BioModular Multi-Scale Systems

1. BioModular Multi-Scale Systems Goal - Design, Model, Fabricate and Evaluate a universal Molecular Processing System to identify a variety of targets for Discovery, Forensics, Homeland Security and Clinical applications

2. Molecular Analysis • In vitro Diagnostics • $2.5T healthcare market, 70% of clinical decisions based on IVD • Homeland Security • Growing market • Forensics • 350,000 case backlog • $2.0B market • Types of Markers • DNA (nuclear, mt) • RNA (messenger, gene activity) • Proteins (over/under-expression) • Challenge • Each marker requires a different assay and hardware platform to analyze • The processing time is typically > 1 day, requiring highly trained operators • Highly expensive ($3,000 per test for BRCA I/II genes)

3. IVD and Cancer • Global cancer cases:12M/yr with7.6M deaths/yr • Total cost of cancer care in US is $171B/yr with productivity losses of ~$1 trillion/yr • Current IVD market is $7B and projected to be $14B by 2013 • IVD potential has not been realized in cancer clinical care area due to; • Lack of “standardized” protocols for generating clinical data from biomarkers • Highly sophisticated equipment and expertise required to implement many existing assays MISSION: Develop a standard assay protocol and instrument platform to provide automated & low-cost target identification.

4. 3’ 5’ G C T A C G T A C T A C C 5’ C C G A T A A A C G T T T A T G G G C 1 2 3 4 5 6 7 8 9 Base calling TGCTACGAT … Next Generation Sequencing(Sequencing-by-Synthesis, SbS) DNA(0.1-1.0 ug) Cluster growth Sample preparation Sequencing Genome Analyzer Image acquisition Cluster Station • Prep gDNA – 6 h • 5 h cluster preparation • 3 d single read run (70 bp reads) • 2b bases per day

5. Single Molecule Sequencing Conformal Seal • 1.1 kbp dsDNA • Gap = 9 nm • Ti/Au nanoelectrodes • Fused quartz substrate • Sputter deposition of metal off-normal Chou, Nano Lett. 8(2008) 1472.

6. To Micro-scale contacts NIL Prepared Substrate Nanowires (d = 5 – 10 nm) Proposed Technology Polymer-based Nanosensor Bioreactor V1 V2 = Enzyme V1 E Nanofluidic Via (5 – 10 nm) Nanopillar Support To Micro-scale contacts V2 E E Enzyme Tether E ssDNA E dsDNA E E Expelled Bases Nanosensor d and ∆t dAMP λ-Exonuclease Length Sensing dTMP Entropic Trap dsDNA

7. Nucleotide ID using Nano-Scale Time-of-Flight Time I C Electrode 1 T ∆t = Flight Time Electrode 2 G A A Histogram C G Frequency ∆t ∆t ∆t ∆t T Threshold Level ∆t (time units)

8. Nanosensor Arrays for DNA Sequencing • Does not require cloning, amplification (PCR, SDA, Cluster), gel electrophoresis or fluorescence • Shear genomic DNA (tight and controllable size distribution) • Hybridize to capture probe • Exonuclease digestion (sequencing, only 1 enzymatic reaction) • Can be done for proteins as well! (proteolytic digestion, PMF) • Can accept any sample and prepare templates for sequencing • Isolate target cells (can sequence single cells with no amplification) and SPE • Prepared via mixed-scale replication technologies with simple modification chemistries (high-scale production at low-cost) • Modular design approach • Polymers used as substrate materials for chips • Can sequence fragments to 50 kbp (or larger) • Simplifies assembly for whole genome sequencing projects • Can re-sequence selective genes for diagnostics (haplotyping) • Throughput and Cost analysis • 1,000 nanosensors per chip • 1,000 bp per second (clipping rate of exonuclease) • 8.6 x 109 per day of raw sequence information (~3-fold redundancy for Human Genome) • COST per genome – <$1,000

9. Modular System for Complete Sample Processing and Sequencing • Process any clinical sample; blood, tissue, urine, saliva, etc. • Universal detector – process any sequence variation • Directly sequence 50 kbp fragments • Sequence one genome in <1 day • Cost for whole genome analysis <$500 Pt Wires

10. Center Organization Council of Deans, VC of Research Director Soper Deputy Directors Rusch Podlaha-Murphy Murphy External Evaluation Team Industrial Advisory Board Administrative Assistants Scientific Advisory Board Cain Center Research Thrust Areas Un./Pre-college Ed. – Pang/Nixon ICI – TBN, Weaver TA2 Functional Materials (McCarley) TA1 Molecular Assays (MaGee) TA4 System Engineering (Murphy) TA3: Multi-Scale Molding (Kazmer)

11. Production Methods Top => Down Bottom => Up Atoms/molecules Bulk domain Atomic structures Micro-/nano- structures Functional System: Mixed-ScaleAssemblies with Top-down & Bottom-up

12. Lab prototypes usually rely on bottom-up techniques, which are not feasible for mass production Layers & components must be redesigned for top-downmanufacturing & assembly Issues: replication, handling, alignment, bonding, … Design forNano-Manufacturing

13. Multi-Station Molding • Layers with different length-scale features will be produced, aligned, and assembled in a multi-station mold: • Internal alignment is grossly setby the tooling and fine-tuned by smart material actuators • Stations includetheir own heaters, actuators, &sensors toperformprocess stages

14. UV & thermal imprint lithography is the process of choice: Provides excellent replication Relatively fast process Amenable to multi-station design Imprint, surface treatment, imprint, bond, … Processing history is vital to layer replication & end-use performance Each processing step provides initial & boundary conditions for subsequent step(s) Residual stresses & layer properties will dictate relaxation, dimensions, and consistency Multi-Layer Processing

15. Simulation aids development of machine design, process settings, and control strategies Simulation of large area, multi-scale molding is daunting An integrated simulation can model the viscoelastic flow and thermal/structural response of multilayer nano-composites across all process stepswith a singleconstitutive model Simulation Multi-Point ImmersedBoundaryMethod(MPIBM)

16. Researchers suggestcontinuum models areinappropriate at nano-scale This is not settled… McKenna, Science, 2005: Continuum Modeling McKenna, 2005 (27 nm PVAc) Plazek, 1980 (2 mm PVAc)

17. Multi-domain characterization Glassy state: DMA Melt state: cone & plate rheometer New constitutive models needed Constitutive Modeling Materialmodelspans15 orders ofmagnitude:from 1 ns to 1 month WLF

18. Multi-Scale Replication:Combinatorial Toolset

19. Resource Capabilities – Patterning Clean room @ LSU EBL @ PSI X-ray Source @ LSU Mask Aligner@ Paul Scherrer Institute (PSI)

20. Resource Capabilities – Molding Blow Molding @ UML Simulations @ UML Injection Molding @ LSU & UML Imprinting @ UML

21. Fabricating Vias in Silicon Stamps at 5-25 nm 25 nm wide

22. Multi-Scale Replication:Surface Modification • Surface chemical and mechanical modification can improve polymer flow, replication fidelity, insert life time, and part demolding • Optimization of coating chemistry, internal structure, and thickness • Different coating methods (i.e., thermal and plasma chemical vapor deposition) and materials (i.e., metal carbides and nitrides) for different mold inserts/polymer substrates • Use of polymer and organic/inorganic composite mold inserts

23. Nanochannelsin PMMA by UVNIL

24. Multilayer Process Testbed

25. Homemade Machine

26. Universal Molecular Processing System sought for in-vitro diagnostics, security, and forensics applications Moore’s law, coupled with advances in top-down nano-manufacturing will enable such devices Significant research barriers must be overcome: Bioreactors for controlled monomer generation Top down manufacturing of mixed scale features Assembly of multilayer composite systems Design methodologies for very large scale integration of mechatronics Conclusions