HP Automatic Liquid Handling (ALH) Hand Pipetted MethodTakes 20 minutes per assay Takes 3 hours by a skilled worker16 pL accuracy 1 µl accuracy (1,000,000 pL)Can be easily adjusted if a mistake is made Mistakes cause large error in data8 orders of magnitude in concentration

3 orders of magnitude in concentration

96 to 384 data points 12 to 30 data pointsHigh resolution in the critical test region Few data points in critical test region. Low error High error

Hewlett-Packard 2010

Hewlett-Packard 2010

SERIAL DILUTIONS WITH AUTOMATED LIQUID HANDLING

[Wortmannin] (nM)

0.01 0.1 1 10 100 1000 10000 100000

RFU

/sec

0

0.2

0.4

0.6

0.8

1

1.2Parameter Value Std. Error

Ki 0.07 0.01

Enzyme conc 4.70 0.07

Background 1.07 2.91e-003

Max Inhibition -1.01 3.45e-003

[Wortmannin] (nM)

1 10 100

Rat

e of

AD

P pr

oduc

tion

(M/s

ec)

-5e-10

0

5e-10

1e-9

1.5e-9

2e-9

2.5e-9

3e-9

Data provided by Hewlett-Packard 2010

2009 Daniele Focosi

ACKNOWLEDGEMENTSOregon State University Chemical Engineering Department

Dr. Philip Harding: ChE 416 Instructor Hewlett Packard: Sponsor Company

Heather Paris: Project MentorMichael Day: Lab AdvisorKen Duda: Equipment DesignerKen Ward: Project Lead

Dispense Effects Using Thermal Inkjet for PharmaceuticalsKayla Pierson, Benjamin Huntington, Paul Dornath

Oregon State University, School of Chemical Biological and Environmental EngineeringSponsor: Hewlett Packard Automatic Liquid Handling Project. Mentor: Heather Paris

Critical RegionCritical Region

Figure 1: Cell assay performed with HP ALH tool. There are many useful data points and the critical region of interest is well defined. The error is greatly reduced (Hewlett Packard 2010)

Figure 2: Cell assay performed by conventional hand dilution method. There are fewer data points with a smaller range of concentrations. The sparsely spaced data causes large error. (Hewlett Packard 2010)

We will characterize jittering polystyrene 384-well plates (3.63 mm diameter)

Use a dispense volume of 0.5 µL of DMSO into 50 µL of buffer

Design a model of the mixing and develop physical understanding

Jittering refers to the shaking of the well-plates in order to induce mixing

Inkjet dispense drops do not have enough momentum to mix

DMSO falls to the bottom of the well, which can cause cell death

Jittering will be implemented to increase convective mass transfer

OUR TEAM’S TASK JITTERING

THEORY

2aLBo

The bond number is a comparison of body forces to surface tension forces on a fluid and is given by Equation 1.

Where a is acceleration, L is the width of the cell, γ is the surface tension, and ρ is the density. The bond number for a 386 well is low meaning that surface tension forces are dominant

(1)

Figure 7: Time lapse of a DMSO and blue dye dispense into buffer filled wells with inkjet tip. This time lapse was taken over one and a half minutes. This sample was not jittered

Inkjet Dispense Head

Well (from 384 Well-Plate)

Buffer

Initially the drop sits on the top layer

DMSO wets the inside of the well

Convective mixing

Figure 8: Jitter while dispensing trial. DMSO is mixed much faster than when it is not jittered. The jitter while dispense lasts 1 second. This photo was taken over 5 seconds

Initially the drop sits on the top layer

Jittering while dispense causes DMSO to mix before it has time to fall.

Tool ALH 384Plate reader

max setting 384 ALH 96 ALH 1536Bond Number 0.232 0.654 0.864 0.052

Incorporate jitter while dispensing controls into the software to allow for specific durations and amplitudes

Install motors with 2-3 times greater acceleration for jittering platform Adjustable frequency and amplitude will allow for wider range of Bond

numbers and accelerations Best jittering results in plate reader are 1 mm diameter at 1500 rpm for 60

seconds Best jittering results for Beta 2 tool jitter while dispense, jitter after for 2

minutes with amplitude of 0.25 mm. Mixing 1536 well-plates seems infeasible with ALH (Bond number ~ 0.058)

RECOMMENDATIONS

METHODS AND RESULTS

Figure 3: Wells that are not jittered are mixed poorly and DMSO sinks to the bottom. This generates a visible color gradient showing that wells are unmixed.

Figure 4: Wells shown were jittered for 60 seconds at 1500 RPM and 1 mm amplitude. Wells are mixed and no color gradient is seen in the wells.

Blue dye was added to the DMSO being dispensed. A visible color gradient indicates poor mixing, Figure 3, and no color

gradient indicates good mixing, Figure 4. After dispense and jittering, one well would be pipette mixed and

shown to another group member who would have to try to determine which well had been mixed

Wells were mixed if the person could not see any difference

No jittering parameters were found to work on the ALH tool Testing continued on a plate shaker with greater jittering capability Working parameters were determined (see Figure 5) Bond numbers were calculated and plotted for plate shaker settings

(see Figure 6)

Speed too low

Amplitude too small

Figure 5: Test parameters for the plate shaker, speed and amplitude, are plotted as y and x axis, respectively. Data points represent either a mixed results, blue, or an unmixed result, red. High speeds and amplitudes correspond with high accelerations and mixing.

Figure 6: Each point plotted represents an experiment done with parameters given on the x and y axis. High bond numbers and accelerations correlate with mixing. A transition region is seen with bond numbers in the range of 0.4 to 0.5 where mixing is intermittent.

MIXING IMAGES

Well-Mixed Not Well-Mixed