High Throughput Bio-Printing with Individualized Piezoelectric Ejectors

Bio-Printing and Bio-Patterning WorkshopSeptember 27-28, 2004, Manchester, UK.

Huangpin Ben Hsieh

Palo Alto Research Center 3333 Coyote Hill Rd.

Palo Alto, CA 94304, USAben@parc.com

Commercial Impact at PARC• Inventions in nearly every Xerox product

– Laser Printing, Laser Diodes, Dual-beam Laser Diodes– DocuPrint NPS (1993-current)– Web XPress services infrastructure – delivered via cross-organizational collaboration– Color RIP for DocuColor 60– Scheduling software for IGen3

• Seeded the Valley– 8 companies based on early PARC research: 3Com, Adobe, VLSI, and KOMAG

• 19 Spin-offs since 1980– 1983: Spectra Diode Laboratories, Inc. – acquired by JDS Uniphase in $41 Billion merger– 1985: Synoptics Communications, Inc. – became Bay Networks, acquired by Nortel in 1998– 1990: Documentum – 2000 Revenue: $197 Million, $829M Market Cap– 1996: Inxight – Information Visualization, closed $29 Million in financing in 2001– 1996: Placeware – leader in web conferencing industry, $200M purchase by Microsoft, 2003– 2000: ContentGuard – digital property rights management, purchased by Microsoft.– 2001: Gyricon Media Inc. – Macy’s and Dow Jones as lead customers for retail signage

• Licensing– Dragon collaboration with Sun Microsystems on SPARC Station's multi-processor

architecture– 3D Visualization techniques licensed to Microsoft Corporation

Biomedical Sciences at PARC

• Leverage PARC technical competencies– Ultra Small Drop Control– MEMS / Electronics / Applied Physics– Information Sciences

• Partner for life science competencies– Scripps Research Institute domain knowledge and

industry contacts– Scripps-PARC Institute for Advanced Biomedical

Science www.scrippsparc.com– Collaborate in securing external funding– Attack big problems

Biomedical Science Efforts

• Bio-Printing1: Individualized Piezoelectric Ejectors – Low cost, high volume DNA arrays

• Universal Assay2: Enthalpy Array– High throughput screening without assay development

• Rare Cell3: Fiber Array Scanning Technology– Early stage cancer detection and fetal analysis

• Image Analysis4

– Protein crystal ID– CryoEM Sample ID

• Mass Spec Data Analysis5

• Bio-Agent Concentration & Detection6

1. Hsieh et al., Ultra High Throughput Microarray Generation and Liquid Dispensing Using Multiple Disposable Piezoelectric Ejectors, Journal of Biomolecular Screening, V9 (2): 85-94, 2004.

2. Torres et al., Enthalpy Arrays, Proceedings of the National Academy of Sciences, 101 (26): 9517-9522, 2004. 3. Krivacic et al., A rare-cell detector for cancer, 101(29): 10501–10504, 20044. Bern et al. Automatic classification of protein crystallization images using a curve tracking algorithm. Journal of Applied Crystallography. 2004 April;

37 (2): 279-287 5. Bern et al., Automatic quality assessment of peptide tandem mass spectra Automatic quality assessment of peptide tandem mass spectra. Twelfth

International Conference on Intelligent Systems for Molecular Biology (ISMB 2004); 2004 July 31 - August 4; Glasgow; Scotland. (to be published in special issue of Bioinformatics).

6. ARO-JSAWM contracts: DAAD19-03-0116, W911NF-04-C-0034

Printing Technologies

Thermal Isolation

Acoustic Single Ejector

Acoustic Multi-Ejector Printhead

Piezo Single Ejector

Piezo Multi-Ejector Printhead (OPB/Tektronix)

Ballistic Aerosol Marking

• Micro-pipetting• Printed organic electronics• Drug inhaler

• Photofinishing• LCD color filter deposition• Printing on pills

• Biological micro-arrays• Printed organic electronics• Micro-pipetting

• Office documents with solid ink• Printed organic electronics• LCD color filter deposition• Decorative & packaging printing• Printing on pills

• Powder deposition/processing

Piezoelectric Single Ejector

• Solve the plumbing problem of traditional inkjet bioprinter• Eliminate cross-contamination• Break the practical limit of number of print heads • Provide low cost, high throughput solutions

EjectorDesign Assembly13 layer

piezo element

Fluid Core of Single Ejector

Aperture=76µmMain Reservoir

Piezo Diaphragm

Fill Hole

Compression Chamber

Purge HolePurge

Reservoir

Refill Ducts Behave Like Check Valves

Reservoir Volume=76µl

4mm

8mm

PARC’s Piezoelectric Ejector Technology

• Printing system with multiple fluids printed simultaneously– Individual piezo ejector for each fluid – Many piezo ejectors or fluids– All stainless steel construction– Non-contact– High fluid utilization– Reconfigurable– Versatile drop control (5pl – 300pl)– Prototype system demonstrated

Initial printing system:– X-Y scanning– Inspections– Building block for larger systems

Bioprinter Prototype

• PARC’s deep printing understanding maps well to bio-fluidics: – Manipulating drops & images– Manufacturing & modeling piezo

ejectors– Precision motion & sensing

systems

Reliable Droplet Ejection

-0.1%

0.0%

0.1%

800

4000

0

800

4000

0

4000

0

4000

0

4000

0

4000

0

4000

0

4000

0

4000

0

4000

0

4000

0

Drops Fired per salvo

% E

rror

- D

rops

Cou

nted

vs

Dro

ps E

xpec

ted

• Counted ≈ 4M drops through 8 apertures• Videos of drop ejection before and after a 4M drop run

Reliability – Idling in Ambient

100 µm

Initial State(surface is clean except for

2 stains from filling)

Ejection after sitting idle at ambient RH for 4 hours:• RH = 19.2% at 23°C• BSA/CAPS deposits formed on ejector surface but do not appear

to interfere with drop ejection• No appearance of drop abnormalities

Features Printed after sitting idle at ambient RH for 4 hours:

(Drops per feature varies from 1000 to 10 drops)

Reliability-Idling in Humidity

100 µm 100 µm 100 µm

Initial State Ejection after sitting idle with RH control for 3 hours

Ejection after sitting idle with RH control for an additional 14 hours

• BSA/CAPS nominal solution

Results:• Surface around aperture remains clean• Drop trajectory remains the same• No appearance of drop abnormalities

Drop Size = 73 umy = 76.076Ln(x) - 13.297

Drop Size = 59 umy = 51.644Ln(x) + 9.832

0

100

200

300

400

500

600

1 10 100 1000

Drop Count (qty)

Feat

ure

Dia

met

er (u

m)

Single Ejector #1610Three strips measured.BSA w ith 0.01 % dye.No Motion Very circular features.

Feature Size With Varying Droplet Qty.

Feature Size Comparison Before and After Hybridization

Bio Materials Successfully Printed• Genomic DNA solution (100ng/uL)

– viscous, long DNA fiber– PCR verified printed DNA

• Aqueous DMSO (5- 50%)– low surface tension– at 40C, 220C, 370C

• Oligonucleotide solutions– arrays hybridized successfully

• BSA and Human IgG protein (1mg/ml)• Human cell line TIB180 (10µm)

– at 40C, 220C, 370C– cell settlement occurred over time– agitation (ultrasound) could reduce settlement

Bio-Array Printing Applications

• Clinical– high volume, low cost: $10 – medium density: 100 to 1000

features– diagnostic, metabolic, reproductive,

forensic

• Research– low volume, high cost: $100 to $500– high density: 1000 to 10,000

features– population arrays, drug discovery,

customized

XY Platen, Dual Speed, 500 um Feature Pitch,

27x46mm Strip

1,6001,7001,800

1,9002,0002,1002,200

2,3002,400

0 5 10 15 20 25 30 35Droplets per Feature

Arr

ays

per 8

Hr S

hift 96 features per strip

384

1536

Web Fed, Dual Speed, 500 um Feature Pitch,

27x46mm Strip

40,00050,00060,00070,00080,00090,000

100,000110,000120,000

0 5 10 15 20 25 30 35

Arr

ays

per 8

Hr S

hift

96 features per strip

384

1536

Strip or Array Production

(from 2002 literature)

Future Work

• “Image” (feature) quality & reliability– In many ways, conventional images demand more accurate

printing than bio applications.– Orderly, consistent bioarrays are within reach, but for large

arrays and lots of ejectors, concerns are:• mechanical issues like alignment• set up time• droplet trajectory changes due to meniscus changes

• Automatic filling station for precious fluids• Inexpensive ejectors: injection molding• Extend ejector performance• Materials studies• System architecture studies

Injection Molded Plastic Ejector

96 Ejector Array

XY Platen, Manual Fill

XY Platen, Auto Fill XY Multi Platen,

Auto Fill, Auto Tune Web, Manual Fill

Web, Auto Fill, Auto Tune

Web, Auto Fill, Auto Tune, PreRacked

0

384

768

1152

1536

10 100 1000 10000 100000

Arrays Produced Per Shift

Uni

que

Flui

ds P

er A

rray

System Architectures

Potential Applications

• Bio-defense– printing small arrays in large quantity– example arrays include bio-agents such as microbial

genomes, toxin Abs, viruses, or chemical arrays– inexpensive

• On-demand printing and dispensing of biomaterials– library of bio-molecules stored in ejectors – computerized, automated pick-and-print

• Handheld nano-liter dispenser• Protein arrays for research and diagnostics• Tissue, organ, or live cell printing

…Continuous Innovation

Thank you! Questions?

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