Polymer Discovery Via Microfluidic Enzymatic Synthesis Prof. Peter Y. Wong Prof. David Kaplan Tufts University - Medford, MA October 3, 2006
Biochemical Science Synthesis 1990- Polymer science 1970- Enzyme Microsystems Technology Mechanical Electronics Micro-fabrication Engineering Overview • Markets • Needs • Problems • Solution • Team • Next Steps • Summary
Many Markets • Any market that benefits from new biochemicals • Improved Foods • Additives, Modification, Nutrition • Green Chemistry • Agricultural, Packaging, Analysis • New Medicines • Topical, Digested, Structural
Markets Needs vs.Wants • New products • Better • Faster • Cheaper • Differentiated • Macro and Micromolecules needed • New material • New processes • Focus on new polymers and processes
Problems and Risks • Current Polymer Discovery Process • Long time with process and people • High costs and large resources needed • FDA, EPA stringent regulations • Limited research to commercialization • Alternate Approaches • Nanoscience/technologies - far in future? • Biomimetics/Bioinspiration - hit or miss? • Microengineering/fluidics - scalability?
Our Solution • Achieve “Green ( ) Polymer Chemistry” through • Enzymatic Synthesis and • Microfluidics • Enzymatic polymerizations can produce products • via mild reaction conditions w/o toxic reagents • in an environmentally friendly synthetic process • that can be scaled from microscale to macroscale • Target macromolecules include • polysaccharides, polyesters, polycarbonates, poly(amino acid)s, polyaromatics, and/or vinyl polymers.
Monomer Enzyme 1 Natural antioxidant Enzyme 2 Antioxidant Polymer Microfluidic Enzymatic Cascade • Universal Lab-On-Chip is very far away • Application Specific Integrated Microfluidic (ASIM) device • Example ASIM – • produce vitamin C enriched polymers (PMMA) polymer • has both scientific and market value.
PMMA Polymer • Disruptive Technology in Packaging • Vitamin C enriched polymers can replace butylated hydroxy anisole (BRA) and butylated hydroxy toluene (BHT) - FDA limits conc. To 0.02%. • New Topical Medicine • Antioxidants are considered important in reducing aging-related phenomena by providing protection against free radicals. • Nutraceutical Supplementation • Ascorbic acid may have an overall positive impact on public health because humans lack the ability to synthesize vitamin C
ASIM • Goals: • Two enzymatic cascade reactions to produce PMMA • low cost devices made of poly(dimethylsiloxane) (PDMS) • efficient method to optimize process with external controls
Translation from Abstract to Hardware ascorbic acid monomer AA-Monomer AA- Ascorbic Acid MMA- Methyl Methacrylate PMMA- Poly (Methyl Methacrylate) P-AA-MMA – Ploy L-Ascorbic Methyl Methacrylate HRP – Horse Radish Peroxidase lipase HRP : hydrogen peroxide ascorbic acid Antioxidant polymer monomer : : Input Input : hydrogen Input Input ascorbic acid ascorbic acid HRP peroxide #1 #2 : : : : Input Input Check Valve Output Output Reaction Vessel #2 Reaction Vessel #1 Reaction Vessel #1 monomer polymer in solvent in solvent AA-Monomer P-AA-MMA AA-Monomer React with lipase React with HRP Hydrogen peroxide : : unreacted unreacted Output Output : : unreacted unreacted Output Output lipase hydrogen peroxide hydrogen peroxide ascorbic acid ascorbic acid
Improved Version 1 Stage I Enzymatic Transesterification Synthesis L-Ascrbyl Methyl methacrylate Reaction vessel 1 50C<reaction temp <60C, 45 min.<reaction time<60 min. Flow rate<0.01 ml/min. 2 Stage II HRP Polymerization L-Ascrbyl Methylmethacrylate Reaction vessel 2 60 min.<reaction time<90 min. 20 min.<shaking time<30 min. Flow rate<0.01 ml/min. Function driven Step Material Quantity used Step Material Quantity used 2,6-di-tert-butyl-4-methylphenol, Dioxane. Functional Substrate (G1.1) L-ascorbic acid (AA)+50% Diox. 150mg, 0.852 mM Mix1 (G2.1) w/G2.2 L-Ascrbyl methylmethacrylate ~0.02 g 0.082 mM Ascorbic acid, Dioxane 1st Vessel >50C 2,2,2-trifluoroethyl methacrylate 0.182 mL, 1.278 mM Tetrahydrofuran(solvent) (THF) N2 flushed 0.11 ml Initiator A B 2nd Vessel HRP 1.6mg/ 0.05ml C TFM, Diox. Lipase, 1.5mlx2 anhydrous Dioxane Dissolve (G2.2) water 2 ul D HRP,THF Enzyme (G1.2) Antarctica lipase (free)+ 40% Diox. 12.5mg Hydrogen Peroxide 9.3ul Hydrogen peroxide E AA_PMMA, /PMMA/ 2.5mg Anti-poly 60C (G1.3) 2,6-di-tert-butyl-4-methylphenol +10% Diox. Mix 2 2 hours (G2.3) Shaking 1 hr 2,4-pentanedione (trigger) 1.77ul Vessel 1 L-Ascrbyl Methyl methacrylate (AA-MMA) (G 1) Vessel 2 Poly L-Ascorbyl Methyl methacrylate (P-AA-MMA) (G.2)
ASIM manufacturing • DRIE Si wafer • PDMS Casting • Thermal Plasma Bonding to glass slide • Embed fluid connectors PDMS on SI PDMS on Glass slide
Pneumatic controlling Micrometer Sample loading Syringes External Hardware
Repeat unit Signal strength Macro Molecular weight/ charge Repeat unit Micro Chemical Analysis • Macro vs. Micro comparison with MALDI-TOF • Need purification but polymer exists
Team • David Kaplan - expertise in enzymatic reactions • Peter Wong - expertise in microfluidics • Jin Zou - PhD graduate in Mechanical Engineering • Martin Son - Tufts Technology Transfer Office • Tufts Capabilities: • Enzymatic synthesis research, development, and production • ASIM - Microfluidic design, analysis, and fabrication • Polymer discovery program – design of experiments and testing
Next Steps • Identify 2 to 3 market products to tackle • 2 months • Initial description of enzymatic synthesis process • 2 months • Convert preliminary patent application to full application with these examples of synthesis • 1 month • Develop next generation of ASIM devices for those specific market products • 6 months • Develop new polymer products • 6 months • Partner with companies to develop new polymers for their markets
Summary • Food/Medicine/Biochem Markets need advantages of new polymers • Microfluidic Enzymatic Synthesis • Make custom polymers • Faster, cheaper discovery • Scalable to mass production • Need partners and funding to • do market analysis, • help secure IP, • develop small number of prototypes, and • expand to market • Contact Martin.Son@tufts.edu