment. Inputs to the living cell can be molecular, including chemical, ionic, nucleic, and antigen molecules [40]. Inputs can also be physical conditions of the environment, such as light, heat, and magnetic ﬁelds [40]–[42].

Once living cells receive inputs to metabolize, they react and respond by producing molecules or other forms of output modalities. In the case of living cells in the human body, we can observe this process and use this information as a biological indicator or biomarker, which reveals insights into human health at the molecular level. This information is critical for personalized medicine that is either prognostic or predictive [12] [43].

40 Figure 15 : Example projects that demonstrated the vision of Living Bits : Researchers have also engineered living cells to produce a wide

1. Mushtari range of output modalities such as visual, olfactory, mechanical, 2. bioLogic 3. Mold Rush and chemical [40]. Examples include producing pigments or 4. Euglena Soccer Game 5. RGB E.Coli ﬂuorescent molecules for visual cues, releasing scents as 6. Breathing Shoes 7. Biota Beat 8. Antibiotic-Responsive Bioart olfactory cues, movements as mechanical cues, and releasing 9. OpenLH 10. My First Biolab enzymes that break down toxins in the environment as a chemical 11. Vespers 12. Carbon Eaters output. A classical example is the engineering of E. coli to support 13. Social Microbial Prosthesis 14. Grown Microbial 3D Fiber Art individuals with type 1 diabetes to produce insulin [44]. 15. Mycelium Artifacts 16.Myco-accessories 17. Growable Robot 18. Biosensing Soft Robot 19. Microbial In the past decade, researchers have used the ability of living cells 20. E. chromi 21. Microbial Perfume to sense inputs and compute outputs as part of interactive 22. Bio-electronic soil sensing device 23. Gut-Brain Computer Interfaces systems ranging from wearable devices [45]–[52] to musical 24. 3D Printed Living Responsive Materials and Devices instruments [53], built environments [54], food [55], games [56] The full descriptions can be found in our research article published in ACM [57], and robots [58] [59], demonstrating a rich spectrum of Augmented Humans 2020 [31] research that demonstrates the vision of Living Bits.

41 By embedding engineered biological systems inside everyday objects, researchers can tap into the biological functions found in living cells to augment the capabilities of materials, objects, and devices. This enhancement ranging from bio-sensing, photosynthesis, and shape changing abilities could lead to a symbiotic relationship between human and biological machines. Nature: Collaborations in Design features diverse examples of projects that aspire to establish this symbiotic relationship [60]. Here, we present a few examples of projects in the theme of human augmentation, which demonstrate the integration of living cells in, with and as computing systems.

Figure 16 : E.Chromi project In the E.Chromi project, researchers demonstrated a proof-of-concept of a biosensing yogurt which changes an individual’s feces color in response to speciﬁc internal disease biomarkers. They accomplished this by engineering two systems: a “Sensitivity Tuner”, a genetic circuit that allows the system to reports meaningful concentrations of the inducer in a sigmoidal “on” or “off” response pattern, and a “colour output”, pigment production via a metabolic pathway inside the bacteria [55]. The project won the Grand Prize at the 2009 International Genetically Engineered Machine Competition (iGEM).

42 Further, researchers demonstrated the use of 3D printed stretchable, robust, and biocompatible hydrogel [61][62] to host engineered bacteria cells that are programmed to sense the signaling chemicals including N-acyl homoserine lactone (AHL), isopropyl -d -1-thiogalactopyranoside

Figure 17 : 3D Printing of Living Responsive Materials (IPTG), rhamnose (Rham), and anhydrotetracycline and Devices (aTc) and respond with ﬂuorescence and chemical Liu, Xinyue, et al. Advanced Materials (2018) secretion. The cells are also capable of performing boolean logic gate computation for creating a multi-chemical sensor that can be attached on the body [63].

Beyond living sensors, Mushtari is a wearable embedded with synthetic microorganisms that can

Figure 18 : Living Mushtari augment human biological functionality for space

Bader, Christoph, et al. 3D Printing and Additive travel. Researchers created a 3D printed wearable Manufacturing (2016) ﬂuidic channels containing two genetically engineered microorganisms: a photosynthetic microbe such as microalgae or cyanobacteria that converts sunlight to sugar, and compatible microbes such as yeast and E. coli that convert sugar into useful materials for the wearer such as drugs, food, fuel and more [45]. Alongside the idea Figure 19 : BioLogic of designing for extreme environments, “Carbon Yao, Lining, et al. ACM Conference on Human Factors in Computing Systems. ACM (2015) Eaters” is a small round button containing algae “Oscillaloria” that absorb and respond to carbon dioxide in the air, changing colour to indicate air quality and the presence of high levels of substances [47]. This allows the wearer to have higher awareness of the surrounding environment.

43 Furthermore, in the project, "Breathing Shoes," researchers collaborated with the athletic ware company Puma to pilot the ﬁrst instance of personalized biofabrication. First, they grew shoes from bacteria which respond to heat. When the heat increases, the bacteria would open air passageways to lower the temperature inside the shoe. Over time, each shoe molds to the proﬁle of their user’s foot [47].

Along with athletic ware, BioLogic is a responsive bio-skin using living actuators created by an automatic deposition method for printing bacteria cells on soft materials [64]. With environmental humidity changes, the cell grows and shrinks, which inﬂuences the material to change its shape. The researchers have demonstrated various use cases of the technology from applying the microbial actuator on fabric to creating a synthetic bio-skin that reacts to body heat and sweat. The design of the bio-skin includes ﬂap structures around heat zones that open and close for cooling down the body [46]. Researchers also have shown the ability to actuate robots using molecular motors assembled into multi-scale ensembles [65].

The ultimate goal for “Living Bits” is to foster the integration of living cells in, with and as computing systems [31]. However, most of the projects presented merely use living cells as biological computers, with minimal efforts in coupling them with digital computation. Thus, there is a missed opportunity for going beyond the current limitations of bio-engineering and digital computation [66]. In the following chapters, we will explore the intersection of bio-digital interfaces further in the context of human-computer integration, and highlight the use of bio-digital interfaces for sensing and actuation in the context of closed-loop human augmentation.

44 Chapter 3 Sensing the Body

45 Chapter 3 Sensing the Body

Figure X : The closed-loop human augmentation cycle In this chapter, we will focus on sensing the body, the ﬁrst half of the closed-loop human augmentation cycle. We can characterize the medium of signals that the body uses for communicating between body parts into two major categories: electrophysiological signals and biochemical signals. The augmentation technology must be aware of the user’s state, both physical and mental, in order to identify when and what just-in-time information or other mode of enhancement is needed.

46 Electrophysiological signaling is used for rapid and real-time communication between neurons and other types of cells such as muscle and cardiovascular cells. It measures the electric current, or the change in voltage between cells caused by action potential activity [1]. Researchers have recently developed a novel electro-encephalography technology, which involves placing electrodes into various parts of the body for electrophysiological recordings. In Figure X : OpenBCI, an open-source brain-computer general, an electrical signal is recorded by the interface platform that allows researcher to measure EEG signals from participants.. electrode, which transmits that signal to an ampliﬁer and a computer for signal cleanup and processing.

This type of signal is currently being used in wearable devices for enabling real-time sensing and computing of human physiological information via digital sensors such as EEG (electro- encephalography - measuring cerebral cortex activity) used for understanding user’s cognitive phenomena such as motor control, emotion, FigureX : Empatica, a wearable device for measuring attention, vision, etc; EOG (electro-oculography - EDA, heart rate, and other physiological signals. measuring eye movement activity) used for gazing user interests and visual attention, EMG (electromyography - measuring the electrical activity produced by skeletal muscles), ECG measuring of heart cardiac muscle activity), skin conductivity for measuring user’s emotional valence and arousal. Further, this digital sensor can also collect behavioral and contextual information such as movement, location, interaction, and more [2], [3].

47 With the advancement of radio and wireless sensing [4], researchers have also demonstrated the ability to monitor human electrophysiological changes without placing any sensor on the human body. This kind of research is enabled by the combination of high-resolution wireless sensing technology that transmits a low-power wireless signal and monitors its reflections off the human body. The distincted physiological changes alter the reflections of the signal, which is analyzed by Figure X : Researchers have demonstrated the applications of wireless sensing to detect body machine learning algorithms for noise removal and movement [5], breathing cycle [6], heart rate [7], emotion [8] and sleep stages [9] without any on-body pattern recognition. Researchers have monitoring devices. demonstrated applications of wireless sensing to detect body movement [5], breathing cycles [6], heart rate [7], emotions [8] and sleep stages [9] without any on-body monitoring devices.

Beyond the electrical sensing of human physiology, biochemical signals are a means of cellular response and communication. These biochemical markers are much harder to sense and less common in current wearable devices. They are used for indicating certain biological phenomena related to behavior, disease, infection, or environmental exposure [10]. Biochemical markers include organic compounds and non-organic compounds that are released or secreted as part of the human metabolism such as metabolites, bacteria and hormones [11]. In medicine, these biochemical markers reveal insights into human health at the molecular level, which could be used for personalized medicine [12].

48 Figure X : The technological waves of biochemical Biochemical sensing platforms have undergone waves of monitoring. The figure is modified from [13] technological breakthrough changing the ways they are deployed in the healthcare system. The first wave of biosensor technology was developed in the 20th century focusing on laboratory instruments for measuring many analyte in fluids (saliva, sweat, urine, or blood) with collection and transfer of the sample to a separate analytical lab [13]. The individual spits, takes a swab, or has a device collecting their body fluid, and places it on the sensor. platforms. Such technology is commonly used in today’s hospitals and healthcare centers.

Advancements in the engineering of integrative digital circuits, microfabrication methods, and sensing technologies enabled the second wave of biosensor technology. This second wave consists of point-of-care diagnostics developed in early 21st century through the miniaturization of the testing process through integrated chip, which puts the laboratory into the hands of medical staff and patients [13].

49 Figure X : The basic components of a biosensor. The The use of colorimetric assays, where the sensor changes color figure is modified from [11] due to a chemical or enzymatic reaction between analyte in biofluid and coated reagents is one of the most widely adopted methods for this second generation platform, and is key for reporting the presence of the markers. This is due to the simplicity and adaptability of such platform for sensing various biomarkers. Researchers have used capillary flow for detection of biomarkers in saliva [14] as well as developing the concept of microfluidics on paper such as Ampli [15], a plug and play set of paper-based blocks that could be arranged in various configurations for testing biochemical reactions in real-time for personalized diagnostics.

We are now entering the emerging third wave of wearable biosensors, where every day people can take the laboratory with them in the form of peripheral devices [13]. Low-cost biosensor platforms have enabled the detection of biomarkers in body fluids by having two basic functional units. First, the receptor (for example, a chemical indicator, enzyme, antibody or DNA) is responsible for selective recognition of the target analyte, which can be coated on materials such as a paper strip or a microfluidic device [16] [17]. 50 Figure X : The evolution of biosensors from invasive to The second part is a transducer (for example, electrochemical, non-invasive. The figure is modified from [11] optical or mechanical) that translates this recognition event into a

useful digital signal for further computation and analysis. One notable example is the use of a glucose monitoring biosensor for diabetes patients, which all three of these technological waves have been applied for. Currently, state of the art glucose sensors are semi-invasive, using a disposable probe coated with glucose oxidase enzyme to sense glucose in the blood sample. This method is becoming the norm for diabetic patients, and it has been deployed and validated through a number of clinical trials, and exceeds over 1 billion in market value [13][18].

Beyond glucose monitoring, researchers have begun to develop new innovative form factors and deployment mechanisms for wearable sensors. These platforms can monitor molecular biomarkers [19] with non-invasive sensors on the human body [11][23].

51 For example, researchers have designed a wearable microfluidic device for capture, storage, and colorimetric sensing of glucose, chloride lactate, and pH from sweat [20]. In addition, researchers have also developed a nanoporous membrane-based wearable sensor that detects cortisol in sweat [21]. Researchers have also developed Dermal Abyss, a smart tattoo created FigureX : wearable microfluidic device for capture, storage, and colorimetric sensing of glucose, chloride with colorimetric and fluorescent biosensors that lactate, and pH from sweat [20] changes color according to the wearer’s interstitial fluid composition [22].

However, these wearable sensor platforms are limited to one-time use as most of the detection reactions between the analytes and receptors are non-reversible. In addition, they also do not provide ﬂexibility in choosing which biomarker they sense [11]. Thus, there is an opportunity for FigureX : A nanoporous membrane-based wearable sensor that detects cortisol in sweat [21]. developing peripheral biomarker monitoring using wearable devices to repeatedly sense analytes in non-invasive bioﬂuids.

We are particularly interested in saliva, as it is an emerging biological ﬂuid for sensing of biomarkers and diagnostics [24]. Saliva is a clear, slightly acidic, complex solution, is produced at about 0.75 − 1.5 L daily in the human body, and is composed of secretions from major pairs of salivary glands Figure 1 : Dermal Abyss, a smart tattoo created with colorimetric and ﬂuorescent biosensors (parotid, submandibular, and sublingual) as well as hundreds of minor salivary glands. Similar to blood, saliva contains various biomarkers including hormones, enzymes, antibodies, antimicrobial compounds, and growth factors [25] [26].

52 Many of these biochemicals enter saliva from the blood by passing through the spaces between cells. Therefore, the major compounds found in blood are also detectable in saliva. Thus, saliva is functionally homologous to serum in reﬂecting the physiological state of the body, including emotional, hormonal, nutritional, and metabolic Figure X : The position of human salivary gland variations [24].

Saliva-based sensors have been widely developed to sense multiple biomarkers such as cortisol [27], glucose [28], nitric oxide [29], pH [30], and more [31]. These biomarkers have been used to detect signs of depression [32], diabetes [33] [34], heart failure [35], and periodontal diseases [36]. This platform mostly exists in a portable swab or spit test platform and a mouth guard form factor [13]. However, most of these sensors are generally limited to one-time use [11], are non-continuous and do not connect with digital platforms for data

Figure X : Sensors for monitoring biomarkers in saliva recording, analysis and building interventions. As including swab or spit test platform and a mouth guard device [13] of 2020, no continuous and wearable saliva-testing products exist [13].

Despite rapid progress in wearable biosensor technology over the past 5 years, we are only at the beginning of realizing the impact of wearable biosensor technologies on improving human health and performance” [11]. Looking at the current literature, most of the biochemical sensing platforms lack the ability to monitor context and activity of the individual, whereas the digital-

53 FigureX : closed-loop bio-digital sensing and actuation. sensing platforms can only sense physiological signals but are not able to sense the molecular dynamics of the human body. We believe that the future bio-digital wearable device must bridges the gap between biochemical sensing and digital sensing. Using digital sensors such as IMU and GPS, the system can recognize the context of the individual such as their location and activity, while physiological sensing such as EEG could help the system to recognize the cognitive performance of the individual in terms of attention and emotion. The biological sensors could help quantify the molecular response of the body, for example by measuring changes in cortisol for stress. The information from both the biochemical and digital sensors can contextualize one another, and provide insights regarding an individual's behavior, which in turn can be used to develop healthier lifestyles, and provide real-time feedback to the individual when unhealthy behavior is recognized. References

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60 Chapter 4 Wearable Lab on Body

61 Chapter 3 Wearable Lab on Body

Pataranutaporn, Pat, et al. "Wearable lab on body: combining sensing of biochemical and digital markers in a wearable device." 2019 41st Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC). IEEE, 2019.

Figure 1 :Overview of the Wearable Lab system including Introduction an on-body device for sensing biomarkers and a mobile application for reading the data Based on a review of the current literature in wearable biosensors and platforms for biomarker monitoring, we believe the future of wearable biosensors is to go beyond specific sensing capability and become a wearable ”lab” on body. The notion of a “laboratory” on the body was inspired by the emerging research on integrated lab on chip [1]–[3], where a small device can have a fully integrated and re-configurable system. This technology mimics several processes usually performed in the laboratory for clinical diagnostics and analysis of human physiological fluids, leading to flexibility in terms of reconfigurability and scalability.

62 To achieve this, the lab on body must have the following Figure X : Analogy of the Wearable Lab to a full scale lab capabilities :

1) The ability to perform continuous measurement of biomarkers: The platform must overcome the challenge of having to change biosensors regularly due to the limited one-time-use capability because of the non-reversible analyte-receptor binding reaction on the sensor [4]. The user should not have to change the sensor every time that they want to use the device, and should have to replace the sensors as little as possible.

2) The platform must have a universal ﬂexibility in scaling up and integrating with multiplexed biosensors and biomarkers. The user should only have to wear a single device that can integrate with multiple biosensors rather than wearing multiple devices that can only sense one or two signals. Based on the user’s needs, the platform should also be able to integrate with new biomarkers as they are being discovered everyday by new research. This is similar to the smartphone paradigm, where a single mobile device can have an inﬁnite increase in capability by having unlimited applications installed on it. 63 3) The platform must be able to contain multiple processes (beyond sensing) similar to steps performed in the laboratory such as sample collection, data storage & transmission, data correction & conversion, and beyond. The device should be able to automate the above processes in an integrated manner, and be able to connect multiple streams including biochemical markers with digital physiological data, as well as behavioral data so as to create actionable insights.

To illustrate our futuristic vision of having a lab on body, we prototyped the “Wearable Lab” a bio-digital platform for sensing of saliva. Our novel platform demonstrates the following capabilities: it 1) performs real-time continuous monitoring of biochemical markers on the body, 2) integrates plug and play paper-based ﬂexible biosensors with digital sensors, and 3) converts colorimetric information from the biochemical sensing reaction to the digital readout.

Technical Implementation

Our platform is designed to continuously sample saliva from the individual and analyze it using a roll of multiplexed biochemical sensors. The platform consists of three main components: a paper-based biochemical sensor cartridge, a saliva acquisition and sensing module, and a smartphone application to store and analyze the data. A small portion of the sensor is exposed to the acquired saliva at a point in time. Once the saliva reacts with a biochemical sensor, the RGB color sensor on the device reads the output and converts the value into the concentration of the biomarker based on the color-concentration model. The deployed sensor is programmed to then advance the roll to expose the next sensor on the tape.

64 Figure 1 : Nunc lacus odio, sagittis quis ligula sit amet, Paper-based Biochemical Sensor Cartridge tincidunt convallis purus. Nulla ut convallis velit. The paper sensor cartridge consists of a roll of paper-based, biochemical sensors that can be installed in the module. In the prototype, we have experimented with a commercially available dipstick that contains 13 biomarker biochemical sensors (glucose, leucocytes, nitrite, urobilinogen, total protein, cacells, ascorbate, ketone, billiburin, albumin, creatinine, pH, and calcium) as well as a single sensor strip for sensing the same chemical at multiple times. The paper sensor type is dependent upon the type of biomarker the individual wants to monitor. For monitoring multiple biomarkers, multiple biochemical paper sensors could be interleaved on one cartridge. The paper sensor strips have a thin ﬁlm of impermeable transparent plastic on the backside to prevent cross-contamination from already sensed or acquired saliva. The width of the paper sensor that the current version of the device supports is 5.5 mm. One can vary the number of samples collected for a speciﬁc biomarker by varying the number of paper-based sensors for that biomarker on the tape.

65 Figure X : The mechanical arrangement of Wearable Lab Saliva Acquisition and Sensing Module platform The saliva acquisition and sensing module consists of a mechanical arrangement for acquiring a small quantity of saliva from the buccal mucosa (inside of the cheek). It consists of two stepper motors, a small PTFE tube, a piece of braided nylon cord, a paper sensor cartridge and the electronics for digital sensing. A PTFE tube of diameter 2mm is held against the inside part of the cheek while the rest of the device sits outside of the cheek. A stepper motor is used to circulate the nylon cord against the cheek and pull it into through the device, collecting a small amount of saliva. The gathered saliva is then transferred onto the paper sensor by contact. Another stepper motor is then used to advance the paper sensor cartridge. A new portion of the paper sensor is rolled out and swabs the saliva from the cord. We also experimented with another mechanism for exposing the sensor

Figure Y1 : Nunc lacus odio, directly to the saliva inside the mouth using a mechanical sagittis quis ligula sit amet, tincidunt convallis purus. Nulla arrangement similar to a conveyor belt as shown ﬁgure Y. ut convallis velit.

66 Figure 1 : The position of Wearable Lab on a user This motion is repeated several times to get the appropriate

amount of saliva onto the paper sensor. The amount of saliva collected on the platform can be programmed and adjusted using the mobile application. The synchronous motion of the paper sensor and the collection mechanism allows the saliva to be transferred from the cord to the sensor without contaminating an unused sensor. This method of collecting the saliva is a work in progress with an ultimate goal for creating a robust and repeatable process for sampling of saliva.

A color RGB sensor is mounted orthogonally to the paper sensor to track the color changes. We used the TCS34725 RGB color sensor with white LED light for illumination. The system is controlled by a BLE enabled microcontroller (BC832) mounted on a PCB inside the casing. The system also has a 9-axis Inertial Measurement Unit (MPU9250) to track orientation as well as actions of the individual. The system is powered by a single cell LiPo battery of capacity 100mAH.

67 We also redesigned the integrated circuit of the TCS34725 RGB color sensor to ﬁt better with the design of our current Wearable Lab form factor.

Smartphone Application The smartphone application accompanying the Wearable Lab device captures and processes the sensed saliva and the IMU data. The app was developed for Android using Android SDK and iOS using Swift library through Xcode. The app communicates and controls the device by connecting with the BC832 microcontroller via bluetooth. It can receive a stream of information from the accelerometer and gyroscope embedded

Figure 1 : The custom redesign of the TCS34725 RGB in 9-axis IMU, which can be used to infer the color sensor individual’s activities including eating, drinking, speaking or no activity using machine learning algorithm.

Using the interface on the phone, the user can actuate the motors to initiate the sampling process, and rotate the strip to expose a new sensor. Finally, the biochemical markers of saliva is acquired using RGB sensor on the Wearable Lab, and later converted into RGB values. These values can be used for correlating the concentration for each of the biomarkers through the RGB-biomarker correlation model available from Figure 1 : The user interface of the Wearable Lab mobile application the manufacturing spec sheet of the sensor on the strip.

68 Figure 1 : RGB-Concentration Correlation Models for the Results Biomarkers We have collected sample data with our prototype to evaluate the performance of the platform as a proof-of-concept. Due to the limited access to the lab and equipment during the COVID-19 pandemic situation, this evaluation is preliminarily and for limited number of subjects, and will require future research to improve and optimize the platform.

First, we demonstrated the use of the optical sensor to quantify the biomarker concentration by developing the colorimetric models of 5 biomarkers: pH, Nitric Oxide, Glucose, Ketone, and Calcium as they vary across the day, and can be used as biomarkers for depression [35], heart failure [36], an diabetes [37]. The model correlating the sensor RGB input with the expected concentration of the biomarker was obtained from the manufacturing spec sheet for each of the sensors using linear correlation graph fitting model. To evaluate the platform on the real saliva sample, we ran a preliminary study, where we used the sensor to sense the variation of biomarkers throughout the day and during different tasks, and compared the concentration using 69 the correlation model. The result is displayed in figure X. Figure X : The variation of biomarkers throughout the day Next, we demonstrate the ability for the Wearable Lab to collect and during different tasks reported by the biochemical accelerometer and gyroscope information using the integrated sensors. IMU. We have the user wear the device, and perform different tasks such as chewing, drinking, speaking, and standing still (as the baseline). Sample signals of these activities are shown in figure y. We can observe distinct patterns of IMU data across different activities, which can be used for training a machine learning model to automatically recognize human oral and facial activities. Together with the biochemical information, we can utilize the information to detect whether the increase in certain biomarkers such as glucose comes from the body background signal or food intake, highlighting the advantages of having an on-face device. Additional activities such as walking, sleeping, and running can be inferred from the Smartphone’s IMU sensor [5] and are also used to augment the data coming from the Wearable Lab by giving additional contextual information. Based on the context, the application models individual behavior and decides to sample the saliva after a certain time point to reduce contamination from an individual’s eating or drinking. Figure 1 : Sample IMU signals collected from the Wearable Lab platform during no activity, speaking, drinking, and chewing

71 Figure 1 : The miniaturized We also evaluated the form factor of the prototype by having a Wearable Lab prototype volunteer wear the device for a period of time. The feedback that we received motivated us to explore the possibility of miniaturizing the device further by reconﬁguring the sensors cartridge and the motors from outside of the cheek to the inside of the user’s mouth. Thus, we reduce the distance for the saliva to travel inside the saliva acquisition module, making the device smaller and more comfortable to wear. We also explored different areas inside the mouth for the device to efﬁciently collect saliva and also different exterior cases for the device to become more socially acceptable. However, the form factor is still a work in progress that requires follow up investigation which we hope to get to after the COVID-19 pandemic. Applications

The ability to continuously monitor both biochemical markers from saliva as well as contextual behavioral data in a non-invasive and minimally obtrusive wearable form factor permits applications that were not previously possible. This section describes novel interventions enabled by the platform.

Diagnosis Previous research has shown the variability of biomarkers used for diagnosing several diseases. Variability in salivary biomarkers such as pH has been shown as an indicator of periodontal disease [6]. Differential cortisol levels have been found between morning and afternoon [7], and new biomarkers are emerging for early detection of Alzheimer’s [8]. Furthermore, salivary diagnostics have been found to provide a diagnosis of cancer, HIV, cardiovascular and possible neurological diseases such as Parkinson’s and Huntington’s disease [9] [10] [11]. The ability to continuously monitor saliva would enable studying dynamic range, frequency and concentrations of biomarkers which have not been studied in longitudinal assays in humans. The method may allow to detect intricacies with the associated biomarker variations. Furthermore, the system being personal and user-specific may enable exploration of the fundamental differences of biomarker statistics between individuals. The trends found in biomarkers could then serve for identification of disease buildup. Given the diagnosis of a disease, similar configuration of the system could potentially also be used for scheduling optimal time and quantities of drug delivery.

73 Wellbeing Researchers have developed various context-aware, closed-loop feedback systems which provide real-time information based on an individual’s physiological signals such as heart rate, respiration, electrodermal activity, and temperature [12][13]. Several biomarkers in saliva allow for comprehensive sensing of an individual’s affective state [14][15]. Our platform enables comprehensive sensing for developing a closed-loop system for mitigating pain, stress, anxiety or enhancing sleep. The biological data coupled with the digital data would allow for better modeling of the individual. Coupling other sensory modalities such as voice of the user with the biomarkers would allow for a better understanding of an individual’s lifestyle and could aid in developing interventions to improve human wellbeing. This research highlights the concept of Living Bits, a vision for the future of interfaces that bridges the gap between human and computer through bio-digital system [16].

Space Exploration The lack of microgravity in space affects the physical and mental impact on astronauts [17] [18]. Different biological systems adapt to spaceﬂight conditions at different rates. In the case of physical problems, without extensive exercise, medical care and a restricted diet, there is a high risk of an astronaut losing muscle strength, endurance, and cardiovascular experience by losing protein in the muscles and calcium in the bones; developing kidney stones due to dehydration and increased calcium excretion, and having sensory disruption caused by changes in body ﬂuids that could put pressure on vision and the eyes [17], [18].

74 In the case of mental health issues, the distance from earth, isolation from family and social support can lead to depression during space ﬂight, which can cause hormone levels to ﬂuctuate and alter the immune system, and the abnormality of the immune system occurs in long-term mission as astronauts are challenged by stress [17], [18]. Astronauts may also experience circadian rhythm disruption due to the noisy environment, heavy workload, and constantly changing schedules, resulting in fatigue and sleep loss [17], [18].

Wearables have the potential to play a critical role in monitoring, supporting, and sustaining human life in space, lessening the need for human medical expert intervention. Early this year, the Canadian space agency started a pilot project with a smart shirt that could monitor astronauts’ vital signals [19]. Further, with the advent of new companies like SpaceX and Blue Origin, which are democratizing access to space, in the near future people transitioning into space may not just be trained astronauts but common people. We think that wearables could play a critical role in this process. The Wearable Lab could be used to keep track of the health of a person during their transition into space, intervene to amend any deﬁciency, and enhance the experience to make it more natural for the body.

75 Limitations and Future Work

Our Wearable Lab is a proof-of-concept platform for continuous sensing of biochemical markers from saliva. It demonstrates the idea of a fully integrated and re-configurable system that mimics several processes usually performed in a laboratory for clinical diagnostics and analysis of human physiological fluids. A lot of work remains to be done in order to translate this work to have real world impact. Here, we discuss the limitations and future work. 1) Form Factor: The current form factor of the device requires the user to have some part of the device hanging outside of his/her mouth. This can be socially awkward in some cultures and scenarios. Thus, in the future we anticipate a miniaturized version of the wearable to be fully enclosed in the mouth and be more comfortable to wear throughout the day. This would require new designs of the sampling and sensing mechanisms that are small and robust. 2) Going beyond Colorimetric Readouts: The colorimetric sensing is one of the most cost effective and simple processes used for biosensors. However, for substrates that cannot be sensed using this approach, we would like to explore how the Wearable Lab can integrate other methods of biochemical sensing such as electrochemistry and more. 3) Machine Learning: We also hope to incorporate machine learning into the smartphone application for classification of individual activities, which would allow us to analyze different streams of data and be able to create closed-loop feedback mechanism for the future. 4) Broader Applications: after discussing our prototypes with peers and fellow researchers that work in the area of biosensors, we realized that the mechanism developed for the Wearable Lab, which has the ability to switch between different biochemical sensors on a roll,

76 and also the capability to renew the sensor roll offers the beneﬁt of continuous sampling and monitoring. This same concept could be applied for sensing biomarkers from other sources such as sweat, urine, and feces. We show an early prototype of such system on Figure X : The adaptation of Wearable Lab continuous monitoring the left.

Conclusion

We presented a novel approach and platform for a combined bio-digital wearable device. The non-invasive, minimally obtrusive platform allows the continuous monitoring of biomarkers from an individual’s saliva. The platform also supports sensing the individual’s biological data to infer individual body responses and digital data (IMU) to infer the individual’s context, thereby bridging the gap between biological and digital sensors in a wearable technology. This platform could be used for various applications such as early diagnostics and creating closed-loop feedback systems. We hope that our bio-digital wearable will inspire future research in wearable biological computing for improving human health and wellbeing. References

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80 Chapter 5 Writing Information to the Body

81 Chapter 5 Writing Information to the Body

“The future of architecture will be soft and hairy” — Salvador Dali

Figure X : The Closed-Loop Human Augmentation Cycle The ability to write information back to the body is the second half

of the closed-loop human augmentation cycle. Augmentation technology must be able to provide just-in-time information and enhancement by stimulating and inﬂuencing positive changes in the human physical and mental state. Similar to the electrophysiological and biochemical signals that the sensors detect from the body, we must create actuators that are capable of producing both electrophysiological and biochemical signals that may be beneﬁcial to the user.

82 Electrophysiological stimulation is an emerging area of research for human enhancement as well as medical therapeutics whereby one purposefully modulates the nervous system’s activity [1][2]. Researchers have developed techniques for electrical stimulation using non-invasive approaches such as transcranial electrical stimulation (tES), transcranial magnetic FigureX : Transcranial electrical stimulation (tES) stimulation (TMS), focused ultrasound (FUS) as well as invasive placement of electrodes deep inside the human body such as deep brain stimulation [2][3].

Stimulation can also be applied to other parts of the body such as muscle stimulation [4], vestibular stimulation [5], taste bud stimulation [6] and more. Even though this is a new area of research, scientists have already shown several positive Figure X : Wearables Based on Electrical Muscle Stimulation [4] applications of body stimulation such as for enhanced language [7], spatial [8] and motor [9] learning through non-invasive brain stimulation, enhancing reﬂect and reaction time through muscle stimulation [10], improving emotional and behavioral responses using haptic stimulation [11], as well as adding proprioceptive feedback in virtual reality experiences through galvanic vestibular Figure X : The use of galvanic vestibular stimulation for enhancing virtual reality experiences [12] stimulation [12]. There exist many efforts in miniaturising the stimulation device in wearable form factor, so the user can have seamless and just-in-time access to the stimulated enhancement [1][4][12].

83 Figure X : Schematic illustration of the biomolecular delivery Beyond writing electrophysiological signals to the human body technology [17] using on-body technology, there is an opportunity for research on writing biochemical signals to the human body. In the past, this has been largely explored for medical applications in the context of drug delivery systems, where researchers developed methods for transdermal drug delivery of macromolecules and vaccines, such as insulin, parathyroid hormone and inﬂuenza vaccine, and more [13]. This delivery approach is wearable, convenient, and painless. They can sustain a longitudinal release proﬁle, which may reduce side effects of a single high dosage. It is estimated that more than one billion transdermal drug delivery platforms are currently manufactured each year [13]. Researchers overcome the physiological barriers of the skin using various mediators and mechanisms such as skin patches [14] and micro-needles [15] [16].

84 Figure X : The four generations of biomolecular delivery Biomolecular delivery has undergone four waves of technological technology [17] milestones, starting with the ﬁrst generation transdermal drug delivery technology. This uses the natural diffusion property of the molecule to passively deliver to the body, but has limitations regarding speed and types of molecules that the platform can deliver. The second generation transdermal delivery technology utilizes stimulation such as from chemical enhancers, heat, electricity, or non-cavitational ultrasound to facilitate the delivery process [17]. Based on advancements in microfabrication technology, the third generation of delivery technology augments the transmission process using micro-needles, which serve as an unobstructed pathway for incoming drugs. Finally, the fourth generation couples the transdermal drug delivery with a wearable sensor for personalized and customized delivery [17].

85 Beyond patch-based transdermal delivery, new innovations in delivering biochemical interventions to the body also include the use of nucleic acids as programmable carriers for biologically active compounds [18], ingestible delivery platform[19], and even inhalable RNA [49] as means of delivering therapeutic agents, expanding the ability Figure X : An ingestible self-orienting system for oral delivery of macromolecules [18] to seamlessly and non-invasively deliver relevant and beneﬁcial biologically active compounds to the body.

The application of writing biochemical signals to the body also extends beyond medical and therapeutic use cases to cognitive and physical augmentation of human capabilities. For example, in our daily life we drink coffee, which includes caffeine molecules that bind to adenosine receptors and act as central nervous system (CNS) stimulant [20]. Similar to caffeine, researchers have also discovered other biologically active compounds that can be used for enhancing various metabolisms that involving human performance if Figure X : Inhaled Nanoformulated mRNA Polyplexes used in appropriate concentration. For example, for Protein Production in Lung Epithelium [] erythropoietin (EPO) is used for stimulating red blood cell production for physical performance enhancement during sports [21], as well as improving cognitive measures of executive functioning and memory [22][23]. Researchers also showed in a study that taking microdoses of psychedelic substances through targeting serotonergic 5-HT2A receptors promotes cognitive ﬂexibility, crucial to creative thinking, and improves convergent and divergent thinking necessary for creativity and problem solving [24]. Similar to other types of stimulation, these compounds need to be used in the right quantities to prevent abuse.

Recent advancements in wearable technology foster the coupling between the delivery of biologically active compounds with the bio-sensing module in a closed-loop system [25][26], where the metabolic and health information from a person inﬂuences and alters the amount of biologically active compounds and drugs being released to achieve personalized intervention and

Figure X : Commercial Glucose Sensor with Wearable overcoming the drawbacks of ﬁxed dosages, such Patch as from oral delivery and injection [27].

One of the notable applications for this area of research is closed-loop insulin delivery systems [14] [28], where researchers develop an integrative device combining an insulin pump and delivery system with an on-body sensor for continuous glucose monitoring, which is controlled by an algorithm that regulates insulin infusion rates into the body. Closed-loop insulin delivery systems have undergone several longitudinal clinical trials, and have shown to be feasible and safe without increasing the risk of hypoglycemia [29], and improving the outcomes of diabetic patients [30] [31]. This demonstrates the revolutionary vision of the concept of ”Cyborg” has real world impact, in the form of a cybernetic organ enhancing a self-regulatory control function in humans [32].

87 Closed-loop insulin delivery technology also led to one of the first open-source patient-design research movements, the ”Open Source Artificial Pancreas Systems (OpenAPS)” group run by more than 100 people worldwide who self-build hybrid closed loop systems with an open source software for monitoring glucose and releasing insulin. The open-source system is shown to be safer than a traditional system in a clinical studied presented at American Diabetes Association Scientific Sessions [33].

Closed-loop systems such as the glucose-insulin monitoring and delivery platform represent the first step toward personalized bio-digital interfaces. However, such systems only focus on a Figure X : ”Open Source Artificial Pancreas Systems (OpenAPS)” single narrow application by monitoring one biomarker (glucose) and delivering one intervention (insulin). Similar to how computing technology started of having one specific application to later become a general programmable platform for a wide range of applications, we imagine that in the future closed-loop systems would be able to holistically sense multiple biomarkers and physiological data, and deliver multiple kinds of interventions to keep the person healthy and augment their capabilities in time of need. This includes the ability to produce and deliver all kinds of beneficial biologically active compounds. In order to do so, we must integrate knowledge regarding wearable computing with biosynthesis and cell-digital interfaces.

88 Table 1. Biologically active compounds that have been With advancements in biosynthesis, researchers have shown that successfully produced using microorganisms pharmaceutically active compounds can be produced with engineered metabolic pathways in microbes [34] [35]. Researchers have used gene editing technology, for instance CRISPR, to genetically manipulate organisms such as E.coli and yeast to produce biologically active compounds in sufﬁcient concentrations for human consumption [36]. In recent years, researchers have additionally demonstrated production of these active compounds in cell-free systems [37]. For example in 2019, researchers demonstrated the production of tetrahydrocannabinol (THC) from a simple sugar such as galactose by engineering baker’s yeast.

89 This is a promising approach for bio-production of drugs in microbial systems requiring only simple sugars as the raw material needed for the process [38]. This enables programmable digital-to- biological conversion for fully automated, versatile and demand-based production of functional biologics [39]. Further, researchers have Figure X : Synthetic biology and microbioreactor platforms for programmable production of biologics at incorporated these engineered living cells in a the point-of-care miniaturized microﬂuidic reactor [40] [41] with Perez-Pinera, Pablo, et al. Nature communications (2016) environmental control capability to achieve point-of-care production of therapeutics and other biologically active compounds.

Finally, to control and switch between different modes of biosynthesis, we must be able to communicate with the cells using digital signals. Researchers have developed an optogenetics approach [42][43] to make living cells express Figure X : Portable, on-demand biomolecular manufacturing optogenetic light-gated ion channel actuators such

Perez-Pinera, Pablo, et al. Cell (2018) as rhodopsin, halorhodopsin, and archaerhodopsin in order to be light sensitive [44]. This allows for reliable, millisecond-timescale light-control of neuronal spiking [42] as well as light-control of cellular gene expression and biological production. For example, researchers engineered an endotoxin-free e.coli to secrete t-deoxyviolacein, an antimicrobial and antitumoral drug using light

Figure X : Digital-to-biological converter for as an on-off switch in a hydrogel patch. The on-demand production of biologics researchers reported that the system maintained Boles, Kent S., et al. Nature biotechnology (2017) levels of drug production and release for at least 42 days [45]. Further, researchers further used quantum dots (QDs) with different excitation ranges coupled with targeted enzyme sites in bacteria to control enzymatic reactions for production of different biofuels and chemicals [46]. Beyond optogenetics, in 2019 researchers were exploring the use of redox-responsive promoter genes which enable Figure X : Optoregulated Drug Release from an Engineered Living Material user-speciﬁed control over biological function

Sankaran, Shrikrishnan, et al. Small (2019) through electrical signals [47]. Even though this techniques is relatively new, researchers have implemented electrogenetics in type 1 diabetic mice for digital control of insulin release from intracellular storage vesicles. The device that incorporates the cells can be wirelessly triggered by an external electrical ﬁeld generator [48].

Through the intersection of research in wearable

Figure X : Towards development of electrogenetics biomolecular delivery, synthetic biology, using electrochemically active bacteria bio-production, and bio-digital interfaces, we see Hirose, Atsumi, Atsushi Kouzuma, and Kazuya Watanabe. Biotechnology advances (2019). a potential opportunity for closed-loop bio-digital interfaces to overcome narrow use cases, and become a general programmable platform for a wide range of applications including health and wellbeing as well as human augmentation

Figure X : Nanorg Microbial Factories: Light-Driven Renewable Biochemical Synthesis Using Quantum Dot-Bacteria Nanobiohybrids

Ding, Yuchen, et al. Journal of the American Chemical Society (2019).

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98 Chapter 6 BioFab on Body

99 Chapter 6 BioFab on Body

Figure X : The system diagram of Wearable BioFab Introduction

While there have been attempts to develop wearable sensors and on-body drug delivery systems that can adjust the biochemical interventions to the user for therapeutics and augmentation purposes, to the best of our knowledge there is no research on on-body programmable synthesis of biological compounds in a wearable device. Such technology combined with sensing, and delivery systems could enable programmable and personalized, on-demand biological interventions.

100 An advantage of wearable bio-production will be that compounds can be produced and administered in smaller quantities and possibly more continuously based on in-the-moment need when other traditional methods for responding to health issues may not be available. Some of the biologically active compounds of interest include: drugs, vaccines, antibodies, hormones, stimulants and other compounds that need to be administered to the human body for health, disease prevention, and performance enhancement. These biologically active compounds could either be directly consumed, inhaled, delivered to blood stream using micro needles, or absorbed through skin based on the type of intervention needed.

To illustrate this vision, we prototyped “Wearable BioFab”, a programmable bio-digital “organ” : an on-body digitally-controlled biosynthesis platform for personalized, on-demand production of biological compounds. This proof-of-concept prototype is accomplished through a milliﬂuidic bioreactor capable of producing multiple biological compounds as controlled by a digital multi-wavelength mechanisms leveraging engineered synthetic gene circuits. To interface biological production with a digital system, we deployed an RGB optogenetics circuit [1], a multi-channel light-activated system that allows for wavelength switching to activate production of different components using digital control. We integrated LED light sources with a microcontroller, liquid micropump, temperature control system, and touch screen display interface to create a compact wearable bioreactor device. To demonstrate the functionality of the prototype, we characterize the components used in the system including: the 3D fabrication of the milliﬂuidic device using a stereolithography method, the growth and engineered bio-production of living cell cultures, and the optogenetics control for switchable pathways of gene circuits.

101 Figure X : The use of stereolithography for millifluidic 3D fabrication of millifluidic device 3D printing The Wearable BioFab serves as the bioreactor for catalyzing the bio-production process of the engineered living cells. To fabricate the bioreactor, we explored the possibility of using an additive manufacturing method/3D printing, which has previously been used for synthetic biology applications [2]–[4] to create the device. Particularly, we were interested in the use of stereolithography 3D printing that uses photopolymerization reaction to form the material [5]. This fabrication method allows for micron scale precision of fabrication for various polymer types with different mechanical and chemical properties. In our fabrication pipeline, we used the CAD software Autodesk Fusion for the 3D design of the fluidic device. The first design that we came up with was simply a single channel millifluidic device with one inlet and outlet. We used a Formlabs printer and software to convert the STL file into a printable layout. After the fabrication, we post-processed the device with a two steps wash, using 90% isopropyl alcohol (IPA), and let the remaining polymer of the 3D printed sample polymerize and dry under UV light.

102 Growth and Bio-production of Engineered Living Cell Culture Next, we performed the biocompatibility test of our 3D printed millifluidic device. We selected non-toxic E. coli, as the strain of choice because it is well established as a model organism in the pharmaceutical industry to produce pharmaceutical and therapeutic compounds [6][7]. We characterized the growth and bio-production using an engineered synthetic circuit ”pGreen”, making E. coli capable of producing green fluorescent protein (GFP) in our 3D printed fluid device. We used flow cytometry (FCM) and microplate readers for longitudinal cell count as a proxy of cell growth and fluorescence measurement as a proxy of bio-production. We used four types of 3D printed resin materials from Formlabs including Clear, Draft, Dental (Dental LT Clear Resin V2), and Elastic.

In this series of experiments, we characterized the following: 1) The cell growth rates as a function of printing material and temperature, 2) The cell bio-production as a function of printing material and temperature, 3) The cell growth as a function of shaking conditions, 4) The cell bio-production as a function of shaking conditions, 5) The cell cell growth as a function of UV cure time, 6) The cell bio-production as a function of UV cure time, 7) The cell cell growth as a function of sterilization method, 8) And, the cell bio-production as a function of its sterilization method.

103 The effect of resin and temperature on cell growth In our experiments we have demonstrated the successful engineered bio-production of fluorescent proteins inside the 3D printed designs. Specifically, the cell growth and bio-production occur at the maximum rate in the following order of type of material: Draft, Dental, Clear, and Elastic in 37 degree Celsius incubation. We believe that this is due to the fact that the Draft and Dental resin were formulated to have low remaining of unpolymerized resin for rapid prototyping and medical compatibility, thus making it less toxic to the cells. We also found that the increase in shaking, which provides aeration and turbulence for the liquid cell culture significantly enhances the cell growth and bio-production compared to other types of interventions. Finally, we found that post-stereolithography processing including UV curing is significant for improving the biocompatibility of the fluidic device as it enhances the growth rate and the bio-production of the cells. However, sterilization using chemicals such as bleach negatively affects cell health. The information from this characterization is used in the final design of the device.

104 Figure 1 : Nunc lacus odio, sagittis quis ligula sit amet, tincidunt convallis purus. Nulla ut convallis velit. 105 Figure 1 : system diagram of the RGB circuit [1] Optogenetics for Bio-Digital Interfaces Next, we demonstrated the use of an optogenetics approach to digitally control and switch the bio-production from one component to another. We used the RGB optogenetic circuit system ﬁrst developed by Rodriguez et al., which uses the array of three photoreceptors sensitive to different wavelengths including blue-light sensor: histidine kinase (YF1) switched off by 470 nm light and on in its absence; red light sensor Cph8, which is switched on by infrared (705 nm) light and off by red (650 nm) light; and green light sensor CcaSR, which is switched on by green (535 nm) light and off by far-red (672 nm) light, to control the expression of genetics pathways [1]. The system allows the cell to distinguish between different wavelengths of light and respond by modulating gene expression, and hence vary the production of compounds. The RGB sensing system is a synthetic 18-gene, 46-kilobase-pair genetic program that was abstracted into four subsystems with the following functions:

106 Figure 1 : The results of the optogenetics system in the 1) Sensor Arrays: for combining the three light sensors (RGB) to light-activated bioreactor produce signals; 2) Circuit: used to integrate signals to an executable dynamic response; 3) Resource Allocator: a way to connect the circuit output to actuators; and 4) Actuators: the set of genes for bio-production and creating outputs [1]. By inserting the desired genes for different bio-production in the actuator with optimization, we can use programmable light to digitally switch between products and control the quantity and concentration of the production. To demonstrate the system and highlight the bio-digital interface, we used three distinct fluorescence molecules : green fluorescent protein (GFP), blue fluorescent protein (BFP), and red fluorescent protein (RFP) as the outputs to corresponding photoreceptors. Thus, if the cell receives a red light signal, it should in theory produce a red fluorescent protein as a response. We tested the circuit in our light-activated bioreactor, which contains RGB LEDs controlled by an Arduino microcontroller. We split the culture into different tubes for testing the cellular response to different wavelengths. The reactor was incubated in a controlled temperature of 37 degree Celsius with shaking. 107 Figure X : The level of bio-production measured by After incubating the engineered microbes in our bioreactor for 24 the normalized level of fluorescence hours, we used flow cytometry to measure the total bio-production of the cell. We shown in the graph above that by changing the wavelength of the light that the microbes were exposed to, we observe the corresponding changes of the bio-production as seen in the changes of fluorescence signals, confirming the switching of gene expression pattern. This demonstrates our success in using an optogenetics switch to digitally control the bio-production process. However, we observed some leakage that occurs when the unselected light is off, where the cell did not completely switch between the mode of production and still produced unexpected molecules. We believe this is due to the impurity of the wavelength that the LEDs were giving to the cell, and could be addressed by adding a wavelength filter to make the wavelength more narrow. The photoreceptor could likely be optimized through protein engineering and directed evolution.

108 Figure 1 : the Wearable BioFab prototype and the system Prototype Integration diagram Based on these characterizations, we created a proof-of-concept prototype of an integrated wearable bioreactor. The prototype contains a 3D printed milliﬂuidic system created using dental resin, which has the highest biocompatibility, while being transparent for the LEDs to be able to penetrate for optogenetic control. The channel contains two open inlets for adding and extracting the engineered cell culture as well as providing the port of connection for future integration with extension modules such as the module for biomolecule delivery. To overcome the shaking condition needed to increase the cell growth, the millifluidic channel was designed to consist of a closed-loop serpentine structure powered by a micro diaphragm pump that periodically aerates and generates flow inside the channel. The pump is controlled by a 1.5A motor driver with PWM speed control. The device also contains an array of dimmable LEDs with wavelengths of 650nm, 532nm, 470 nm for optogenetic activation

109 and a heating pad underneath the millifluidic channel as well as a breakout high precision temperature sensor board for temperature control. The heating pad, sensor, and motor driver are then integrated with an ESP-32S Development Board with 2.4GHz Dual-Mode WiFi + Bluetooth Dual Cores Microcontroller Processor for the control using the PWM channel, I2C channel and digital pin. The ESP-32S board allows the Biofab system to wirelessly connect with other wearable sensor devices for closed-loop interventions. However, an optional touch screen can also be added on the device and connected to the ESP-32S via a digital pin for manual control of the system. The system is powered using a small lithium ion battery. Finally, the exterior case of the device is printed with non-transparent resin to prevent outside light to interfere with the optogenetic process.

To activate the device, the user must ﬁrst load the engineered living cells with optogenetic compatibility in the millifluidic device. We imagine that, in the future, this starter cell culture will come pre-programmed with genetics circuit for producing speciﬁc compounds. The device can receive input on what molecules to produce from the user’s manual input via a touch screen or over wireless communication. The system will activate the LEDs to start the optogenetic induction to start the bio-production. As the cell culture starts the bio-production process, the user must rejuvenate the cells every 24-48 hours by introducing fresh growth medium for the cells to grow, similar to the recharging of a digital device such as smartphone. The device would calculate when the bio-production would be ready using the cell growth model and notify the user when to harvest the biomolecules, or in the future be able to deliver it via a delivery platform into the user’s body. Figure X : Potential use cases of Wearable BioFab Applications

To the best of our knowledge, Wearable BioFab is the ﬁrst prototype for digitally-controlled biomolecular production on the body, which goes beyond the current research in biomolecular delivery systems. It represents a proof-of-concept for a promising vision of closed-loop bio-digital interfaces that could have an impact in pharmaceuticals, personalized medicine, human augmentation, and beyond.

Pharmaceuticals and Personalized Medicine Building on top of closed-loop systems such as the glucose-insulin monitoring and delivery platform, which has proven to have tremendous impact on human health by allowing patients to receive personalized dosages and just-in-time biochemical interventions, we foresee that our technology, which enables on-body bio-production and digital control will expand the medical capability of closed-loop systems by making it possible for the device to store “biological blueprints” of multiple beneﬁcial compounds in the form of a genetics circuit, and be able to execute them in time of need simply with a digital signal. 111 In the future, this blueprint could be designed beforehand to be highly speciﬁc for one person, enhancing the effect of treatment. The ability to produce biomolecules on the body also opens up the possibility for smaller dosages of intervention, administered over a longer period of time, which could reduce the lethality and negative side effects of certain treatments. Finally, the ability to switch between the production of different molecules using simply a digital signal allows the wearable bioreactor to react and respond quickly to various diseases that might arise. Once new treatment become available, future users imight be able to update the living cells with new genetic circuits for new bio-production capability similar to updating a phone app.

Human Augmentation As discussed in the previous chapter, the application of using biochemical compounds extends beyond medical and therapeutic to cognitive and physical augmentation of human capabilities. Molecules such as caffeine that stimulate alertness [8], erythropoietin (EPO) which stimulates red blood cell production for physical performance enhancement during sports [9] and cognitive and memory enhancements [10], [11], as well as psychedelic substances that have recently been reported to help promote cognitive ﬂexibility, crucial to creative thinking [12] are part of the growing list of biological interventions that enhance human capabilities. The ability to safely produce these molecules in the right quantities on-demand will allow humans of the future to have augmented capabilities beyond our imagination.

Space Exploration One of the big challenges for space travel is staying healthy in a constrained environment. Since astronauts need superior physical and mental ﬁtness to survive in space, only a few individuals can so far have access to space travel. The work we propose can lead to wearable enhancements that can lower the entry level for humans to travel to 112 space. Further, human physiology in the microgravity of space is different from earth. We ultimately envision a “closed-loop health enhancement device” integrating different functions onto the body or in a space suit used for diagnosis, production and administration in a continuous feedback loop. Successful development of our device design would help astronauts in maintaining their health in a closed-loop cycle by continuously monitoring health status, producing the appropriate active compounds that help in restoring their health, administering these active compounds into the body, and integrating these three processes in a feedback loop.

This continuous feedback system would cut down the time for health evaluation and allow crew members to focus on their mission as aspects of astronaut health are continuously being monitored and addressed. For instance, a potential situation on Mars or the Moon might involve pain and inflammation induced by a physically demanding journey collecting soil and rock samples farther from the space shuttle. A closed loop system could detect the pain and inflammation, produce an anti-inflammatory (such as ibuprofen), and administer it into the astronaut’s body without human intervention. The astronaut can also program the device to regularly synthesize specific biochemicals that the body constantly needs to stay healthy in space such as the production of bisphosphonates, a class of drugs used to counter bone density loss.

Finally, the astronaut can also produce certain metabolic factors which can potentially enhance performance without risk of overdosing or the burden of administering it themselves. Therefore, the device will assist astronauts in focusing on their work by maintaining their health status with minimal involvement. This would be beneﬁcial for missions such as NASA 2033 Mars mission where crew members have to travel farther from their station for various complicated missions. 113 Figure 1 : Nunc lacus odio, sagittis quis ligula sit amet, tincidunt convallis purus. Nulla ut convallis velit.

Figure X: We envision integrating different modules that could be used for diagnosis, production and administration in a continuous feedback loop.

114 Limitations and Future Work

Our Wearable BioFab is a proof-of-concept prototype that demonstrates a futuristic vision of bio-digital interfaces. There are several limitations that require future research to address:

1) Biological Circuit: the current RGB genetic circuit only allows for the switching between three different pathways. This could potentially be scaled up to expand the fabrication capability by engineering new photoreceptors that are more optimized, and highly speciﬁc to a narrow wavelengths, or by using other methods for bio-digital interfaces such as electrogenetics [13] or the coupling of proteins with nano particles [14]. Further, the current circuit also has a leakage expression when the system is supposed to be off, due to the foundation of biological system which is stochastic. The goal of synthetic biology is to overcome such challenges, and create digital-like computation in living cells. Thus, this problem could potentially be solved through advancements in synthetic biology and engineering of synthetic minimal cells [15], [16] to be more digital-like. Finally, the living cells in the Wearable BioFab require the reintroduction of growth medium to stay alive and operative. We see this as similar to the process of recharging a digital device at the end of the day. In the future, as engineered living system becoming more integrated into human life, there should be research on designing novel infrastructure such as a ”charging stations” for a biological device.

115 2) Form factor: Our Wearable BioFab is wrist-worn, which provides the advantage of being easily put on the body and being reachable. However, such a system is not limited to the wrist and could be manifested in other form factors, such as cloth or a space helmet.

3) Integration: to fully realize the vision of closed-loop bio-digital enhancement, wearable Biofab must be integrated with sensors and a biomolecular delivery platform. We foresee multiple ways to administer the biomolecules produced by the system. First, we can genetically engineer strains that are used for fermentation of simple sugars, yogurt, and kombucha which are good for human consumption. For the biomolecule could not be consumed directly with the fermented product, another possibility could be to design and develop a miniaturized puriﬁcation module in the wearable where it lyses the cells and extracts the biomolecules produced. The biomolecules can either be directly injected into the blood using micro needles, inhaled through the nose, or absorbed through the skin.

4) Safe usage: The most important aspect and consideration of the Wearable BioFab is to ensure its safety. In the future, a safety mechanism must be put in place to prevent people from using the platform to overproduce certain compounds beyond the safety threshold or produce dangerous molecules that can cause negative effects on human health. This safety mechanism can come in the form of distributed digital authentication like blockchain systems that allow every decision of the user to be recorded and verified before the production happens, or potentially in the form of a biological mechanism in the engineered living cell that prevents it from accepting non-verifiable genetic code. Conclusion We presented the proof-of-concept "Wearable BioFab", an on-body, digitally-controlled biosynthesis platform for production of biological compounds. We prototyped an integrated millifluidic bioreactor to produce example biological compounds through the use of a digitally controlled multi-wavelength mechanisms leveraging engineered genetic circuits. We interfaced biological production with a digital system using an RGB optogenetic circuit, a multi-channel light-activated system that allows for wavelength switching to activate production of different components using digital control. We presented our characterization on the 3D fabrication of millifluidic devices using a stereolithography method, the growth and engineered bio-production of living cell culture, and the optogenetic control for switchable pathways of gene circuits, and discussed the limitation of the platform. To the best of our knowledge, Wearable BioFab is the first prototype that demonstrates the idea of digitally controlled biomolecular production on the body that goes beyond the current research on biomolecular delivery systems. We hope that our research will inspire researchers to think about a vision of closed-loop bio-digital interfaces that could have impact in the areas of pharmaceuticals and personalized medicine, human augmentation, and beyond. References

118 Chapter 7 Conclusion

119 Chapter 7 Conclusion

“I just invent. Then I wait until (hu)man comes around to needing what I've invented.” — Buckminster Fuller

This thesis explores the vision of closed-loop, bio-digital interfaces for human augmentation, where the bio-digital devices allow for both reading and writing of biological information to the human body. We demonstrated this vision through two proof-of-concept prototypes: "Wearable Lab", an integrated bio-digital platform for continuous sensing of biomarkers, and "Wearable BioFab", an on-body, digitally-controlled biosynthesis platform for personalized, on-demand production of biomolecules. We presented the technical implementation as well as the characterization and evaluation of these platforms. We discussed the limitations of these technologies as the two prototypes are far from perfect. However, they are the very ﬁrst prototypes that demonstrate the vision of bio-digital interfaces. They challenge the boundaries of current wearable technology by showing how biological systems can be integrated with digital computation.

This research is situated in the emerging paradigm of human-computer integration that suggests that the next step of human evolution will be fusing human unique capabilities with technological enhancements. Simultaneously, this thesis pushes the conversation forward by proposing that the term "computer" in human-computer integration is no longer limited to digital computers, but can also include engineered living systems that can be used as a complement to a digital system.

120 We introduced the framework of ”Living Bits”: the integration of living cells in, with and as computing systems, which extend the capability of digital systems to intimately tap into biological information and its functionalities.

As technological augmentation becomes the new nature of human beings, one critical question remains: How do we safely and ethically elevate humanity to the next level? And with Clines and Kline suggesting that being a technologically enhanced human is to be free, what goal is worthy of that “augmented freedom”? I believe that researchers in human augmentation must always pose this question to themselves regarding their work.

In my opinion, based on conversations that I have had over the years with my fellow researchers, I suggest that we think of this question at three different scales: the personal level, societal level, and global level. At the personal level, the technology that we design should empower rather than enforce, which means that the agency and ownership must remain with the person, not the technology. The technology should assist, inspire, and create the condition for individual to become the best version of themselves if they chose to do so. The device should become an augmented “organ” that is integrative with the body and enhance existing capability cognitively and physically. However, safety mechanisms must also be implemented to prevent the user from harming themselves as the augmentation also means more capability to negatively affect the person.

At the societal scale, we must also consider the interaction that the person might have with other people that may or may not prefer to use the augmentation technology. With current technologies, we have already seen the social inequality created through the unequal access of information and technology and should aim to avoid it with new technologies we create.

121 Science ﬁction ﬁlms such as GATTACA depict a future society where biologically-augmented, genetically-engineered individuals are highly celebrated over “normal” people. The scenario in the movie reflects a distorted society that puts extreme emphasis on achieving perfection through technological means. Thus, in return, the society forces the individual to become augmented through enforcement rather than empowerment, which goes against the principle that we discussed earlier. The question is how can we prevent such future to become our future, and prevent the gap of inequality from getting wider?

I believe that this question has no easy answer or solution, and it requires more than researchers that work on augmentation technology alone to solve it. However, I believe that we can start by recognizing the fact that beyond the physical impact that our technology has on people, our research can also reinforce, or unintendedly support certain ideologies or social thoughts that could be harmful to society. Therefore, it is very important for researchers to declare a clear vision as well as ethical guidelines for how they foresee their work becoming integrated in society in the future. Throughout history, countless inventions has unintendedly contributed to destructions of society beyond the imagination and intention of the inventors. Therefore, researchers should not be blinded by the excitement of new discovery and invention, but also learn from history, and participate in the broader ethical discussion with other scholars and the public.

In term of inequity, even though the uneven distribution of technology and resources is an ongoing struggle for centuries, where the geographical boundaries, racial injustice, social classes, and other factors determine and discriminate people who have access to technology from the one who do not. Real-time augmentation technology, on the other hand, shifts the conversation on this topic to the next level as it creates -

122 "just-in-time inequity" moment between the augmented and non-augmented human, as a piece of offered information, molecular injection, or brain stimulation that enhances human performance could lead to very different outcomes for two people talking in the same room.

I am afraid that the increasing use of technological enhancement could become a new type of “privilege” that certain groups have over one another. Similar to other privileges that humanities scholars have characterized in race, age, gender, class, and sexual orientation, they can intersect with one another and lead to unbalanced power dynamics and discrimination, as well as perpetuate systemic inequity. Researchers that study social justice suggest that engaging in self-reflection and open discussions about privilege is an essential step to addressing individual and systemic inequities in our society [1]. Augmented human researchers should be encouraged to think outside of the technological realm and engage with scholars of other disciplines that study the relationship between social equity and technology, and keep this practice central to their research.

Finally, thinking about the impacts and implications of augmented human research at the global scale is very important as it requires us to consider the "human" less as a focus, but more as a part of the larger complex ecosystem. We must constantly remind ourselves that "human-machine symbiosis" is always in a larger symbiosis with nature and the environment. In fact, the term "symbiosis" was borrowed from the study of ecology. Thus, the augmentation technology that solely enhances human capability to extract and utilize resources beyond balance, and does not consider environmental factors, would not be sustainable for the long run of human survival.

123 The concept of Living Bits presented earlier in this thesis, which integrates biological systems as an alternative or a counterpart of digital computation presents an exciting opportunity to rethink augmentation technology using organic and regenerative resources [2]. Having a symbiotic relationship with "living computers" on the human body that enhance our capabilities should remind us of the deep connections that we share with other species and forms of life inhabiting the planet.

I hope that these three scale of considerations, the personal level, societal level, and global level can serve as a primer for future discussions around human augmentation and can help broaden the perspective of augmented human research, and steer the ﬁeld into making positive impact on society.

As I am about to conclude this thesis, I thought about the global crises that the world is currently facing, and our role as researchers and inventors of the future. I recalled a quote that I introduced early on in this thesis from my mentor Professor Pattie Maes, where she says "We like to invent new disciplines or look at new problems, and invent bandwagons rather than jump on them". I believe that as a researcher, we have an important obligation to society to use our curiosity and skills for discovering and applying knowledge for the betterment of humankind. But not just for solving the problems that are knocking at our door, but also to help shine the light toward the exciting new possibilities of tomorrow, and inspire us all to live through this darkest time. I believe this is the magic of research, and the reason for me pursuing these works. I hope it will continue to inspire future researchers as much as it inspires me.

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