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Design, Fabrication, and Testing of Miniaturized Protein Expression Systems

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Title:
Design, Fabrication, and Testing of Miniaturized Protein Expression Systems
Creator:
MEI, QIAN ( Author, Primary )
Copyright Date:
2008

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Subjects / Keywords:
DNA ( jstor )
In vitro fertilization ( jstor )
Initial value theorem ( jstor )
Miniaturization ( jstor )
Polymerase chain reaction ( jstor )
Protein synthesis ( jstor )
RNA ( jstor )
Sensors ( jstor )
Signals ( jstor )
Toxins ( jstor )

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University of Florida
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University of Florida
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Copyright Qian Mei. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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8/31/2007
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649814545 ( OCLC )

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DESIGN, FABRICATION AND TESTI NG OF MINIATURIZED PROTEIN EXPRESSION SYSTEMS By QIAN MEI A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Qian Mei

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iii ACKNOWLEDGMENTS It is a pleasure to express my gratitude to all those people without whom this thesis would not be possible. First and foremost I would like to gratef ully acknowledge my supervisor, Dr. Z. Hugh Fan, for his inspiration, guidance, ment oring, and support throughout my thesis. Dr. Fan has been cultivating an interdiscip linary environment by populating the laboratory with people from all different backgrounds, wo rking on a number of different projects and introducing all sorts of new ideas from va rious fields. I thank him also for providing me an opportunity to grow as a student and e ngineer in the unique research environment he creates. I would like to thank Drs. Malisa Sarn tinoranont and Shouguang Jin for their time to serve on my supervisory committee and provide valuable comments and advice. I would like to thank Dr. Brian Cain fo r the access of a luminometer and Dr. Weihong Tan for the access of a fluorescence spectrometer. I would like to acknowledge Drs. Nancy De nslow, Quangfan Chen, Philip Laipis, Wei Lian, and Xinchun Shen for useful discussion. My deep appreciation also goes to Carl Fredrickson and Andrew Simon for their contributions to the developmen t and fabrication of devices. I would like to thank to entire Dr. Fan’ s Research Group, both former and current members, for their support and friendship. I sincerely cherish the moments we worked together as colleagues and friends.

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iv I would like to express my thanks to Dr. Hong Wang, my husband and best friend, whose constant love, encouragement, endless patience and passion enable me to complete this work. Lastly, and most importantly, I wish to thank my parents and entire extended family for providing a loving environment during my life.

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v TABLE OF CONTENTS Upage TACKNOWLEDGMENTST.................................................................................................iii TLIST OF TABLEST............................................................................................................vii TLIST OF FIGUREST.........................................................................................................viiiT ABSTRACT....................................................................................................................... ..x CHAPTER 1 INTRODUCTION........................................................................................................1 1.1 Miniaturized Device and Its Application...........................................................1 1.1.1 MEMS..........................................................................................................2 1.1.2 BioMEMS, Lab-on-a-Chip and MicroTAS..................................................3 1.2 Protein Expression..................................................................................................8 1.2.1 DNA, RNA, Protein and Central Dogma.....................................................8 1.2.2 In Vivo and I n Vitro (Cell Free) Protein Expression..................................10 1.3 Overview and Organization of This Thesis..........................................................12 2 IN VITRO PROTEIN EXPRESSION IN A MICROWELL DEVICE AND ITS USE FOR TOXIN DETECTION...............................................................................14 2.1 Introduction...........................................................................................................14 2.2 Background...........................................................................................................15 2.2.1 Prokaryotic IVT System.............................................................................15 2.2.2 Eukaryotic IVT System..............................................................................16 2.2.3 Choice of IVT Systems..............................................................................17 2.3 Experimental.........................................................................................................18 2.3.1 Reagents and Materials...............................................................................18 2.3.2 Device Fabrication......................................................................................19 .3.3 DNA Template for Cell-Free Protein Synthesis...........................................20 2.3.4 Cell-Free Protein Synthesis........................................................................21 2.3.5 Toxin Inhibition Assay...............................................................................23 2.3.6 Detection.....................................................................................................23 2.4 Results and Discussion.........................................................................................25 2.4.1 Toxin Detection Scheme............................................................................25 2.4.2 Plasmid-based Protein Expression.............................................................27

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vi 2.4.3 Linear DNA Template-based Protein Expression......................................29 2.4.4 Inhibitory Effects of T oxins on Protein Synthesis.....................................31 2.4.5 Miniaturized IVT Array.............................................................................34 2.5 Conclusions...........................................................................................................37 3 MICROFLUIDIC REACTORS FO R IN VITRO PROTEIN SYNTHESIS AND RICIN DETECTION..................................................................................................38 3.1 Introduction...........................................................................................................38 3.2 Background...........................................................................................................39 3.2.1 Ricin...........................................................................................................39 3.2.2 Continuous-Flow Cell-Free Pr otein Expression Systems..........................41 3.2.3 Continuous-Exchange Cell-Free System....................................................43 3.3 Experimental.........................................................................................................45 3.3.1 Materials and Reagents...............................................................................45 3.3.2 Device Fabrication......................................................................................45 3.3.3 In vitro Protein Expression.........................................................................46 3.3.4 Ricin Detection...........................................................................................47 3.4 Results and Discussion.........................................................................................48 3.4.1 Device Design............................................................................................48 3.4.2 GFP Expression..........................................................................................49 3.4.3 Luciferase Expression................................................................................51 3.4.4 Biological Signal Amplification.................................................................52 3.3.5 Detection of Ricin by In Vitro Protein Synthesis.......................................53 3.5 Conclusions...........................................................................................................55 4 SUMMARY AND FUTURE WORK........................................................................56 4.1 Summary...............................................................................................................56 4.1.1 Device Design and Fabrication..................................................................56 4.1.2 Protein Expression Systems.......................................................................57 4.1.3 Applications of Protein Synthesis Array....................................................58 4.2 Future Work..........................................................................................................58 4.2.1 Device Design and Fabrication..................................................................59 4.2.2 Protein Expression System.........................................................................59 4.2.3 Applications................................................................................................60 LIST OF REFERENCES...................................................................................................62 BIOGRAPHICAL SKETCH.............................................................................................72

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vii LIST OF TABLES UTable U Upage U 1-1 RNA codon table......................................................................................................11 2-1 Principal composition of prokaryotic IVT systems..................................................16 2-2 Pricinpal composition of eukaryotic IVT systems...................................................17 2-3 Prokaryotic versus eukaryotic IVT systems.............................................................18 2-4 Oligonucleotide primers used for mutant GFP........................................................22 3-1 Summary of methods fo r detection of ricin.............................................................41 3-2 Summary of IVT system...........................................................................................44

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viii LIST OF FIGURES UFigure U Upage U 1-1 Materials, fabrication and applications of BioMEMS devices...................................4 1-2 Structure of DNA and RNA.......................................................................................9 1-3 The central dogma of molecular biology..................................................................10 2-1 A picture of two devices fo rming an array (3x4) of 12 wells...................................20 2-2 Vector maps............................................................................................................. .22 2-3 A device with an a rray (12x8) of wells for in vi tro expression of a group of proteins.....................................................................................................................26 2-4 In vitro protein ex pression in microcentrifuge t ubes. (a) WT-GFP expression confirmed by Western blot.......................................................................................28 2-5 PCR analysis by agarose gel el ectrophoresis. Lane 1: PCR product after purification; Lane 2: DNA mark er; Lane 3: raw PCR product................................30 2-6 Excitation and emission spectra of the MonsterPTMP GFP...........................................31 2-7 Inhibitory effects of TC and CH on cell-free protein expression in microcentrifuge tubes...............................................................................................33 2-8 The response pattern of the 3x4 IV T sensor array for two toxin simulants, tetracycline (TC, a) and cycloheximide (CH, b)......................................................36 3-1 Schematic representations of current formats for IVT system.................................42 3-2 Schematic repres entation of the device....................................................................46 3-3 A novel microfluidic array device for in vitro GFP synthesis..................................51 3-4 Production yield of luci ferase as a function of the expr ession time in the device or in a microcentrifuge tube......................................................................................52 3-5 Signal amplification as a result of the inhibitory effects of ricin on the production of every copy of protein.........................................................................53

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ix 3-6 Calibration curve for ricin detection.........................................................................54 3-7 Synthesis of luciferase and detection of ricin in a few minutes...............................55

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x Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science DESIGN, FABRICATION AND TESTI NG OF MINIATURIZED PROTEIN EXPRESSION SYSTEMS By Qian Mei August 2006 Chair: Z. Hugh Fan Major Department: Mechanic al and Aerospace Engineering Recent development in miniaturization provides an opportunity for performing chemical and biomedical research in a novel, fast and unique way. This thesis presents two miniaturized plastic devi ces for in vitro protein expressions, as well as their applications for toxin detection. Biological synthesis of a protein includes th e steps of gene transcription and protein translation, which are typically carried out in host cells. However, some proteins are difficult to be synthesized in cells due to th eir insolubility, degrad ation and cytotoxicity. To address some of these challenges, cell-free protein synthesis, a pr ocess called in vitro transcription and translation (IVT), has been developed. In this thesis, IVT is first demonstrated in a microwell array for expres sion of proteins, incl uding green fluorescent protein (GFP), chloramphenico l acetyl-transferase (CAT) a nd luciferase. One of the major downsides of the microwell format is that it does not consist of any fluid manipulation. As a result, nut rients can not be supplied a nd inhibitory byproducts can

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xi not be removed, significantly reducing protein expression yield. To address this, a novel microfluidic array device consisting of a mechanism for fluid manipulation is then developed. Each unit in the device is composed of a tray and a well for expression of an individual protein. The tray chamber is for the IVT reaction and the well functions as a nutrient reservoir, and they are separated by a dialysis membrane. The microfluidic device possesses a mechanism to supply nutrients continuously and remove byproducts, leading to higher protei n expression yields. A concept of using these devices for toxi n detection is presented; the method is based on their inhibitory effects of toxins on protein synthesis. The concept is first demonstrated by detecting toxin simulants, tetracycline (TC) and cycloheximide (CH). The differential response patte rns from an array device for two toxin simulants suggest the feasibility of toxin det ection. This newly established method was also used for detecting a real toxin, ricin.

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1 CHAPTER 1 INTRODUCTION In this chapter, miniaturization and the potential applications of miniaturized devices will be discussed first. It is followed by a brief intr oduction of basic principles and terminologies in molecular biology that ar e related to this interdisciplinary research. 1.1 Miniaturized Device and Its Application Richard Feynman presented two famous ta lks about the miniaturized tools and devices: one is titled “There is plenty of room at the botto m— an invitation to enter a new field of physics” at the annual meeting of the American Physical Society at the California Institute of Technology on December 29th, 1959;P1P the other is “Infinitesimal Machinery—Revisiting ‘there is plenty of room at the bottom’ ” at the Jet Propulsion Laboratory in Californi a on February 23th, 1983.P2P These talks invited scientists and engineers to explore the realm of ultra-sm all structures and systems. Though Feynman never coined any fashionable word, like “nano” or “MEMS,” he is now widely credited as the first visionary in the field of miniaturized devices, and more recently, nanotechnology. Eleven months after his first talk, $1000 pr ize offered in Feynman’s talk for “the first guy who makes an operating electric mo tor in 1/64 inch cube” was claimed by William McLellan.P1P Since then, the engineering community has made exponential progress in miniaturization in microelectr onics, micro-electro-mechanical systems and nanotechnologies. The miniaturi zed computers were partially realized when he gave his second talk in 1983.

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2 But he must be very disappointed by the slow progress in miniaturized biological systems, which he speculated in his talk:P1P (p. 62) The marvelous biological system. The biological example of writing information on a small scale has inspired me to thi nk of something that should be possible. Biology is not simply writing information; it is doing something about it. A biological system can be exceedingly sma ll. Many of the cells are very tiny, but they are very active; they manufacture various substances ; they walk around; they wiggle; and they do all kinds of marvelous things---all on a very small scale. Also, they store information. Consider the possibi lity that we too can make a thing very small which does what we want---tha t we can manufacture an object that maneuvers at that level! Chemists and biologists are beginni ng to understand the potential of miniaturization and starting to work closel y with engineers to develop miniaturized devices.P3-5P 1.1.1 MEMS Micro-Electro-Mechanical Systems (MEM S) are the miniaturized systems or devices that integrate electrical and mechanical components on a common silicon substrate through microfabrication technology.P6-10P The sizes of these components are from the submicrometer level to the millimeter level; and there can be any number, from a few to millions, in a particular sy stem to complete a specific function.P11-16P Traditionally, MEMS devices are achieved by repeating the depos ition (thin film), patterning (photolithography) and etching st eps on two major materials, silicon and glass.P8, 17, 18P Besides the fabrication technologies directly port ed from the integrated circuit industry, new protocols ha ve been developed in last three decades to manufacture mechanical elements, like gears, beams, springs, etc. in MEMS devices.P19P During the last two decades, the MEMS devi ces shift rapidly from research lab to industry. Nexus market analysis predicted the market for microsystems will grow from $12 billion in 2004 to $25 billion in 2009 at the rate of 16% per year.P20P Examples of

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3 applications of MEMS devices ar e read/write head of hard driv e, inkjet printer cartridge, automotive accelerometers and pressure sensors. The topic of this thesis is about BioM EMS, the most recent and dramatically expanding biological applications of MEMS. 1.1.2 BioMEMS, Lab-on-a-Chip and MicroTAS BioMEMS (biological or biomedical micro-electro-Mechanical system) gets its name from MEMS, but BioMEMS devices rare ly use silicon or i nvolve a moving part (like beams or gears), which are hallmarks of MEMS devices. It may be one of least precise terms to describe th e application of MEMS and/or micro/nano technologies in biological system or bioanalytical chemistry. BioMEMS is now much more than a subset or division of MEMS, it is becoming a fiel d into itself, uniquely identified by its materials, fabrication technol ogies, chemistry and physics. While most researchers from engineering like to use BioMEMS to emphasize the microfabrication techniques used for manufact uring these miniaturized devices, chemists prefer to use Lab-on-a-chip (LOC) or micro total analysis system (MicroTAS or TAS) to indicate that more than one processing step s are integrated on to a single platform to perform a well-defined analysis task or a specific function.P21P More and more researchers are now using these terms in terchangeably. They sometime s also categorize devices without mechanical and/or electronic compone nts, such as DNA or protein microarrays, under BioMEMS. In the following part of this section, the material, fabri cation, applications (Figure 1-1) and advantages of BioMEMS or LO C devices will be briefly reviewed.

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4 BioMEMS devices are mainly fabricated wi th three categories of materials. They are (1) traditional MEMS materials, like sili con and glass; (2) plastic materials and polymers; (3) biomaterials, such as DNA, protein, cell and hydrogel. Figure 1-1. Materials, fabrication and applications of BioMEMS devices. At the inception of BioMEMS, silicon wafers and glass slides were used extensively because the MEMS fabricati on technologies, such as lithography, bulk micromachining, surface machining, could be directly ported to fabrication such devices.P17, 22, 23P However, the cost of such a device is very high. As reported by Manz et al. in 1998, the cost for each etched glass chip for continuous PCR amplification was $500 and for the apparatus was $4000.P24P In last couple of years, more and more research groups started to use plastic materials and polymers. The main reasons of using plastic materials including: (1) low cost. The material itself and the compatible fabrication process are much less inexpensive than the silicon or glass and they can be used in disposable devices, reducing the

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5 possibility of cross-contamin ation of the re-used devices;P25-28P (2) multi-functionalities. There are different plastic materials and pol ymers with different chemical, physical and biological properties. A hard and glassy or a soft and rubbery chip with different chemically functionalized surface can be easil y chosen from a doze of frequently used materials; (3) super biocompatibility. It’s easier to interface poly mer materials to the biological tissues. The lower toxicity, less immuno-inflammatory and bio-degradability permit in vitro application of such devices. The fabrication methods for manufacturing plastic devices include soft -lithography, mold injection, ho t embossing, laser ablation and micromilling.P29, 30P Most of these technologies still re quire a stamp, a mold, or a master fabricated with traditional MEMS technologies . Once the stamp is fabricated, the pattern on it can be mechanically transferred to hundreds to thousands of plastic chips. It is very cost-effective. The miniaturized devices used in this thesis were made from PMMA (poly methyl methacrylate) by direct micromilling. The third category is biomaterials, incl uding biomolecules (DNA, protein, etc.) and hydrogel. Hydrogel is widely used in scaffold fabrication for artifi cial organ and other implantable medical devices. Lots of welldesigned hydrogel may swell in responding to the environment, such as pH, temperature, et c., which makes it as a perfect component in closed-loop “smart devices”.P31, 32P While the above mentioned two categories of materials are normally fabricated with top-down methods (from large scale to small scale, like photolithography), the bottom-up approaches (f rom atoms or molecules to a specific pattern), such as self assembly, are widely used for biomaterials. “Nature is the best engineer”.P33P Combining the top-down and bottom-up approaches, much more complex systems can be manufactured.

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6 There are three categories of applications of BioMEMS devices: micro analysis, on chip synthesis and hybrid devices (Figure 1-1). The micro analysis devices are typically classified as eith er microarrays or microfluidic separation devices. A DNA or pr otein microarray usually refers to a collection of miniaturized test sites, which are composed of DNA, protein, small molecules, probes, self-assembled or in situ synthesized on a solid substrate that permits thousands or more tests to be performed at the same time. Developed in early 1990s, electrophoresis microchip is a typical mi crofluidic device for micro analysis.P27, 34-50P It is still one of the most active research topics in LOC and/or BioMEMS, and lots of efforts have been devoted to the integration a nd parallelization of microchip by different research groups. Burns et al. fabricated an integrated nanoliter DNA analysis device with microfluidic channels, heaters, temperature sensors and fluorescence detectors that could perform sample loading, mixing, PCR, electr ophoresis and detecti on in a single chip.P51P Mathies group pioneered in the deve lopment of 16, 48 and 96 microchannel electrophoresis microchips for genotyping and DNA sequencing.P49, 52, 53P A recent success of parallelization is the 768 lane DNA seque ncing system reported by Ehrlich et al.. Within this system, two sets of 384 lane s could be alternatively cycled between electrophoresis and regeneration an d the productivity was 4M bases.P54P While majority research reports are about the micro analysis system, considerable efforts have been devoted to the on chip synthesis or microre actors since later 1990s. Take advantages from the fast mixing,P55P improved mass and heat transfer from the microstructures in the microreactors,P56-58P and on chip (bio)chemical synthesis studiesP59, 60P showed great success as pred icted from the very beginning. One typical system is

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7 continuous-flow PCR microchip repo rted by Manz et al in 1998.P24P Recent research focuses on the integration of multiple reaction steps, on line analysis feedback, the solution-based combinational synthesis which has the potential for oligonucleotide, and peptide library construction and gene assembly.P61-70P There are full of challenges and opport unities to investigate the hybrid BioMEMS devices, such as implantable therapeutic devices and artificial organs. An ideal implantable therapeutic device composed of a sensor for monitoring level of biomolecules and controlling an actuator for dr ug or other therapeutic reagents delivery. A model system is glucose biosensor and in sulin delivery system to relieve diabetes patients from repetitively injection of insulin.P71P Although there are lots of success cases in either in vivo glucose det ection or in vivo insulin deliv ery system, but no integrated system has been reported by now. This categ ory of BioMEMS devices is not within the scope of this thesis. Further inform ation can be found in recent reviews.P72, 73P After brief review of the materials, fa brication and applic ations of BioMEMS devices, we can answer this question: w hy we need miniaturized BioMEMS devices? Because there are several advant ages of such devices, including: (1) small dimension of the components will reduce the weight, si ze and energy consumption of the device, leading to the high throughput, parallel and automatic comp lex system; (2) small amount of reagents may reduce the cost of the assa y and small amount of analyte may save the precious sample consumption; (3) the pe rformance (ultra-fast reaction rate, high resolution and sensitivity, etc.) of the devi ce is greatly improved because of the fast mixing, heat exchange, and mass transfer; (4) the reliability of the assay is improved because the batch fabrication processes make the low price, disposable device possible,

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8 and the confined miniaturized reactors almo st eliminate the cross-contamination from different samples. These advantages and the potential commercial interest pull the miniaturized BioMEMS devices from emer ging to burgeoning in last 15 years. 1.2 Protein Expression In this section, the basic prin ciples and terminologies of molecule biology related to this interdisciplinary work will be introduced. 1.2.1 DNA, RNA, Protein and Central Dogma DNA or deoxyribonucleic acid, usually in the form of double strand helix, is the carrier of genetic information of all HcellularH forms of HlifeH and most HvirusesH.P74P Each monomer of DNA is made from simple units, ca lled nucleotides, which consist of a sugar (deoxyribose) with a phosphate group attached to it, and a base, which may be either adenine (A), guanine (G), cytosine (C), or thymine (T). The chemical bond between one phosphate to the next sugar form a backbone that link one nucleotide to the next. The bases protruding from the bac kbone can pair with bases from another strand of DNA to form double strand helix, as shown in Figure 1-2. RNA or ribonucleic acid is slightly differe nt from DNA. The sugar in the backbone is ribose and one of the four bases is diffe rent – uracil (U) replaces thymine (T). Unlike DNA, RNA is almost always a single-stranded molecule and ha s a much shorter chain of nucleotides. The hydroxyl group attached to the ribose is prone to hydrolysis, which makes RNA less stable than DNA. But RNA al ways contains secondary structure to prompt the stability. Protein, also called polypeptide, is a complex, high-molecular-mass, Horganic compoundH that is made from a long chain of amino acids, each linked to its neighbor through a peptide bond. Proteins constitute most of the dry mass of a cell and they are

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9 involved in every function perf ormed by a cell, including regulation of cellular functions such as signal transduction and metabolism. Life, chemically speaking, is nothing but the function of proteins. Figure 1-T2. Structure of DNA and RNAT. Figure 1-3 shows the cent ral dogma of molecular bi ology firstly enunciated by HFrancis CrickH in H1958H and re-stated in a HNatureH paper published in H1970H.P75P It summarized internal relations between DNA, RNA and prot ein and can be expressed in one sentence: DNA is transcribed to RNA, which is then transl ated to protein; but protein is never backtranslated to RNA or DNA. The central dogma can be represented by th ree major steps: (1) DNA replication: it transmits the genetic information from the pare nt cell to each daughter cell. It is carried out by a complex group of proteins that unwind the double stranded helix, and using DNA polymerase and its associated proteins to faithfully repl icate the original template; (2) transcription: the genetic information contained in DNA is transferred to mRNA.

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10 Figure 1-3. The central dog ma of molecular biology. mRNA is synthesized and catalyzed by th e RNA polymerase using DNA as a template; (3) translation: after transcri ption, mRNA will be transported out of nucleus and bound to ribosome. The sequence information of mRNA is translated to an amino acid chain (polypeptide) by tRNAs (transfer RNAs) and associated enzymes according to the three letter codons (Table 1-1). Af ter translation, the peptide chain may require additional process or directly folds to a mature protein with a specific conformation. Since Crick’s paper published, there ar e a number of new facts have been discovered.P76-79P For example, the retroviruses can transcribe RNA to DNA and some viruses only have RNA genome.P76P But central dogma is still a very useful theory for guiding most experiments. 1.2.2 In Vivo and I n Vitro (Cell Free) Protein Expression In vivo protein expression process refers to protein synthesis in cells, which is already discussed in above section. A wealth of genetic and biochemical knowledge has been accumulated both in prokaryotic cells (like E. coli ) and in eukaryotic cells (like insect cells and mammalian cells). Severa l companies provide commercial cloning and protein expression kit with improved protein expression level and simplified protocols.

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11 Table 1-1. RNA codon table Second Position First Position U C A G Third Porsition U phenylalaninetyrosine cysteine C stop stop A U leucine serine stop tryptophan G U histidine C A C leucine proline glutamine arginine G U asparagine serine C isoleucine A A * methionine threonine lysine arginine G U Aspartic acid C A G valine alanine Glutanic acid glycine G * Start codon. However, there are still some drawbacks, including tedious labor in transfection, cell culturing and cell lysing , instability of mRNA and expr essed protein, improper folding, and non-active protein. In vitro or cell free protein synthesis is an attractive alternation of in vivo protein expression. As early as in 1950s, several groups independe ntly demonstrated protein synthesis machinery can work in absence of cell integrity. The limitation of the first generation cell-free expression system based on crude cell extract is the short lift time, and as a consequence, low productivity. The lim itation comes from tw o facts: one is the rapid depletion of high energy phosphate pool ; the other is the accumulation of the byproducts during the protein s ynthesis that inhibit the re action. In 1988, Spirin et al overcame the bottleneck of the in vitro pr otein synthesis system by demonstrating a continuous flow system to continuously supply the consumable substrates and remove the byproducts.P80P Following this work, several groups used different reagents exchange

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12 scheme to produce hundreds to thousands of microgram of proteins per milliliter of expression solution.P80-87P After testing the production of various function proteins (enzyme, antibody, hormone, etc) in more than 40 laboratories, Roche introduced a Rapid Translation System (RTS) in 2001.P88P All commercialized expression systems are those based on E. coli , wheat germ and rabbit reticulocyte. These systems will be summarized in chapter 2. Compared with in vivo protein expre ssion system, the main advantages and potential application of cell free protein synthesis systems include: (1) Absence of the cell membrane elimin ates steps associated with introducing DNA into cells, lysing cells a nd clearing lysate. More purif ied protein can be easily obtained. (2) Being compatible with the miniaturi zed and automated instrument, which can fulfill the high-throughput protein expression requirement in protein engineering and proteomic area. (3) The composition of the reaction mixture can be easily modified to favor post translational modificatio n and protein folding. (4) The non-natural and ch emically modified amino ac ids can be cooperated into the protein, which provides the possibility of protein engineer ing, protein function research and pharmaceutical studies. (5) The cytotoxic proteins and unstable or low expressed protei ns can be obtained. (6) The procedure is extreme faster, making it highly suited for screening expression templates and conditions. 1.3 Overview and Organization of This Thesis The objective of this work is to fabric ate a miniaturized device for protein synthesis. The miniaturized device is composed of an array of units; each unit consists of a reaction chamber for protein expression a nd a feeding chamber for nutrient supply and byproduct removal. Different in vitro protein expression system s are evaluated in the device. This device has also demonstrated fo r screening anti-biotic inhibitors and ultrafast ricin detection. The rest part of this thesis is outlined as follows.

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13 In chapter 2, different in vitro protein expression systems are evaluated in the miniaturized device consisting of an array of reaction chambers. As a proof of the concept for toxin detection, the expression yiel ds of different proteins are measured in presence of antibiotic inhibitors. In chapter 3, the performances of protein synthesis in the static and the continuous exchange reaction chamber are compared. Ultr a fast and sensitive detection of a toxin, ricin, is employed as an example of the appl ication of the continuous exchange protein synthesis device. In chapter 4, conclusion and futu re directions will be discussed.

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14 CHAPTER 2 IN VITRO PROTEIN EXPRESSION IN A MI CROWELL DEVICE A ND ITS USE FOR TOXIN DETECTIONTP*PT 2.1 Introduction Rapid and parallel synthesis of multiple pr oteins in an array format will greatly accelerate the development in medical, biologic al and environmental sciences. Biological synthesis of a protein includes the steps of gene transcription a nd protein translation, which are typically carried out in host cells. However, some proteins are difficult to be synthesized in cells due to their inso lubility, degradation and cytotoxicity.P90, 91P To address some of these challenges, cell-free protei n synthesis, a process called in vitro transcription and translation (IVT), has been developed.P91-103P In IVT systems, a DNA template consisting of a coding sequence is transcribed into messenger RNA using RNA polymerases and an appropriate promoter; either eukaryotic or prokaryotic lysate is then exploited for providing ribosomes and addi tional components necessary for protein translation. The transcription a nd translation steps can be coup led together and take place in the same reaction mixtures. IVT has b een demonstrated for various applications, including in situ immobilization of e xpressed proteins onto solid surfaces,P66, 104P synthesis of drug transporters,P105P and high-throughput screening.P106P In vitro protein expression has also been implemented in miniaturized devicesP68, 107-112P because miniaturization of analytical instruments has been one of major developments in the past decadeT. TP*PT A part of this chapter has been published in Mei, Q.; Fredrickson, C. K.; Jin, S.; Fan, Z. H. Anal. Chem. 2005 , 77 , 5494-5500.[89]

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15 In this chapter, we describe a device cons isting of an array of miniaturized wells. The device was used for in vitro expression of three proteins, includ ing green fluorescent protein (GFP), chloramphenicol acetyl-tran sferase (CAT), and luciferase. Two expression systems were used: a prokaryotic and a eukaryotic system. Differential inhibitory effects of two toxin simulants, te tracycline (TC) and cy cloheximide (CH), on the protein expression yield were observed, providing a unique response pattern of the array device for each toxin. In addition, the quantitative relationship between the yield of expressed proteins and the amount of a toxin provide a calibra tion curves, leading to true analysis. 2.2 Background In vitro protein synthesis systems are based on the cellular protein synthesis machinery to perform protein synthesis outsi de intact cells, and are divided into two types: prokaryotic and eukar yotic expression system. In v itro protein synthesis systems can be carried out either in a coupled manner, which requires an exogenous DNA as a template, or as an uncoupled system, in which messenger RNA (mRNA) is used as a template. Here we will focus on the coupled transcription-translation system (IVT). 2.2.1 Prokaryotic IVT System The most commonly used prokaryo tic system is derived from Escherichia coli lysate, including all the necessary en zymes and machinery for translation.P113P After two groups (Zubay; Gold and Scheiger) achieved some major improvements,P92-98P IVT has been widely used. The principal component s of Zubay and Gold-Schweiger system are listed in Table 2-1. The major difference be tween them is Zubay’s system contains 30,000 g centrifugation supernatant (S30) E. coli lysate depleted of endogenous RNA and DNA, while Gold-Schweiger system is ba sed on the ribosome-free and nucleic acid

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16 deprived of S100 fraction. In both systems, they contain an endogenous or phage T7 RNA polymerase, which transcribes mRNA from an exogenous DNA template. This RNA molecule is then used as template for translation. The DNA template may be either a gene cloned into a plasmid vector or a P CR amplified template. A ribosome binding site (RBS) is required for template translated in prokaryotic systems. Du ring transcription, the 5’ end of the mRNA becomes available fo r ribosome bind and translation initiation, allowing transcription and transl ation to occur simultaneously. Table 2-1 Principal composition of prokaryotic IVT systems.P114P Zubay’s system Gold-Schweiger S30 of E. coli containing RNA polymerase, ribosomes, tRNAs, ARSes, and translation factors S100 of E. coli containing RNA polymerase, ARSes and translation factor Amino acids ribosome ATP, GTP, UTP and CTP ATP, GTP, UTP and CTP Energy regenerating system: PEP+PK, or CP+CK Energy regenerating system: PEP+PK, or CP+CK Sulfhydryl compounds: ME or DTT Amino acids cAMP Total tRNA Salts: MgP2+P, CaP2+P, KP+P and NHB4PB+P Sulfhydryl compounds: ME or DTT DNA template Salts: MgP2+P, CaP2+P, KP+P and NHB4PB+P DNA template Abbreviation: ARSes, aminoacyl-tRNA synthetases; PEP, phophoenal pyruvate; PK, pyruvate kinase; CP, creatine phosphate; CK, creatine kinase; ME, mercaptoethanol; DTT, dithiotreitol 2.2.2 Eukaryotic IVT System Eukaryotic IVT systems, such as rabbit re ticulocyte lysate and wheat germ extract, are prepared from the cytoplasmic frac tion. However, they lack endogenous RNA polymerase activity because the process of tran scription only takes pl ace in the nuclei of the cel,l while protein synthesis is carried ou t in the cytoplasm of the cell, which is the major difference between the prokaryotic a nd eukaryotic cell. Th is limitation can be addressed by addition of an exogenous RNA polymerase. Purified E. coli RNA

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17 polymerase was first used to couple transcri ption and translation in eukaryotic IVT systems.P115-119P Bacteriophage T7, T3 and SP6 RNA polymerases were then isolated to use in IVT systems and they were introduced into eukaryotic system to produce mRNA, using DNA template with corresponding promotersP87, 120, 121P (Table 2-2). Table 2-2. Pricinpal composition of eukaryotic IVT systems.P114P Rabbit reticulocyte lysate Wheat germ extract Lysate containing ribosomes, tRNAs, ARSes, and translation factors Extract containing ribosomes, tRNAs, ARSes, and translation factors SP6,T3 or T7 RNA polymerase SP6,T3 and T7 RNA polymerase Amino acids Amino acids ATP, GTP, UTP and CTP ATP, GTP, UTP and CTP Energy regenerating system: CP+CK Energy regenerating system: CP+CK Sulfhydryl compounds: DTT Sulfhydryl compounds: DTT MgP2+P, KP+P and polyamine MgP2+P, CaP2+P, KP+P and polyamine Hemin, glutathione and cAMP DNA template with SP6, T3, or T7 promoter DNA template with SP6, T3, or T7 promoter 2.2.3 Choice of IVT Systems Although any organism could potentially be used as a source to prepare an in vitro protein synthesis system, E. coli lysate, rabbit reticulocyte lysate and wheat germ extract are widely used and commercially available.P64, 88, 101, 122-124P The choice of a synthesis system is based on the origin and biochemical nature of synthesized proteins and their downstream applications. Generally, prokaryot ic IVT systems can provide higher yields and are suitable for structural analysis. However, eukaryotic IVT systems can allow a better platform for functional studies, especially for posttranslational proteins. The comparison of prokaryotic and eukaryotic systems is summarized in Table 2-3.

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18 Table 2-3 Prokaryotic versus eukaryotic IVT systems.P91P Prokaryotic Eukaryotic Ribosomes 70S 80S Initiation factors IF: 1, 2 and 3 eIFs 1, 1A, 2, 2A, 2B, 3, 4A, 4B, 4E, 4F,4G, 5, 5A,6 Elongation factors EF-Tu, EF-Ts, EF-G eEF1A, eEF1B, eEF2 Termination factors RF1, RF 2, RF3 eRF1, eRF3 Energy-regeneration system PEP+PK, or CP+CK, or pyrucate+NAD+CoA CP+CK Sulfhydryl compounds ME or DTT DTT Advantage (1)Simplicity and university of DNA template design; (2)High translation rate. (1)High stability; (2) Better compatibility with the synthesis of eukaryotic proteins Disadvantage (1)shorter reaction time; (2)High rate of degradation of DNA, mRNA and protein; (3)Aggregation of protein products (1)Lower translation rate; (2)Complexity of design of DNA template Abbreviation: IF, initiation factor; EF, el ongation factor; eEF, eukaryotic elongation factor; RF, release factor; eRF, eukaryotic release factor. 2.3 Experimental 2.3.1 Reagents and Materials The RTS 100 E. coli HY kit, two expression vectors containing the genes encoding green fluorescent protein (GFP) and chloramphe nicol acetyl-transferase (CAT), and anti6xHis were obtained form Roche Diagnosti cs GmbH (Mannheim, Germany). TNT Quick Coupled Transcription/Transla tion system, T7 luciferase DNA vector, luciferase assay reagent, phMGFP vector, PCR Master mix, PCR clean-up kit, DNA marker and nucleasefree water were from Promega Corporation (Madison, WI). RNA tr anscription kit was bought from Stratagene (La Jolla, CA) and RNA quantitation kit was from Molecular Probes (Eugene, OR). Primers were synt hesized from Integrated DNA technologies (Coralville, IA). Acryla mide-bisacrylamide (electr ophoretic grade, 5% C), tetramethylethylenediamine, sodium dodecyl sulphate (SDS), a mmonium persulfate, tris(hydroxymethyl)aminomethane (Tris), glycine, sodium chloride, glycerol,

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19 bromophenol blue, -mercaptoethanol, Tween-20, tetrac ycline, and cycloheximide were purchased from Fisher Scientific (Atlan ta, GA). Polyvinylidene difluoride (PVDF) membranes (0.2 m), and filter papers were from Bio-Rad Laboratories (Hercule, CA). Molecular weight standards, biotinylated secondary antibody and streptavidin-alkaline phosphatase were from Amersham Bioscience s (Piscataway, NJ), while recombinant Green Fluorescent Protein (rGFP) and rabbit anti-GFP polyclonal antibody were from BD Biosciences (Palo Alto, CA). The phos phatase staining solution (Bromo-chloroindoryl phosphate/Nitro Blue Tetrazoliu m, BCIP/NBT) was obtained from KPL (Gaithersburg, MD). 2.3.2 Device Fabrication Polymethyl methacrylate (PMMA) is widely used in bioMEMS device fabrication. Its excellent optical properties, very low fluorescence background, and compatibility with chemical or biological reagents make it as the first choice of the materials for microfluidic device. Moreover, the fabrica tion steps are straightforward and cheap. A miniaturized device with an array of 2x3 wells was designed and fabricated for demonstrating the toxin detection concept. Tw o of such devices in Figure 2-1 form an array of 3x4 wells, which is the format for demonstrating toxin detection as discussed below. The device was made from PMMA (L ucite International, Cordova, TN) and the wells were created by a milling machine (F lashcut CNC, Menlo Park, CA). The distances between wells (center to cente r) are 9 mm according to the standards for microplates defined by the Society for Biom olecular Screening and accepted by the American National Standards Institute; this arrangement conforms the device to the alignment of 96-well plates and ensures compatibility with a variety of commercial fluid dispensing systems and plate readers. Th e diameter and depth of each well are 2.7 mm

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20 and 2.3 mm, respectively, providing the total we ll volume of ~13 L. This is about 25 times smaller than the wells in conventiona l 96-well microplates. The decrease in the well size will significantly reduce reagen t consumption for high-throughput assays.P68P After fabrication, the device wa s sterilized by exposing to UV light for 30 minutes that insured the consistency of the protein expression. Figure 2-1. A picture of two devices forming an array (3x4) of 12 wells. A US penny is also pictured for comparison of the size. The wells in each device were laid out according to the standards of 96-we ll plates, but with ~25 time reduction in the well volume. .3.3 DNA Template for Cell-Fr ee Protein Synthesis GFP vector (Figure 2-2a), CAT vector (Fi gure 2-2b) and lucifera se vector (Figure 2-2c) were used as the templates for the plas mid-based cell-free protei n synthesis. For the experiments using linear DNA template produce d by PCR for cell-free protein synthesis, the coding region of mutant GFP was amplif ied from the plasmid phMGFP (Figure 2-2d) using the forward and backward primers gi ven in Table 2-4. To generate linear DNA template, PCR Master Mix containing Taq DNA polymerase, was reconstituted per the manufacturer’s recommendations . A PCR tube with 50 l of the prepared solution was

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21 placed in a PTC-100 programmable thermal c ontroller (MJ Research, Waltham, MA). PCR amplification consisted of 25 cycles, in which denaturation (94 C) and annealing (57 C) steps were 45 s, and the extension step (72 C) was 1 min. The PCR products were purified using PCR-clean up kit (as manual instruction) prior to their use for protein expression. The amount of PCR products was determined by agarose gel electrophoresis and spectrophotometry. To prepare gel, agar ose was dissolved in tris-boric acid-EDTA (TBE) buffer solution and cast into a gel sla b. When solidified, it was immersed in TBE buffer containing ethidium bromide dye. Th e PCR products were added to the sample wells in the slab, which was placed in the se paration cell and voltage was then applied. When separation was completed, the gel was removed from the cell, de-stained and rinsed to remove excess ethidium bromide, and imaged using a UV transilluminator. 2.3.4 Cell-Free Protein Synthesis For the prokaryotic expression system, 50 L of RTS 100 reaction solution was composed of 12 L E. coli lysate, 10 L reaction mix (proprietary composition, supplied in the kit by the manufacturer), 12 L amino acids without methionine, 1 L methionine, 5 L reconstitution buffer (proprietary composition, supplied in the kit by the manufacturer), and 10 L nuclease-free water containing 1 g GFP or CAT vector. The reaction solution was then incubated in a micr ocentrifuge tube at 30 C for 4 hours. For GFP, the reaction solution was stored at 4 C for additional 24 hours for the maturation of GFP. For the eukaryotic expression system, ra bbit reticulocyte lysate or wheat germ extract was used. The reaction mix of 50 L for luciferase synthesis was prepared by combining 40 L TNT Quick master mix (proprietary composition, supplied in the kit by

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22 Figure 2-2. Vector maps. (a) pIVEX GFP v ector map. (b) pIVEX CAT vector map. (c) Luciferase control DNA map. (d) phMGFP vector map. Additional description: ori, origin of plasmid replication; AmpR, b-lactamase gene (resistant to ampicillin). Table 2-4. Oligonucleotide primers used for mutant GFP. Orientation Sequence (5’-3’) Forward CAA CAG TCT CGA ACT TAA GC Backward AAA ACC TCC CAC ATC TCC the manufacturer), 1 L methionine, and 9 L nuclease-free water containing 1 g luciferase vector. Wheat germ reaction mixt ure for monster GFP synthesis consisted of the following components: 25 L wheat germ extract, 2 L reaction buffer, 1 L T7 RNA polymerase, 1 L of 1 mM amino acid, 1 L Ribonulease inhibitor and 19 L nucleasefree water containing 2 g of linear MonsterPTMP GFP template or 1 g of phMGFP vector. Incubation was performed in a microcentrifuge tube at 30 C for 1.5 hours. In certain

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23 experiments, the transcription and translati on reactions were imple mented sequentially. The plasmid template or PCR-product was inc ubated in a transcrip tion mixture and then supplemented with wheat germ extract to initiate the tran slation reaction. 2.3.5 Toxin Inhibition Assay For the toxin inhibition assay in a micr ocentrifuge tube, a stock solution of tetracycline and that of cyclohe ximide were prepared at 15 g/ L and 10 g/ L, respectively. A series of amounts of tetrac ycline or cycloheximide were added into protein expression mixture. The concentrations of toxins used are listed in the figures or text. To save reagents and match with mini aturized devices, 8 L of prokaryotic or eukaryotic expression solution was used, ma king the total volume of each inhibition assay at 10 L. For each set of experiments, a positive control (without inhibitor) and a negative control (without the expression vector) have also been included. When the protein expression and toxin inhi bition assays were implemented in the miniaturized device, the volume of th e reaction mixture was reduced to 6.5 L. Films with pressure-sensitive adhesive, so-calle d “PCR tape” (3M, Minn eapolis, MN), were used to seal the wells to prevent evaporation. 2.3.6 Detection We used Western blot, fluorescence or lu minescence for measuring the yield of protein expression, depending on the property of the proteins expre ssed. Detection of expressed WT-GFP and CAT wa s achieved using Western bl ot. A reaction product solution of 1 L was mixed with 15 L of gel-loadi ng sample buffer, which contains 50 mM Tris-HCl, pH 6.8, 2% W/V SDS, 0.01% W/V bromophenol blue, 10% V/V glycerol, and 5% V/V 2-mercaptoethanol. The mixt ure was then separated in a 15% SDSpolyacrylamide gel in the Mini -Protean III Cell system (BioRa d). After electrophoresis,

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24 the gel was removed from the glass plates and then equilibrated in the transfer buffer, which is comprised of 48 mM Tris, 39 mM glycine, and 20% V/V methanol. PVDF membrane was pre-soaked with methanol, followed by soaking in the transfer buffer for 1 hour. The Mini Trans-Blot system (BioRad) wa s set up with pre-wetted fiber pad, filter paper, gel, and PVDF membrane according to the instruction from the manufacturer. The cassette and ice-cooling unit were placed in the tank that was filled with the transfer buffer. After blotting, the PVDF membrane was re moved from the trans-blot apparatus and blocked with 5% w/v non-fat dried milk in Tris-buffered saline (TBS) solution (with 0.05% Tween-20) for 1 hour at room temperat ure. After being washed three times (5 minutes each time) with TBS solution, the membrane was incubated for 1 hour at room temperature with 1 g/mL anti-GFP polyclonal antibody for GFP product or 0.3 g/mL anti-6xHisB Bmonoclonal antibody for CAT product, respectively. At the end of conjugation, the membrane was washed three times and then incubated with 1.5 g/mL biotinylated secondary antibody at room temp erature for 1 hour. Upon completion of the incubation, the membrane was rinsed again w ith TBS solution for th ree times, followed by incubation at ambient temperature for 30 minutes with a soluti on of streptavidinalkaline phosphatase (1:2000 dilution from th e stock solution). After being washed, the membrane was immersed in chromogenic s ubstrate (BCIP/NBT) for 3 minutes, followed by rinsing with water (to stop reaction). Im ages of protein bands were acquired with a color laser scanner (Canon); proteins bands were quantified using ImageJ from the National Institute of Health.P125P

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25 Detection of synthesi zed WT-GFP and MonsterPTMP GFP was also carried out by Spex Fluorolog spectrofluorometer (Jobi n Yvon Inc, Edison, NJ). The excitation spectrum of WT-GFP was scanned from 350 nm to 500 nm with emission wavelength at 550 nm; and the emission spectrum was scanned from 450 nm to 580 nm with excitation wavelength at 470 nm. The emission spectrum of MonsterPTMP GFP was collected from 450 nm to 580 nm with excita tion wavelength at 480 nm. Detection of luciferase expressed by IVT was achieved by SIRIUS luminometer from Berthold (Pforzheim, Germany). The luminometer was programmed to have a twosecond delay, followed by a five-second meas urement of luciferase activity. The expression product of 2 L was added to a luminome ter tube containing 40 L of luciferase assay reagent and mixed evenly. Th e tube was then placed in the luminometer; and the data were acquired. 2.4 Results and Discussion 2.4.1 Toxin Detection Scheme Multiplexed sensor array is a unique appro ach to obtain the fingerprint of a new agent. The concept of the sensor array for detecting toxins using IVT is illustrated in Figure 2-3. The device consists of an arra y of IVT wells; each well is designed to express one protein and thus functions as a se nsor. The number of IVT sensors can be as high as 96 or its integral multiples, in the format of traditional microplates and can be easily adapted to commercial plate readers. Multiple wells (e.g., 3x4 wells circumscribed within the dashed lines and shadowed with diag onal lines) form one set, in which the top row is for the positive controls to express ea ch of three proteins, the second row for the negative controls, and the third and fourth rows are for the sample, allowing one repeat to

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26 enhance the precision. Use of the positive a nd negative controls and comparison of the protein DNA toxin RNA toxin transcription translation Figure 2-3. A device with an ar ray (12x8) of wells for in vi tro expression of a group of proteins. The area shadowed with dia gonal lines indicates a subset (3x4) of 12 wells, among which the top row of 3 wells are for expression of three different proteins as the positive cont rols, the second row for the negative controls, and other two ro ws for the samples with one repeat for enhanced precision. Total of 8 series of such a 12-well set may be made for detecting different toxins. The insert in th e expanded view show s transcription and translation steps in each well; and the processes may be inhibited by toxins. signal from the sample wells with those in the control wells will reduce false positives and negatives. The set will express a group (t hree in this case) of pre-characterized proteins in different expression systems; the proteins and expression systems will be judiciously selected so that protein synthesis in each we ll is inhibited or affected differentially by different type of toxins. Therefore the unique response pattern (or signature) of a toxin due to different inhibito ry effects will be registered and used as a

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27 tool for detection and identification. New agents will be identified by comparing the response pattern with signatures of known ag ents in a pre-acquired database. In the particular format illustrated, the rest of we lls in the 96-well array can be designed to detect additional seven types of toxins if 12-well set is proved to be enough for the identification. 2.4.2 Plasmid-based Protein Expression To demonstrate the concept of toxin detection by protein e xpression, we first synthesized three proteins in two types of expression syst ems. The first protein is wide type green fluorescent protein (WT-GFP), a widely-u sed fluorescent molecule with known DNA sequence and crystal structure.P126P Protein expression was carried out by using an expression vector as a DNA template, whic h consists of GFP coding sequence and the necessary regulatory elements includi ng T7-RNA polymerase promoter, ribosome binding site, start codon, stop codon, and T7 terminator. The expression vector was mixed with E. coli lysate and a reaction mix consisting of T7-RNA polymerase, nucleotides, amino acids, and other re agents. WT-GFP product was confirmed by Western blotting and fluorescence spectrometry. The result of Western blotting is shown in Figure 2-4a. A clear band in lane 4 indicates the presence of WT-GFP in the expression product. According to pre-stained protein markers, the molecular weight of WT-GFP expressed is estimated ~31 KD. E xpressed WT-GFP contains a stretch of additional six histidines (6xHis) at its C-te rminal, causing its molecular weight slightly larger than recombinant GFP (rGFP) purchased commercially. The negative control in the experiment contains all reagents ex cept for the expression vector. The WT-GFP expression also can be quantified by fluores cence spectrometry. When it was excited with 488 nm light, the excitation and emission spectra of synthesi zed WT-GFP were shown in

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28 Figure 2-4b, indicating the synthesized WT-GFP folds properly and has biological activity. (a) (b) (c) (d) Figure 2-4. In vitro protein expression in microcentrifuge tubes. (a) WT-GFP expression confirmed by Western blot. Lane 1: pr e-stained protein markers; 2: negative control; 3: recombinant GFP (rGFP) pur chased; 4: GFP expressed. (b) WTGFP confirmed by fluorescence spectrom etry. Top: excitation spectrum; Bottom: emission spectrum. (c) CA T expression confirmed by Western blotting. Lane 1: pr otein markers; 2: negative co ntrol; 3: CAT expressed. (d) Luciferase expression confirmed by lumi nescence detection. Lane 1: negative control; 2: luciferase expre ssed. The intensity of luminescence in the y axis is in log scale. The second protein is chloramphenicol acetyl-transferase (CAT), an enzyme responsible for bacterial resi stance to an antibiotic drug, chloramphenicol. CAT was expressed in the same E. coli expression system; success of the protein expression was also confirmed using Western blot as shown in Figure 2-4c . According to pre-stained protein markers, the molecular weight of CAT expressed is estimated ~26 KD, which agrees with the value reported in the literature. The third pr otein is luciferase, an enzyme from firefly tails that catalyzes the producti on of light in the presence of luciferin , adenosine triphosphate (ATP), molecular oxygen and MgP2+P. Synthesis of luciferase was carried out using rabbit reticul ocyte expression system as de scribed in the experimental

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29 section. Detection of the expression product was achieved by monitoring the intensity of luminescence after mixing 2 L of the product with luciferin , ATP,OB2B and MgP2+P. As shown in Figure 2-4d, the luminescence signal of the product is 5 orders of magnitude higher than that of the negative control. 2.4.3 Linear DNA Template-based Protein Expression The WT-GFP gene originally cloned from Montastrea cavernosa can be expressed into the protein which tends to photobleach and is very dim. In addition, the correct folding of WT-GFP requires thre e distinct physical processe s: the attainment of the correct threedimensional structure, cycli zation of the chromophore, and oxidation of the cyclized intermediate.P127P So the folding of WT-GFP can not complete during transcription/translation in a cell-free system and it takes addi tion time to have biological activity. The mutants of GFP wh ich have brighter fluorescen ce and fold more efficiently at 37 C were developed, such as MonsterPTMP GFP, enhanced GFP, blue fluorescent protein and yellow fluor escent protein. MonsterPTMP GFP can produce fluoresces at least 20% brighter than other comme rcially available GFPs and the spectral properties of the MonsterPTMP GFP are also slightly re d-shifted. Peak excitation occurs at 505 nm, with a shoulder at 480 nm, and the peak emission occurs at 515 nm. MonsterPTMP GFP coding sequence was first amp lified from phMGFP vector by PCR with corresponding primer. PCR products were purified by a PCR clean-up system and confirmed by the agarose gel electrophoresis (Figure 2-5). Th e results indicated that the length of PCR products is about 1190 bp, as same as we designed. The synthesized MonsterPTMP GFP from PCR-amplified DNA or the v ector using coupled transcription and translation synthesis was confirmed by fl uorescent spectrometry (Figure 2-6). The excitation and emission spectra of the synthesized MonsterPTMP GFP are consistent with

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30 those reported by Promega. The results also sh owed that the protein yield expressed from plasmid is higher than that from PCR amplified DNA, because the linear DNA are easily degraded by the endogenous nucleases, thus, the amount of mRNA mol ecules is limited. Unfortunately, the expression of MonsterPTMP GFP can not be re produced. Then PCR products were first transcripted to mRNA and then mRNA molecules were translated to protein but protein can not be detected by fluorescent spectrometry. The reasons may include: (1) the amount of expressed MonsterPTMP GFP is too low to be detected using fluorescent spectrometry; (2) no MonsterPTMP GFP was expressed due to rapid degradation of mRNA, thus terminats the translational reaction quickly. Figure 2-5. PCR analysis by agarose gel el ectrophoresis. Lane 1: PCR product after purification; Lane 2: DNA mark er; Lane 3: raw PCR product.

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31 (a) (b) Figure 2-6. Excitation and emi ssion spectra of the MonsterPTMP GFP. (a) The excitation spectra of expressed MonsterPTMP GFP. (b) The emission spectra of expressed MonsterPTMP GFP, excited at 480 nm. Top curve: MonsterPTMP GFP synthesized from phMGFP vector; Middle curve: MonsterPTMP GFP synthesized from PCRamplified DNA; Bottom curve: negative control. 2.4.4 Inhibitory Effects of Toxi ns on Protein Synthesis To illustrate the detection of toxins, we used tetracycline (TC) and cycloheximide (CH) as toxin simulants to study their inhibito ry effects on protein expression. TC is an antibiotic substance produced by Streptomyces species.P128P It acts only on prokaryotic cells and it blocks binding of aminoacey l-transfer RNA to A-site of ribosomes.P129P CH acts specifically on eukaryotic cells and it inhibi ts the activity of pep tidyl transferase, an enzyme needed in the transl ocation reaction on ribosomes. Figure 2-7 shows the effects of a series of concentrations of TC or CH on the expression yields of GFP, CAT, and luciferase synthesized in two protein expres sion systems. As illustrated in Figure 2-7a, GFP synthesis was completely inhibited wh en 3000 ng/L of TC was used. Partial inhibition was observed when a series of lo wer concentrations (300 ng/L to 0.3 ng/L) of TC was added. The experiment also in cluded the positive control, in which no inhibitor (TC) was added. The negative cont rol contained no expr ession vector, thus

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32 representing the background signal. Thes e results suggest th at a qualitative and quantitative relationship exists between the expression yield and toxin amount, a critical figure of merit for a sensor. The comparison between TC and CH for GFP production in the E. coli expression system is shown in Figure 2-7b and 27c. The expression yield fo r each protein was normalized against the expression yield of the positive contro l (without toxin), so that it is easier to compare the toxic effects. The results clearl y indicate that TC has inhibitory effect on GFP production and the degree of inhibition is proportional to the amount of TC in the sample, whereas CH has a negligible effect on the yield of GFP production and the level of minor inhibition remained the same in the range of amount of TC we used. These results suggest that the diffe rential inhibitory effects of a toxin on the expression of different proteins are possibl y used for toxin detection. Figure 2-7d and 2-7e exhibit similar di sparity between TC and CH for CAT production in the E. coli expression system. Again, TC has inhibitory effect on CAT production and the degree of inhibition is propor tional to the amount of TC in the sample, whereas CH has a negligible effect on the yield of CAT production a nd the level of minor inhibition remained essentially same in the range of amount of TC we used. Furthermore, comparison of Figure 2-7b a nd 2-7d indicates that although TC has inhibitory effect on both GFP and CAT pr oduction, the degree of inhibition per unit amount of TC differs between these two prot eins, evident from the difference in the slopes of respective linear regression lines. Thes e results further suggest that each toxin’s differential inhibitory effects on expression of different protei n can be used as a signature for toxin detection and identification.

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33 0.0 4.0 8.0 12.0 16.0 -4048 0 0.2 0.4 0.6 0.8 1 -4048 0 0.2 0.4 0.6 0.8 1 -4048 0 4 8 12 16 -30369 0 0.2 0.4 0.6 0.8 1 -30369 0 0.2 0.4 0.6 0.8 1 -30369(c) CH effect on GFP expression (d) TC effect on CAT expression (e) CH effect on CAT expression (f) TC effect on luciferaseexpression (g) CH effect on luciferaseexpression123456789(a) (b) TC effect on GFP expression Figure 2-7. Inhibitory effects of TC and CH on cell-free protein expression in microcentrifuge tubes. (a) Western bl ot analysis confirms the inhibitory effects of TC on the expression yield of GFP in E. coli expression system. Lane 1: pre-stained protein markers; lanes 2-6: expression of GFP with 3000, 300, 30, 3, 0.3 ng/L of TC, respectively; lane 7: positive control; lane 8: negative control; lane 9: rGFP purchased. (b-g) Inhibitory effects of TC (b, d, and f) or CH (c, e, and g) on the expr ession yield of GFP (b, c) in E. coli expression system, of CAT (d, e) in E. coli expression system, and of luciferase (f, g) in rabbit reticulocy te expression system. The expression yields of GFP and CAT were quantifie d by Western blotting while that of luciferase was measured by luminescen ce. All x axes are the concentration (ng/L) of toxin, in log scale. Y ax es are the amount of expressed protein either normalized to the positive control (b-e) or in log scale of luminescence signal (f, g).

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34 The comparison between TC and CH fo r luciferase production in rabbit reticulocyte expression system is shown in Figure 2-7f and 2-7g. An opposite effect was observed; TC has a negligible effect on th e luciferase producti on in the eukaryotic expression system, whereas CH has a significa nt inhibitory effect and the degree of inhibition is proportional to th e amount of CH present in the sample. The result indicates that different expre ssion systems can be used to ex pand the variability, so that a unique response pattern can be obtained for t oxin detection by using a set of proteins produced in different ex pression systems. These results are significant because they indicate not only the feasibility of the concept of toxin detection presented in this work, but also the possi bility of using IVT assay for high-throughput screening of drug candidates. We confirmed differential inhibitory effects of antibiotic inhibitors such as TC and CH on protein expression in vitro , in a way very similar to th eir effects on protein expression in vivo .P130P Therefore, an IVT array device may provide a great plat form for searching for the best antibiotic drug candidates. 2.4.5 Miniaturized IVT Array After the feasibility of toxin detection us ing protein expression is demonstrated in microcentrifuge tubes, we attempted IVT a nd toxin detection in a miniaturized well device. The design of the experiments was th e same as in the shadowed area of the 96well array in Figure 2-3, in which a set of 3x4 wells is assigned for detecting one toxin. Two of the 2x3 well devices in Figure 2-1 were combined to form a 3x4 well device. As illustrated in Figure 2-3, the first row of 3 wells was used as the positive control, expressing GFP and CAT vector in the E. coli expression system and luciferase vector in the rabbit reticulocyte expression system. Th ese wells were free of toxins. The second

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35 row of 3 wells was used as the negative control without DNA vectors added. The third and forth rows of 3 wells were added with a certain amount of a toxin stimulant, either CH or TC, into the protein expression system . Figure 2-8a shows th e response pattern of the IVT sensor array when 25 ng of TC was used whereas the response pattern of the same IVT array for 17 ng of CH is illustrated in Figure 2-8b. Although there is slight difference between two sample repeats for rows 3 and 4 for each toxin, the response pattern is reproducible as exp ected. The significant differe nce in the response patterns between CH and TC clearly indica tes that it is feasible to use IVT sensor array to detect and identify toxins. Although we used Western blot and lumi nescence detection to monitor protein production in this concept demonstration, it sh ould be feasible to use a common method for toxin detection. One of such methods is to use GFP as an indi cator for the detection of protein expression due to its green fluorescence. GFP has been used for visualization, tracking, and quantification of a variety of proteins in cells after they are fused together.P126P An increase of fluorescence signal in an IVT well will indicate the production of GFP or GFP-fused proteins. Quantitative information may be obtained by comparing the fluorescence signals of sample wells and of reference wells, which include both positive and negative controls in the arra y device. Any variation or adverse effects will be cancelled out between control and sample wells. The magnitude of the signal can be correlated to the amount of proteins pr oduced in the device. Indeed, we used a fluorescence spectrometer to confirm the pr oduction of GFP expressed as mentioned above. However, we did not use it for CA T and luciferase because we have not yet expressed them by fusion with GFP.

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36 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1p o s i t i v en e g a t iv eT C T CGFP CAT luciferasesignal (normalized)(a)p o s i t iv e n e g a t iv eC H C HGFP CAT luciferasesignal (normalized)(b) Figure 2-8. The response pattern of the 3x4 IV T sensor array for two toxin simulants, tetracycline (TC, a) and cycloheximide (CH, b). The experiments were carried out in two of the 2x3 well devices in Figure 1. The signals for the positive control were from the first row of 3 wells in the device, in which GFP, CAT, and luciferase were expr essed in their respective expression systems. These wells were free of toxi ns. The signals for the negative control were from the second row of 3 wells in the device, in which the expression vector was not added. The signals for the samples were from the remaining two rows of 3 wells in the device, in which either 17 ng of CH or 25 ng of TC was added into the protein expression system. Rather than using GFP, we may also desi gn the expression vector containing a coding sequence for expressing an additi onal stretch of six histidines (6xHis) at the C-terminal of the protein of interest. Ma ny proteins produced by recombin ant techniques are designed to contain a 6xHis tag, so that they can be purified through intera ctions between 6xHis

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37 tags and Ni-nitrilotriacetate chromatographic columns.P131, 132P Both GFP and CAT proteins produced in this work contain 6xHis tag, even though we did not need the purification step. A variety of biological assays are availa ble for detecting the amount of 6xHis tag fused with a protein. There are many other tags that may be fused with proteins as reviewed in the literature.P131P In addition, luminescence detection can also be used by fusing luciferase with proteins of intere st, as demonstrated in the present report. 2.5 Conclusions A novel concept for toxin detection is pr esented based on toxin’s inhibition of biological protein synthesis— in the step of either DNA transcription or protein translation. We demonstrated the feasibility of the concept by (1) in vitro expression of four proteins either from plasmid or li near template, including wide type green fluorescent protein, chloramphenicol acetyl -transferase, luciferase, and MonsterPTMP GFP; (2) confirming differential inhibitory effect s of two toxin simulants, tetracycline and cycloheximide, on the expression yields of th ese proteins in either prokaryotic or eukaryotic expression system; (3) obtaining unique response pa ttern (or signature) of the 3x4 IVT array device for each toxin simulant.

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38 CHAPTER 3 MICROFLUIDIC REACTORS FOR IN VI TRO PROTEIN SYNT HESIS AND RICIN DETECTIONTP*PT 3.1 Introduction Detection and identification of toxic agen ts are important for medical diagnostics, food/water safety testing, and biological wa rfare defense. Methods to detect them include immunoassay,P134, 135P sensors,P136, 137P mass spectrometry,P138P and genetic analysis.P139142P Nucleic acid-based genetic analysis, e .g., polymerase chain reaction (PCR), involves DNA amplification that offers high sensit ivity and unambiguous identification. However, it is not applicable to toxins th at contain no nucleic acids. One example of such toxins is ricin, which is listed as a Category B bioter rorism agent according to the Centers for Disease Control and PreventionP143P and was used as an agent in the letter sent to US congress in Feb. 2004. The structure, toxicity, mechanism of action, and current detection methods of ricin will be di scussed in section 3.2. Immunoassay is advantageous over other methods due to its high sensitivity. However, it requires an antibody that is specific to the agent of interest and it often involves labor-intensive sample preparation. Therefore, a simple, fa st, and highly sensitiv e detection method is essential. In this chapter, we describe an approa ch that exploits the mechanism—by which ricin causes toxic effects—as the sensing sche me, that is, ricin kills people by blocking protein synthesis in cells of human body. As discussed in ch apter 2, protein synthesis has TP*PT A part of this chapter has been submitted for publicati on in Mei, Q.; Fredrickson, C. K.; Lian, W.; Jin, S.; Fan, Z. H. Anal. Chem. 2006 .[133]

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39 been realized in microwell IVT array. One of the major problems of IVT preformed in this microwell device is its short reaction time, thus significan tly reduces protein expression yield. The problem was addressed by the invention of c ontinuous flow cellfree and continuous exchange cell-free systems, in which nutrients can be refurbished and inhibitory byproducts can be removed (summarized in section 3.2). The array device in this chapter consists of a mechanism for fluid manipulation. As a result, higher protein expression yields can be obtained, leading to larger detection signals (lower detection limit) when ricin is present. More importantly, this detection approach accomplishes signal amplification. For each copy of RNA, thousands of copies of proteins can be produced. The inhibitory effects of ricin on the production of each copy of protein are accumulated, leading to a si gnificantly enhanced detection signal. In addition, ricin detection using th e one-step operation of prot ein expression simplifies the detection procedure compared to ELISA that consists of many steps of applying reagents and washing.P144P The detection can be achieve d in as short as 5 minutes. 3.2 Background 3.2.1 Ricin Ricin, extracted from the seeds of the castor bean plant ( Ricinus communis ), is one of the most toxic and easily produced plant toxins. Ricin cons ists of two chains, A and B, connected by a disulfide bond. The B chain bind s to galactose or N-acetylgalactosamine of glycoproteins and glycolipids on the surface of a eukaryotic cell, stimulating the endocytic uptake of the ricin. The A chain is an enzyme that binds and depurinates a specific adenine of the 28S ribosome RNA (rRNA). The adenine ring of the ribosome becomes sandwiched between two tyrosine rings in the catalytic cleft of the enzyme and is hydrolyzed by the enzyme N-glycosidase action. As a consequence, aminoacyl-tRNA

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40 can no longer bind to trancated 28S rRNA and pr otein synthesis is blocked in the cell, which lead to cell death and tissue image.P145, 146P The target adenine is a specific RNA sequence that contains the unusual tetran ucleotide loop, GAGA. The B chain has two binding sites for galactose, and about 106 ri cin molecules may bind per cell. However, just a single ricin molecule that enters th e cytosol can inactivate over 1,500 ribosomes per minute and kill the cell. P147, 148P Although ricin has been shown to inhibit tumor growth a nd it has been applied for cancer therapy,P149, 150P it is considered for biological weaponization and is listed as a Category B bioterrorism agent due to its ex treme ease of producti on and high toxicity. The lethal dosage of ricin, which is le thal to 50% of the exposed population, is approximately 3-5 g per kilogram of body weight.P151P Since ricin has no selectivity for specific cells, the clinical si gns and symptoms of ricin into xicity depend on the dose and the route of exposure. For example, inhalation causes cough, fever, nausea and pulmonary oedema; oral injection results in vomiting, diarrhea, hydration, low blood pressure, or necrosis of spleen, liver and kidneys.P152P The methods to detect ricin at low concentration levels typically include enzymelinked immunosorbent assay (ELISA)P153P and immunoassay using radioactive labeling.P154P Although offering high sensitivity, they involve many steps that are labor-intensive and time-consuming. For radio-immunoassay, the ha ndling and disposal of radioisotopes are always environmental challenges. Recently, Li gler’s group reported a fluorescence-based multianalyte immunosensor that has a de tection limit of 25 pg/L of ricin.P137, 155P Stine and Pishko exploited specific interacti on between ricin and glycosphingolipids and developed a quartz crystal microbalance sensor with the detection limit of 5000 pg/L of

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41 ricin.P156P Shankar et al. depicted an immunoassaybased magnetoelastic sensor that shows a detection limit of 5 pg/L of ricin.P157P The detection limit and time required for detection of ricin are summarized in Table 3-1. Table 3-1 Summary of methods for detection of ricin. Research groups Methods Lables Limit of Detection Detection time Reference Ramakrishnan Immunoassay P125PI 50 pg/tube 30 h P158P Leith ELISA Avidin/biotin immunoperoxi dase 0.2 pg/L 30 h P159P Narang Immunoassay Cy5 0.1 pg/L 1 h P160P Dill Immunoassay Horseradish Peroxidase 0.3 pg/ L 4 h P161P Rubina Immunoassay Cy3 0.1 pg/ L 30 h P162P Yu Surface plasmon resonance phase sensor No label N/A N/A P163P Shyu Immunoassay Gold particles 50 pg/ L 10 min P164P Shankar Immunoassay Alkaline phosphatase 5 pg/ L 30 h P157P Wadkins Immunoassay Cy5 25 pg/ L 1.5 h P155P Stine Quartz crystal microbalance sensor No label 5000 pg/ L 6 h P156P 3.2.2 Continuous-Flow Cell-Free Protein Expression Systems In an IVT system performed in the fixed volume of a microcentrifuge tube (Figure 3-1a), the main limitation is its short lifetime because the re action terminates as soon as any essential substrate is exhausted, or by-pr oducts accumulate to th e level of inhibition, usually within 60 minutes. As a result, this leads to poor yield of protein. The major breakthrough was achieved by Spirin and coworkers with the invention of the continuous-flow cell-free (CFCF) system , in which the reaction products were continuously removed and the nutrients were continuous supplied.P80P This can be achieved

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42 by a semi-permeable membrane, which maintains the high molecular weight components (ribosome, mRNA, ARSases, etc.) in the react ion chamber and separates it from feeding chamber containing the small molecular components (amino acids, ATP, GTP, etc.). In this case, the nutrients are continuously s upplied into and all th e products including synthesized proteins are removed from the chamber by the forced flow of the feeding solution through an ultrafiltra tion membrane (Figure 3-1b). Figure 3-1. Schematic representations of curre nt formats for IVT system. (a) batch (a microcentrifuge tube); (b) continuous -flow cell-free (CFCF) system; (c) continuous exchange cell-free (CECF) system. The first CFCF system developed by Sp irin was a modified Amicon 8MC mircoultrafiltration chamber.P80P Typically, the volume of the r eaction mixture was 1 mL and the feeding solution was pumped in with a constant flow rate of 1 mL/hour. The ultrafiltration membranes used in their report were Am icon PM-10, PM-30, XM-50, with respective molecular weight cut-offs of 10 KDa, 30KDa, and 50KDa. Both E. coli lysate and wheat germ extract were tested in this reactor for translat ion of various mRNA, resulting in extended reaction time (20-40 hr) of synthesis and a si gnificantly high yield of products. The products are relatively small proteins including MS2 coat proteins (16 KDa), brome mosaic virus coat protein (19kDa) and calcitomin polypeptide (3.4KDa).P80P

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43 Since then, several other groups reprodu ced and optimized the CFCE systems and realized coupled transcriptiontranslation in the CFCF format. For examples, Endo et al. reported dihydrofolate reductase (DHFR) with enzymatic function was synthesized up to 20 hr using the same reactor as Spirin’s.P81P Kigawa improved Spirin’s design by using a HPLC pump instead of peristal tic pump to make better contro l of a constant flow rate.P82P In addition, Kigawa used a coupled E. co li IVT and produced active chloramphenicol acetyltransferase (CAT). Ot her similar reports are summarized in Table 3-2. 3.2.3 Continuous-Exchange Cell-Free System Another simple way to allow continuous supply of nutrients and removal of products is to employ a dialysis bag or a flat dialysis memb rane for the passive exchange of low molecular components, named as continuous-exchanged cell-free (CECF) systemsP86P (Figure 3-1c). Kim reported 1.2 mg of CAT was synthesized in 1 mL dialysis bag and the reaction can extend up to 14 hr.P83P Kigawa developed a high-productive E. coli CECF system and the yield of CAT a nd RAS protein increased to 6 mg/mL over 21 hours of incubation.P90P More recently, Madin presente d a highly productive wheat germ CECF system which can synthesize an amount of 0.5 to 1.5 mg/ml of several proteins, such as DHFR, GFP and luciferase in 24 hr.P165P As an alternative, a hollow fiber reactor with increased filtration area was proposed by Yamamoto.P166P This modified configuration also enabled in situ concentration of extract, thus speeding the reaction rate. The commercial CECF instrument with a programmable control ling temperature, supplemented with well-developed reagent k its, was launched to the market by Roche Applied Science; this is called Rapid Transl ation Systems which use an optimized E. coli lysate or wheat germ extract. The production of various proteins can be synthesized up to

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44 5 mg/mL in 24 hours.P88P Table 3-2 summarized nearly all published data so far reported on CFCF and CECF protein synthesis. Table 3-2 Summary of IVT system. Expression Systems Reaction Systems Protein synthesized Duration hours Yield (mg/mL) Reference E. coli CFCF MS2 coat protein 20 0.1 P80P Wheat germ CFCF BMV coat protein 20 0.18 P80P E. coli CFCF Calcitonin 40 0.06 P80P E. coli CFCF CAT 25 0.1 P167P Rabbit reticulocyte CECF CAT 34 0.1 P87P Wheat germ CFCF DHFR 24 0.2 P87P Wheat germ CFCF DHFR 20 0.917 P81P E. coli CECF CAT 14 1.2 P83P E. coli CECF CAT 15 1.5 P82P E. coli CECF CAT 8 3.5 P168P Wheat germ CECF DHFR 60 4 P165P Wheat germ CECF Luciferase 60 1.1 P165P Wheat germ CECF TMV RNA replicase 72 0.6 P165P E. coli CECF GFP 21 4.4 P169P Wheat germ CECF DHFR 30 0.8 P170P E. coli RTS 500 CAT 24 0.9 P171P E. coli RTS 500 DHFR 24 0.2 P88P E. coli RTS 500 GFP 24 5.9 P172P E. coli RTS 500 Erythropoietin 24 0.3-2.2 P173P Wheat germ RTS 100 GUS 24 0.05 P174P Wheat germ RTS 100 SHR 24 0.794 P174P Wheat germ RTS 100 SRC 24 0.95 P174P Wheat germ RTS 500 SHR 24 1.07 P174P Wheat germ RTS 500 SRC 24 1.34 P174P Although CFCF systems have several obvious advantages (purity and high yield of protein products), the operat ional complexities (pump and valve) limit the potential for practical use. Due to the simplicity and lo w cost, CECF systems became attractive to scientists during the past decade. In addition, CECF systems could be achieved in a miniaturized format and become a powerf ul tool for high-throughput expression of proteins, as discussed in following sections.

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45 3.3 Experimental 3.3.1 Materials and Reagents The RTS 100 wheat germ CECF kit, RTS 500 E. coli kit, and the expression vector containing the gene of green fluorescent protein (GFP) were obtained from Roche Diagnostics GmbH (Mannheim, Germany). T7 luciferase DNA vector , luciferase assay reagent, and nuclease-free water were ac quired from Promega Corporation (Madison, WI), while ricin A chain was purchased from Sigma (St. Louis, MO). Acrylic sheets with thickness of 0.25” (6.3 mm) and 0.10” (2.5 mm) were from Lucite-ES (Lucite International, Inc., Cordova, TN). The dialysis membrane with the molecular weight cutoff of 8 KDa was obtained from Spect rum Labs (Rancho Dominguez, CA) while a biocompatible epoxy (353ND-T) was bought fr om Epoxy Technologies (Billerica, MA). 3.3.2 Device Fabrication The design of the array device is shown in Figure 3-2; it co nsists of two parts. The top part, tray, was fabricated by drilling an arra y of holes in a 2.5 mm-thick acrylic sheet. The diameter of the hole is 3 mm. The pitch (the distance between the holes center) is 9 mm, following the microplate standards de fined by the Society for Biomolecular Screening (SBS) and accepted by the American National Standards Institute. The sheet with holes is further milled from the botto m side to create a flange using a CNC-mill (Flashcut 2100, Menlo Park, CA), resulting in a 1mm-thick wall for the tray chamber. The dialysis membrane was then glued usi ng the epoxy to the bottom of each hole to form the tray chamber. The bottom part, well, was created by milling an array of 4 mm deep wells into a piece of a 6.3 mm-thick acry lic sheet. The diameter of the wells is 7 mm; each well is concentric with the co rresponding tray chamber when they are

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46 assembled. Both tray and well plates were sterilized by exposing to a UV light for 30 minutes. Well tray membrane (a) (b) tray well 9 mm sample positive control negative control Figure 3-2. Schematic representation of the device. (a) Three dimensional view of a miniaturized, 2x3 solution array for rici n detection. The units were laid out according to the standards of 96-well plat es (i.e., 9 mm pitch). (b) The crosssectional view of one unit of the array in (a). 3.3.3 In vitro Protein Expression For the prokaryotic expression system, CF CF GFP synthesis in the 500 E coli kit was carried out using the vector GFP with a concentration of 1 g/L according to the instruction manual. In brief, the reaction solution containe d 0.525 mL E.coli lysate, 0.225 mL reaction mix, 0.27 mL amino acid without methionine and 30 L methionine. The feeding solution consisted of 8.1 mL feeding mix, 2.65 mL amino acid without methionine and 0.3 mL methionine. To run th e protein synthesis in the miniaturized membrane device, the tray was filled with the 8 L of the reaction solution containing GFP vector while 80 L of the feeding solutio n was added to each well in the well plate. A biocompatible sealing tape was used to seal the tray to prevent evaporation. The device was placed on a shaker with a speed 200 rpm an d incubated at room temperature for 4, 6, 10, 14 and 20 hrs. As for comparison, 8 L of reaction solution was dispensed into microcentrifuge tube and incubated the same time period as that of the membrane device.

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47 After GFP synthesis, the reaction tubes were storde at 4 C overnight for the maturation of GFP before analysis and the expressi on yield was quantified by Western blotting.P89P For the eukaryotic expression system, lu ciferase was synthesized using RTS 100 wheat germ expression kit. The reaction solu tion for trays was prep ared by mixing 15 L wheat germ extract, 15 L reaction mix (provided in the kit), 4 L amino acids, 1 L methionine and 15 L of nuclease-free water co ntaining 1 g of luciferase vector. For each tray chamber, 8 L of the reaction solu tion was used. The feeding solution for wells was prepared by combining 900 L feeding mix (provided in the kit), 80 L amino acid and 20 L methionine. In each well, 80 L of the feeding solution was introduced. The tray and well plates were assembled and th en placed on a shaker (at room temperature) for a period of time (e.g., 0.5 hour). The amount of luciferase synthesized was determined by mixing the expression product w ith luciferase assay reagents, followed by luminescence detection in a luminometer (Ber thold, Germany), as described in chapter 2P89P. When luciferase was synthesized in a microcentrifuge tube, the same reaction solution (8 L) was used wit hout the feeding solution. 3.3.4 Ricin Detection A series of concentrations of ricin A solutions, ranging from 0.001 ng/ L to 0.02 ng/ L, were prepared from a stock solution of 1 g/ L. Denature of Ricin A was achieved by heating the ricin A samples at 95 PoPC for 5 minutes. To demonstrate ricin detection, 6 L of the reaction solution (discussed above) is pipeted into the tray, followed by 2 L of ricin samples. The volume of the feeding solution remained at 80 L. For the positive controls in the same device, 2 L of water was added. The negative controls contain no luciferase vector, providing with the ba ckground signal. To achieve

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48 lower detection limit, 4-hr protein expressi on was used, though ricin detection can be achieved in as short as 5 minutes. 3.4 Results and Discussion 3.4.1 Device Design As mentioned above, protein expression can be produced in a cell-free medium employing IVT. It is also well-recognized that high-yield protein expression of IVT can be attained in the flow of a feeding solu tion, but not under static conditions in a fixed volume.P80, 91P As a result, commercial bench-top instruments inco rporate the principle of continuous flow with a magnetic stirrer.P88, 171P However, the bench-top instruments often employs milliliters of reagentsP88P and it is difficult to achieve the high-throughput format as discussed by Angenendt et al.P68P We fabricated an array device consisting of a mechanism for fluid manipulation; it also has a potential to implement protein synthesis in a high-throughput format due to miniaturization. As illustrated in Figure 32, IVT was implemented in an array of units; each unit is for expression of one protein (e.g., lu ciferase). The units on the left of the array are for the positive controls (free of ri cin), the units in the middle for the negative controls (no DNA vectors), and the units on the right are for samples. Two rows of each unit are for repeat experiments to enhance the precision. The positive and negative controls are used for quantific ation as well as for reducing false positives and negatives for toxin detection as discussed later on. Th e solution array is designed to conform with 96-well microplates, ensuring compatibility with a variety of commerc ial fluid dispensing systems and commercial plate readers for detection. Each unit in the device consists of a tr ay and a well (Figure 3-2b). The tray chamber is for the IVT reaction; the well is concentric with th e corresponding tray

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49 chamber and functions as a nutrient reservoir. The well contains amino acids, adenosine triphosphate (ATP), and other reagents. The tr ay contains the cell-fr ee expression mixture extracted from wheat germs, as well as the same reagents in the well. A dialysis membrane is glued to the bottom of the tra y, connecting the tray and well and providing a means to supply nutrients and remove th e reaction byproducts. The incorporation of membrane is criticalP80, 101P because of two facts: (1) the fl ow of a nutrient-feeding solution will lead to higher expression yield compared to static conditions, because protein synthesis will not terminate earlier due to fa st depletion of the energy source (ATP); (2) removal of small molecular byproducts is also critical to high yield ex pression of proteins in a cell-free medium, because possible inhi bition of protein synt hesis by the byproducts (e.g., hydrolysis products of triphosphates) will not take place. Continuous supply of nutrients and rem oval of small molecular byproducts are achieved by osmosis, which results from th e concentration difference of chemicals between two sides of the membrane. In addi tion, the flow to supply fresh solution from the well to the tray is augmented by a hydr ostatic pressure, which is caused by the difference in the solution level between the tray and well. When the well has slightly higher solution level than the tray (~1 mm), the pressure difference resulting from the height difference will drive nutrien ts from the well into the tray. 3.4.2 GFP Expression To demonstrate the protein expression capaci ty of this miniaturized IVT membrane device, we first synthesized two proteins in two types of expression systems and compared the protein yield between microcentr ifuge tube and membrane device. The first protein is wide type GFP, which was synthe sized using commercial E coli system with a GFP coding sequence. GFP product was conf irmed by microplate re ader and Western

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50 blotting. The result of Western blotting is s hown in Figure 3-3a. According to prestained protein markers, the molecular weight of GFP expressed is estimated at ~ 31 KDa. The quantified data as shown in Figure 3-3b indi cated the device design is proper. When IVT was implemented in a microcentrifuge tube, GFP was synthesized in the first 4 hr and then the reactions ceased. In contrast, when it was in the device with continuous feeding of nutrients and removal of byproducts, G FP was continuously produced up to 20 hr. The amount of GFP synthesized in the mini aturized device was about 14 times larger than that with the microcentrifuge tube. The GFP expression also can be quantif ied by fluorescence emission. Excited with 488 nm light, the emission spectrum of synthesized GFP and commercial recombinant GFP are shown in Figure 3-3c, indicating the G FP synthesized in miniaturized device has biological activity. While Western blotting can detect all expressed GFP, no matter what the spatial conformation is, onl y the correctly folded GFP shows a fluorescence signal. As shown in Figure 3-3d, the fluorescence inte nsity increased with the incubation time. After 20 hours incubation, the fluorescence intens ity reached the plateau. Fluorescence is a highly sensitive and widely used analytical method. But there are potential limitations when we try to use the fluorescence intens ity to quantify the GFP expression in the microwell device. First, the long folding tim e of GFP lead slow response time. Even though lots of mutagenesis studies have im proved the folding properties, the slow maturation time is still a big obstacle of prac tical application of G FP. Second, integrating the excitation light source into the microde vice will involve a lot of microfabrication time-consuming and costly steps. Moreover, it is very difficult to completely filter the

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51 excitation light in the chip, which may indu ce high background and greatly decrease the sensitivity of the system. (a) (b) (c) (d) 0 10000 20000 30000 40000 50000 0481216202428 folding time (h)fluorescent intensit y Figure 3-3. A novel microfluidic array device for in vitro GFP synthesis. (a) Western blot analysis of expressed GFP in th e microfluidic array device and microcentrifuge tube; the arrow indicat ed the cell-free expressed GFP. (b) Time courses of the microcentrifuge t ube and microfluidic array device for GFP synthesis in E coli system. (c) Th e emission spectrum of expressed GFP and recombinant GFP (rGFP). (d) Fluor escent intensity of GFP corresponding to the folding time. 3.4.3 Luciferase Expression Because of the limitation of fluoresce nce detection on the chip, we further investigated a low background bioluminence sy stem. A similar result (Figure 3-4) was obtained for another protein, lu ciferase, and the production yi eld increased more than 2.6 M 0 4 20 24 4 6 11420 ( hrs ) 6 10 1

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52 fold in the device than that in a microcentr ifuge tube. In addition, luciferase folding can be processed during the process of translation.P175P These results suggest that we achieved the desired fluid manipulation in the device. Figure 3-4. Production yield of luci ferase as a function of the expression time in the device or in a microcentrifuge tube. 3.4.4 Biological Sign al Amplification As discussed above, ricin causes toxic effects by inactivating ribosomes and inhibiting protein synthesis in biological cells and then le ading to cell death and tissue damage.P145, 146P We exploit its toxicity mechanism as the sensing scheme to detect ricin. This detection method possesses in herent biological signal amplif ication, as illustrated in Figure 3-5. One copy of DNA will be transcri bed into one copy of messenger RNA. However, for each copy of RNA, thousands of copies of proteins can be produced. This is estimated by the amount of DNA vector used and the amount of the corresponding proteins produced in IVT. The inhibitory e ffects of ricin exist on the production of every copy of protein, as illustrated in Figure 3-5. As a result, the detection signal (i.e., the difference between the sample and the positiv e control) is accumulated, leading to an amplified signal.

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53 Figure 3-5. Signal amplification as a result of the inhibitory effects of ricin on the production of every copy of protein. Thousands of copi es of a protein can be expressed from each copy of RNA. This signal amplification is similar, to some degree, to the enzyme-enabled signal amplification in ELISA. Intrinsic to ELISA is the addition of reagents conjugated to enzymes; assays are then quantified by the bui ld-up of colored products after the addition of the corresponding product.P144P The signal amplification results from the enzyme that catalyzes a certain amount of substrates to detectable products. Two widely-used enzymes are horseradish peroxidase and alkaline phosphatase, which transfer ophenylene diamine and p-nitrophenylphosphate , respectively, and generate colored products.P144P Therefore, we expect the detecti on method based on prot ein inhibition has comparable sensitivity with ELISA. 3.3.5 Detection of Ricin by In Vitro Protein Synthesis To demonstrate the detection of ricin, we studied its inhibitory effects on luciferase expression by adding a series of concentrations of ricin into the IVT reactions in the array device. The calibration curve is shown in Figure 3-6 , in which the expression yield of luciferase (indicated by the luminescence) was plotted as a function of ricin concentration. The error bar of each data point indicates the standard deviation that was obtained from three repeat experiments. A lin ear relationship exists from 1 to 20 pg/L. The detection limit is at least 1 pg/L, since the signal-to-noise at this concentration is

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54 larger than 3. We also observed that there wa s a negligible inhibitory effect on luciferase expression when ricin was heat-denatured and its biological activity was disabled (Figure 3-6). In other words, there was no significant difference between the positive controls and the one with denatured ricin. We can in fer from these results that the IVT method can detect the toxic ity level of ricin after physical and/or chemical treatments. Luciferase can be synthesized within a few minutes, as shown in Figure 3-7a, in which the comparison of expression yields, wa s made between the miniaturized device and a microcentrifuge tube. A s hort IVT time is critical for t hose applications that need a quick response. We also confirmed that we we re able to detect ricin within 5 minutes, as shown in Figure 3-7b. 5.0E+06 1.0E+07 1.5E+07 2.0E+07 05101520Luminescence (Arb. Units)Ricin Concentration (pg/ L) ricin heat-denatured ricin Figure 3-6. Calibration curve for ricin detection. Protein expression yield, indicated by luminescence, decreased with the concen tration of ricin (solid circles). However, the expression yield remained the same when the ricin was heat denatured and its toxicity was deactivat ed (open circles). Experiments were carried out in the device. The error bars are the standard deviation obtained from three repeat experiments. Lines ar e the best fit of lin ear regression of the experimental results.

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55 0.0E+00 4.0E+05 8.0E+05 1.2E+06 00.020.040.060.080.1 0E+00 2E+06 4E+06 6E+06 0102030Luminescence (Arb. Units)Time (min.)device tube Luminescence (Arb. Units)Ricin Concentration (ng/ L)ricin heat-denatured ricin (a) (b) Figure 3-7. Synthesis of luciferase and detectio n of ricin in a few mi nutes. (a) Luciferase production as a function of the expression ti me in a miniaturized device or in a microcentrifuge tube. The expression yi eld is indicated by the luminescence. (b) Protein expression yield, indicated by luminescence, decreased with the concentration of ricin when the expressi on time was fixed at 5 minutes (solid circles). However, the expression yield remained the same when the ricin’s toxicity was deactivated by heat denature (open circles). 3.5 Conclusions We have developed a novel miniaturized IVT device consisting of a dialysis membrane, which offers a means to supply nutrients continuously and to remove byproducts of protein synthesis. We demonstr ated the capacity of our device by: (1) In vitro expression of two proteins, GFP and luciferase, (2) ricin detection by the mechanism of protein expression pathway. Protei n expression in our device can sustain a translation reaction as long as 20 h, thereby the expression efficiency is high. Moreover, the miniaturized device has the further adva ntage of reducing the volume of reagents, thus the costs. The miniaturized device a ppears to perform better for ricin detection, offering better sensitivity, larger dynamic ra nge and shorted detection time. The device has potential for high throughput an d parallel toxin detection.

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56 CHAPTER 4 SUMMARY AND FUTURE WORK 4.1 Summary 4.1.1 Device Design and Fabrication Two types of micro-well array device ha ve been developed for parallel protein synthesis and they are discussed in chapter 2 and chapter 3, respectively. A micro-well array device fabricated from one piece of PMMA sheet was presented in chapter 2 for protein synthesis. The majo r shortcoming of the micro-well array device is the limitation of reaction lifetime, thus lead s to a low yield of s ynthesized proteins. Expression of CAT and GFP ceased after 4 h and 1.5 h, respectively To addresses this challenge, a two-piece de vice is designed to consisting of a tray and a well separated by a dialysis membrane and it can exploit was employed continuous exchange cell free (CECF) technology as di scussed in chapter 3. In this device, the reaction can take place under the conditions, in which the reaction byproducts (Pi and NMPs) are removed, and the consumable substrates (amino acids, NTPs, and energyregenerating compounds) are continuously s upplied. In addition, the trays and the wells are arranged in 96-well plate format, which is compatible with a variety of commercial fluid dispensing systems and plate reader s. The two-piece device allows protein expression to continue for up to 20 h w ith maximal protein yields, because the accumulation of inhibitory byproducts or th e exhaustion of substrates is avoided.

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57 4.1.2 Protein Expression Systems As discussed in chapter 2, either the pr okaryotic or the eukaryotic expression systems possess their own advantages. The choice of an expression system is based on the origin and biochemical nature of s ynthesized proteins and their downstream applications. In this thesis, both prokaryotic prot ein expression systems, like RTS 100 E. coli HY kit, and eukaryotic protein expression systems, like RTS 100 wheat germ CECF kit and TNT Quick Coupled Transcri ption/Translation system were tested in miniaturized array devices. Three proteins: CAT, GFP a nd luciferase, were successfully expressed using these expression systems. After expression, the synthesized a protei n was detected and quantified by one or more methods, including Western blotting, fluorescence, or luminescence, according to the property of the protein. CAT and GFP were quantified by western blotting. GFP was also detected by fluorescence whereas lucifera se was detected by luminescence. Western blotting is the most commonlyused method for specific protei n detection. However, it takes long time and it is difficult to be in tegrated with the miniaturized device. Fluorescence detection based on GFP is a good ap proach, since GFP is a relatively small protein and many proteins have been succe ssfully fused with GFP. Compared with fluorescence, luminescence based on lucifera se requires no excita tion light source, simplifying the design and fabrication of the mi niaturized device. However, luciferase is a relatively large protein (62 kD a) and there is no report about luciferase fused protein by now.

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58 4.1.3 Applications of Protein Synthesis Array The miniaturized protein synthesis array wa s demonstrated in this thesis for toxin and drug detection. In chapter 2, tetracycline (TC) and cy cloheximide (CH) were used as toxin simulants to study their inhibito ry effects on protein expressio n. It was confirmed that TC has an inhibitory effect on the expression of GFP and CAT in the prokaryotic expression system, whereas CH has a negligible effect. In contrast, TC has a negligible effect on the production of luciferase in the eukaryotic expression system. In addition, the response pattern of TC and CH in a 3 X 4 array microw ell deive. The results show the potential of toxin detection based on the mechanisms of toxin actions. In chapter 3, the use of the device for ri cin detection was demonstrated. Compared with traditional la bor-intensive and time-consumi ng ELISA method, this detection method is simpler and faster, with the detection limit of 1 pg/L of ricin which is at least comparable to the reported ELISA method a nd at least one order of magnitude more sensitive and faster than most other reported detection methods. Moreover, any bioterrorism agent that inhibits biological pr otein synthesis, in the step of either DNA transcription or protein transl ation, could be poten tially detected by this method, which is extremely important for large scale and ultra sensitive pre-screen of a potential bioterrorism agent and public health threat. 4.2 Future Work As summarized in the previous section, an in vitro protein expression system was successfully established and its potential applicat ions were demonstrated in this thesis. In this section, the possible future work is proposed and discussed.

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59 4.2.1 Device Design and Fabrication As for device fabrication, a range of pl astic materials, such as polycarbonate, PDMS and poly cyclicolefin will be studied. In current device, a dialysis membrane was glued to tray, which may potentially cause several problems: (1) the glue needs to be carefully chosen and tested of biocompatibility; (2) it is difficult to ensu re the uniformity, such as volume of a microwell, contact area of the membrane, or of a different microwell; (3) it may cause leakage; (4) it is not compa tible with automation of devi ce fabrication. New membranes are selected according to the following crit eria: (1) the pore size is large enough for nutrient supplies and byproduct removal a nd small enough to keep the synthesized proteins; (2) the membrane is compatible with the large–scale fabrication method, such as lamination. The pore structures must be ma intained during the sealing. Nanoporous PC membranes with the pore size of 10 nm and 15 nm are under testing. Furthermore, an integrated device can be designed with a positive flow system to enhance mixing effects because the principle of CECF depends on passive diffusion via a dialysis membrane. The relationship between geometric variables (e.g. length and depth of channel), material variables (e.g. fl uid viscosity), opera ting variables (e.g. displacement volume and flow rate) and th e protein synthesis efficiency will be investigated. 4.2.2 Protein Expression System In this thesis, we employed E.coli extract and wheat germ extract as demonstration of prokaryotic and eukaryotic protein expression system. Theo retically, any organism that makes a large amount of proteins can be used for in vitro protein expression, but we will continuously stick with above commercial systems in future work.

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60 However, we will test different DNA te mplates. Successful in vitro protein expression depends on a correct starting DNA template, which consists of a gene encoding a target protein and the necessary regulatory elements including promoter, terminator, ribosome binding site and tags. A lthough GFP, CAT and luciferase have been expressed from corresponding vect ors provided by kit, a variety of different T7 driven plasmids, such as pIVEX, pET, and pEXP fo r in vitro protein expression are available and DNA coding sequence can be inserted in to these plasmids by standard cloning procedures. However, it is necessary to cu lture cell to clone and amplify plasmids. An alternative method is using PCR to generate linear DNA template to avoid timeconsuming and labor-intensive steps of plas mid preparation. The advantages of this method include: (1) easy deletion and inser tion of sequences; (2) easy introduction of point mutations and random mutations; (3) easil y and fast fuse of di fferent proteins. The advantage of this method is that the yield of protein synthesis is lower than using plasmid. The low yield may be caused either by th e fast decay of linear DNA templates by the exonuclease presented in the cell-free extr acts or by the limited accessibility of transcription and translation factors due to the secondary structure in DNA or RNA. In chapter 2, we tried to use a PCR amp lified template for protein expression, but failed to obtain detectable amount of protein. We susp ect the sequence of mRNA preceding the translation factor binding site may form the secondary structure and the bind sites are embedded. New sets of primer s with much shorter preceding sequence are being designed and we are going to test their performance. 4.2.3 Applications In this thesis, the feasibility of using in vitro protein expression system for detecting inhibitory effects of different toxin simulants/dr ugs was demonstrated in the

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61 proof-ofconcept experiments. Other toxin simu lators and/or drugs will be tested and the response pattern (or signature) database of the array device will be constructed, which can be used as a tool for detection and identification of known and unknown toxins. Other potential applications of in vitro protein expression arra y, which is depend on the availability of supporti ng funding, include (1) high th roughput, cloning independent screen of mutant and engineer ed proteins; (2) construction of the protein library from cDNA library for functional genomic and proteomic research. Inspired by Feynman’s talk “There is plen ty of room at the bottom— an invitation to enter a new field of physics” , I accomplish this research. I would like to end this thesis with an invitation, “there is plenty of ch emistry and biology in miniaturized devices”. The door is open; scientists and engineers from different disciplinary are welcome to explore such a marvelous small world.

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72 BIOGRAPHICAL SKETCH Qian Mei was born in China, in 1977. She was raised in Luoyang, a beautiful city in central China, known as the “city of pe onies.” In 1996, she entered Southeast University in Nanjing, China, and earned her Bachelor of Science degree in biomedical engineering four years later. Then she proc eeded to her graduate studies in the same University and in 2003 she received a Master of Science in biomedical engineering. In the same year, she enrolled in biomedical engineering at the Health Science Center in University of Tennessee at Memphis. In th e spring of 2004, she relocated to Florida and started her graduate study in mechanical and aerospace engineering in University of Florida. Upon the completion of master’s degree, she will keep on pursuing her Ph.D. degree in the same program.