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A Reconfigurable Pipette for Customized, Cost-Effective Liquid Handling Daniel J Wilson, and Charles R. Mace Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02556 • Publication Date (Web): 27 Jul 2017 Downloaded from http://pubs.acs.org on July 29, 2017

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Analytical Chemistry

A Reconfigurable Pipette for Customized, Cost-Effective Liquid Handling

Daniel J. Wilson and Charles R. Mace*

Department of Chemistry, Tufts University, 62 Talbot Ave., Medford, MA 02155

* corresponding author email: [email protected]

ACS Paragon Plus Environment


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Abstract We have developed a multichannel air displacement pipette with reconfigurable channels for non-standard liquid handling applications. While linear multichannel pipettes enable many established assays, they do not support analytical tools with customized liquid holding geometries, specifically paper-based microfluidic devices. Using our pipette, complex paper-based microfluidic devices can be fabricated without requiring multiple, time-consuming motions with a single-channel pipette or device designs limited to the configurations of traditional multichannel pipettes. We created this tool by modifying a commercial 8-channel pipette using machined and 3D-printed components. We demonstrate the quantitative capabilities of our tool by comparing its performance to that of a calibrated, single-channel pipette in volume delivery experiments. Our reconfigurable pipette supports the advancement of custom analytical tools with non-standard liquid handling requirements and provides an ergonomic alternative to commercial equipment for developers of paper-based devices.


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Introduction The air displacement pipette, one of many volumetric tools that enable the quantitative transfer of liquids between different containers,1,2 is used universally in chemical and biological laboratories for handling low viscosity solutions.3 Depending on the application, these pipettes can be purchased as fixed or variable volume tools, and in single-channel or multichannel configurations. The linear configurations of multichannel pipettes are based on standard geometries of industrial liquid handling equipment, which have not changed in design for decades.4,5 These designs are standardized, and must meet specific requirements describing dimensions for the overall footprint of microwell plates,6,7 and the heights,8 position,9 and elevation of individual wells.10 More recently developed multichannel pipettes allow the user to adjust the spacing between tips by employing sliding11 or cranking12 mechanical components. These tools are useful for moving solutions between containers of different geometries (e.g., moving from microcentrifuge tubes to a microwell plate), but are more expensive and less commonly used than fixed-geometry multichannel pipettes. While adjustable-spacing multichannel pipettes offer spatial control in one dimension, they do not permit simultaneous introduction of liquids to multiple zones in a nonlinear or arrayed configuration. Operators are therefore required to perform numerous, repeated motions to conduct assays. For example, a single enzyme-linked immunosorbent assay (ELISA) can require tens to hundreds of pipetting steps over the course of a protocol.13 As a result, there are substantial health and safety risks associated with the ergonomics of using pipettes repetitively.14 Upper extremity cumulative trauma disorders (CTDs) that can result from pipetting include tendinitis, tenosynovitis, and carpal tunnel syndrome, among others.15,16 Automated liquid-handling robots can alleviate challenges associated with performing labor-intensive assays manually, but they come with several practical


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disadvantages: (i) They can only accommodate liquid containers and dispensers arranged in conventional geometries (e.g., for 96- or 384-well plates),17 which may constrain the development of new assays or instruments. (ii) They are expensive. For example, the Rainin BenchSmart costs $18,000 for each range of adjustable volumes (e.g., 0.5–20 µL or 5–200 µL).18 Solutions for automated, user-defined dispensing rely on, for example, piezoelectric,19 acoustic,20 and valve21 methods to create “droplets on demand”.22 Commercial dispensers employing these technologies are similarly expensive, with costs increasing with performance capability and desired number of channels, and are designed to dispense sub-µL volumes. As a result, automated dispensers are better suited for clinical laboratories, core facilities, or high-volume production facilities that are equipped to perform routine tasks. However, a significant gap still exists for laboratories, particularly those with limited access to resources, that need to facilitate new research ventures and assay prototyping. One expanding area of research affected by limitations in reagent dispensing is the development of low-cost diagnostics using paper-based microfluidic devices.23,24,25,26 The production of these devices requires independent, hydrophilic zones patterned on layers of paper to be treated with reagents. The spatial distribution of these zones—on a single layer of paper27 or across multiple layers of a three-dimensional device28—can be designed entirely by the user to enable a number of analytical tests including immunoassays,29,30 electrochemical assays,31,32,33 and nucleic acid amplification tests.34 However, for these devices to be produced in the laboratory, one must either (i) perform repetitive motions with a single channel pipette or (ii) place stringent constrictions on the device design to facilitate compatibility with conventional multichannel pipettes. For example, while multiplexed analyses can be completed using layers of paper patterned to resemble a microwell plate,35,36,37 requiring a user to perform multiple timed steps may preclude the application of such devices at the point-of-care where ease-of-use is


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essential.38 Instead, in order to create field-ready devices that simplify user operation, paper-based

assays

may

rely

on

devices

with

unique,

non-standard

architectures.39,40,41,42,43,44,45 If these, or similar, device prototypes are to be developed and translated out of the academic laboratory, then methods to manufacture them must also be identified. Low-cost options for the automated dispensing of aqueous solutions include commercial inkjet printers that have been modified to dispense reagents.22 However, these printers require careful consideration of the rheological properties of the dispensed solution,46 powered computers to design arraying patterns and enable operation, and are typically limited to printing pL- or nL-volumes. Consequently, these printers may not be suitable for laboratories in low-income economies, applications where reagents are scarce or difficult to formulate, or the manufacture of paper-based microfluidic devices, which require zones to be treated with µL-volumes of reagents. Two approaches to the fabrication of low-cost liquid dispensers were reported recently in the literature. The first is a modified syringe designed to operate traditional microfluidic devices.47 While this hand-held syringe replaces the need for external pumps to drive flow through a microfluidic device, it is intended to deliver mL-volumes over time and is not capable of dispensing µL-volumes accurately. The second report presents an open source design for a 3D-printed single-channel micropipette.48 While the performance of this inexpensive micropipette compared favorably to an ISO standard49 and commercial pipette, it ultimately does not address the limitations related to ergonomics we have identified here. Instead, we sought to develop an approach to build reconfigurable pipettes to facilitate the quantitative and simultaneous transfer of liquid from a stock container to multiple, non-conventionally arranged zones. Unlike any comparable option, such pipettes would have the potential to simplify the production of intricate or multiplexed paper-based microfluidic devices, enable numerous user-defined device architectures,


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and reduce both requirements of the user and the potential for occupational health dangers. This manuscript reports the development of an approach to fabricate customizable and reconfigurable multichannel pipettes for cost-effective liquid handling applications. We modified a

commercially

available

multichannel

pipette50

to enable

the

reconfiguration of eight independent pipette channels in any two-dimensional array. We fabricated this custom pipette from a readily adaptable multichannel pipette with machined and 3D-printed components. We characterized the accuracy and precision of a customized pipette with three channels arranged equilaterally by measuring the mass of the water delivered by each channel. We show that this user-defined multichannel pipette performs equivalently to a calibrated, single-channel pipette. The accessibility of modern rapid prototyping tools makes our approach feasible for laboratories with multichannel pipettes and access to additive manufacturing equipment.

Experimental Design Design of reconfigurable pipettes We fabricated our reconfigurable pipette by modifying a commercially available multichannel pipette that was on-hand in our laboratory. We made the decision to repurpose an existing pipette to eliminate mechanical engineering challenges associated with building this tool from scratch, and also to introduce a procedure that could make such devices more broadly accessible to other users. To begin, we selected an 8channel pipette because its 2–20 µL volume range was appropriate for treating reagent zones of paper-based microfluidic devices (Figure 1A). This approach could also be applied to other volume ranges or channel numbers. For example, a 12-channel pipette would enable additional two-dimensional channel configurations and larger pipetting geometries than an 8-channel pipette.


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Multichannel pipettes consist of separate channels, which are each composed of a plunger and barrel that mate to form a functional unit. These channels are affixed by two injection-molded plastic holders, which connect the plunger and barrel components of all eight channels and allow each channel of the pipette to operate in unison with the rest (Figure 1B). These two holders are attached to a common pair of guide rods and separated from each other by springs on these rods. The top piece is mobile (i.e., mechanically actuated against the springs by the pipette’s plunger button) and holds the channel plungers. The stationary bottom piece secures the channel barrels and is mounted to the bottom of the guide rods. Upon depression of the plunger button, the channel plungers are moved into the channel barrels by a distance specified by the setting on the pipette’s volume dial. Upon release of the button, these plungers return to their original positions due to expansion of the guide rod springs against the holder. By this mechanism, actuation of the pipette button facilitates aspiration and dispensing of liquid. To fabricate our custom, reconfigurable pipette, we separated the eight linearly arranged channels of the pipette into independent assemblies (Figure 1C–E). We oriented these channels in two-dimensional space using application-specific 3D-printed clips (Figure 2A,B). These clips attached to the pipette’s guide rods using the same hardware as the manufacturer’s holders (Figure 2C), and allowed the pipette to be operated in a conventional and ergonomic fashion, without any further modifications (Figure 2D, Figure S1).

Machining of independent channel components In the linear configuration of the commercial multichannel pipette, the plunger and barrel hardware thread directly into the injection molded holders. Threaded caps (i.e., screws) keep the plungers attached to the molded mobile holder, while springs contained underneath these caps keep the plungers in position (Figure S2A), stabilizing


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interactions with their companion barrels. The stationary holder is more complex than its mobile counterpart, with threaded inputs on top and bottom for each channel (Figure S2B). On the top of this holder, threaded caps at each channel position contain a spring that presses a plastic insert downward inside the holder. This insert seals around the plunger when the two holders are joined together. Channel barrels thread directly into the bottom of the stationary holder, sealing due to compression of an internal gasket. The airtight seals between the plastic insert and plunger, as well as the pipette barrel and plastic holder (Figure S2C), enable consistent pressure transduction across all channels during pipette use. To separate the linearly arranged hardware into independent channels, we designed individual plastic pieces to hold the plunger and barrel components. This was done according to the exact dimensions of the manufacturer’s holding pieces (Figure S3), which were acquired with a digital caliper. From these designs, we had individual holding pieces machined from Delrin stock for use in our reconfigurable pipette. After machining, we attached the original hardware (i.e., plungers, barrels, fasteners, springs, etc.) to the Delrin holders. Because our channel holders were independently machined, a minimum wall thickness (i.e., between the outer and inner diameters of the threaded Delrin tube) was required to maintain holder strength, durability, and ease of handling during channel reconfiguration. For this reason, we could not replicate the linear tip-to-tip spacing necessary for 96-well plates. For applications that require this spacing, we recommend commercially available pipettes. After the machined pieces were used to assemble our 8 independent channels, application-specific pipetting geometries requiring up to 8 channels could be realized using custom 3D-printed holding clips (Figures S4–S6).


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3D printing of holding clips Individual channels in a custom micropipette were held in 3D-printed clips that substituted for the manufacturer’s injection-molded pieces. Machined Delrin channel holders snap directly into these clips to enable simple and rapid device reconfiguration. Modifications to the 3D-printed clip design allow channels to be held in user-defined twodimensional patterns. The mobile top clip has two holes that match the spacing of the pipette’s guide rods. When the pipette button is pressed, the master plunger moves this clip and each channel plunger downward into the channel barrels. The channel barrels are held by a stationary bottom clip, which has two mounting holes for the pipette guide rod screws. The guide rod springs rest against the flat surfaces of the top and bottom clips, and return the plungers to their original positions upon release of the button. By basing the design of our clips on the manufacturer’s holders, we allow our pipette to be used in the same manner as a conventional multichannel pipette and without any modification to the handle hardware (Figure 2D). In our design, the geometries of the top and bottom clips are mirrored so that the plungers and bodies are always are aligned with each other. To prevent the plungers from pushing the pipette bodies through the bottom clip and out of the device, a flat-bottomed rim was added to each hole in the bottom clip. In the clip designs (see Supporting Information), the channel holders are movable units within a square (1.55 in. x 1.55 in.) template. This enables simple manipulation of the pipetting geometry for reasonably sized paper-based microfluidic devices. However, this template is not restrictive for other pipette channel arrays.

Fabrication of accessories To make the use of our device reagent efficient and compatible with existing liquid containers, we 3D-printed custom microcentrifuge tube racks with the same geometry as our pipette’s channels to simplify the user experience and enable rapid reconfiguration of


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stock containers. The holes of these racks were sized to fit standard 1.5-mL microcentrifuge tubes (Figure S7A–C, S8A–C) and PCR tubes (Figure S7D–F), but the design can be easily adjusted to accommodate other liquid containers for other custom multichannel pipette arrays (see Supporting Information). The slip-fit of tubes within the rack allows reagents to be conventionally prepared, stored, and replaced as needed.

Statistical analysis We used equivalence testing to compare the performance our single-channel and custom pipette in volume delivery experiments. Common significance tests (e.g., student’s t-test, ANOVA) aim to reject the null hypothesis, which is that two means are similar, and demonstrate that there is a significant difference between two means. Failure to reject this null hypothesis is not sufficient evidence to claim that two means are statistically equivalent.51 In equivalence testing, the null hypothesis is that the difference between two means is slightly larger than a prescribed value. This difference threshold is not large enough to have any practical consequences. If this null hypothesis is rejected, the alternative hypothesis is accepted and the two means can be considered statistically equivalent.49,52 The difference threshold can be set from previous experience, or calculated as a percentage of the parameter of interest.53 We chose a difference threshold of 5% of the nominal maximum volume of a calibrated singlechannel pipette (i.e., 0.5 µL for a Rainin Pipet-Lite LTS Pipette L-10XLS+).

Results and Discussion Accuracy and precision of a custom, reconfigurable pipette To evaluate the performance of a custom pipette, we selected a range of volumes useful for treating patterned zones in paper-based microfluidic devices. We compared the volume delivery capabilities of our “gold-standard,” a calibrated single-channel


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pipette (0.5–10 µL), to our multichannel linear pipette (2–20 µL) and its reconfigured three-channel form (Figure 3). The delivered volume was calculated from the delivered mass and measured density of water. The density of water was measured using an oscillating u-tube density meter (Anton Paar DMA4100M), and the mass of delivered water was measured using an analytical balance (Mettler Toledo MS304TS). To determine the accuracy of our pipette, we compared the volume of water dispensed from each channel to the intended volume selected on the pipette dial. Channel precision was calculated as a coefficient of variation from ten replicate measurements. Metrics of pipette performance for all three device configurations are available in Table 1. For the reconfigured three-channel pipette, the average accuracy and coefficient of variation for all channels were 98.1% and 4.9%, respectively. Generally, channel accuracy and coefficient of variation improved with increasing intended volume. This trend, exhibited by both the “gold-standard” single channel pipette and reconfigured pipettes, mirrors manufacturer specifications54 as well as industry standards.55 Additionally, to further demonstrate practical equivalence and the quantitative capabilities of our approach, we used this multichannel pipette to treat reagent storage zones in a paper-based microfluidic device to perform an assay for glucose (Figure S9–S12, Table S1). Using the two one-sided test (TOST),49 we demonstrate that the reconfigured pipette is practically equivalent to the single-channel pipette within 5% of its nominal volume (Table 2). This indicates that our tool performs the same as the standard device for constructing paper-based devices, and also that channel reconfiguration did not negatively affect the pipette’s performance. Equivalence testing failed in only 3 of 44 cases (6.8%), all of which were for linearly-arranged pipette channels. These failures could be attributed to a number of potential factors. For example, prior to the start of this project, the multichannel pipette that we selected for reconfiguration had not been used or subjected to routine maintenance (i.e., inspection or calibration) for some time (ca.


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five years). It is possible that the mechanical components of this pipette were not up to the manufacturer’s standards at the time the device was deconstructed for reconfiguration. However, disassembly and modification of the pipette did not cause any additional hardware issues. TOST results indicate that each channel of our reconfigured pipette can be considered practically equivalent to the calibrated single channel pipette that is used routinely in our laboratory. To evaluate sources of error in our pipettes, we performed linear regression analysis (using Prism 7) on the volume data for each channel. Linear regression plots (Figure S13) and fits (Table S2) indicated that error in volume dispensing was not exclusively systematic or random. A slope of 1 and non-zero intercept would indicate purely systematic error, while an intercept of 0 and a slope other than 1 would indicate purely random error. All linear regression fits for the single-channel, linear, and reconfigured pipettes can be characterized by some interplay of both random and systematic error. Comparisons of fit for linear and reconfigured channels indicate that the plungers in our reconfigured pipette have a range of motion and barrel fit sufficiently comparable to those of the plungers in the original linear device.

Conclusions Our reconfigurable pipette is a versatile and efficient tool that provides users with a customizable and ergonomic alternative to conventional liquid handling equipment. By using this pipette in volume delivery experiments, we demonstrate that user-defined, reconfigurable pipettes perform equivalently to gold-standard, calibrated pipettes. This equivalence has only been established for the single pipetting geometry that we tested here. Future iterations of this tool for different paper-based device geometries will require similar comparison to conventional pipettes, but the accuracy and precision of each individual channel suggests that other pipettes arrays should perform similarly.


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Additionally, our possible pipetting geometries are limited by the sizes of the hardware components of the original multichannel pipette. Miniaturization of these components will enable closer spacing of pipette tips and subsequent reduction of sizes of paper-based devices. This shortcoming could be addressed through careful selection of the original commercial pipette, or design and fabrication of a new pipette mechanism. While there are opportunities for expansion and improvement upon this preliminary tool, our reconfigurable pipette serves as a demonstration that customizable liquid handling equipment has the potential to enable the prototyping of multiplexed paper-based microfluidic devices and enhance their production on the laboratory-scale.56

Acknowledgements This work was supported by Tufts University. D.J.W. was supported by a DOE GAANN Fellowship. We thank Kassandra Spiller for discussions of statistical analysis, Syrena Fernandes and Max Goder-Reiser for assistance with the glucose assay, Rayleigh Parker for help cleaning 3D-printed pieces, and the Tufts University Machine Shop for fabrication of machined components and technical discussions.

Conflict of Interest Disclosure The authors declare no competing financial interest.

Supporting Information Materials and methods; paper-based glucose assay; schematics and images detailing the design and fabrication of custom pipettes; experimental details regarding data analysis for pipette performance, equivalence testing for volume delivery experiments, and equivalence testing for glucose assays; Figures S1–S13; Tables S1–S2.


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Figure 1. Breakdown of a commercial, multichannel pipette as a foundation for reconfigurable pipette arrays. (A) Image of the 2–20 µL linear, 8-channel pipette used to construct the reconfigurable pipette. (B) Removal of the tip ejection cover reveals a fixed linear channel assembly. Machined Delrin components were used to create independent barrel (C) and plunger (D) units that mate to form functional channels (E).


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Figure 2. Construction of a custom configuration for a three-channel pipette. (A) Independent plunger units snap into a 3D-printed clip that replaces the plunger holder of the original pipette. (B) Similarly, independent barrel units snap into a 3D-printed clip that replaces the barrel holder of the original pipette. (C) When assembled, the mirrored clips mate to produce a functional pipetting head. The mobile plunger clip is driven downward by the master piston upon depression of the pipette button, and is returned to its original position upon release of the button by the guide rod springs separating the two clips. (D) Independent zones of chromatography paper patterned with the same geometry as the pipette can be treated with reagents in a single pipetting motion.


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Figure 3. Bar graph comparing the performance of three different pipette geometries (single channel, linear configuration, and reconfigured three-channel) at delivering four selected volumes of water. Error bars represent the standard deviation from 10 replicate measurements.


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Table 1. Accuracies (ACC) and coefficients of variation (CV) of dispensed volumes of water for all pipette channels and configurations.

dispensed volume (µL) 2.5 5.0 7.5 10.0 single channel channel 1

ACC (%) 94.2 CV (%) 9.1

97.8 7.7

98.0 2.5

99.3 3.8

86.2 7.4 90.6 6.0 95.0 10.0 89.8 7.0 93.0 6.7 94.2 7.8 95.8 8.5 95.0 9.5

89.8 4.7 93.2 5.0 96.4 4.5 93.8 7.5 95.0 5.6 98.2 4.3 97.4 4.9 97.2 6.2

95.4 1.8 95.4 2.4 98.8 2.8 97.9 3.3 97.6 2.5 100.0 2.4 99.1 2.9 100.0 3.1

94.9 2.1 94.7 1.9 98.3 2.9 97.5 2.2 99.8 2.8 99.3 2.6 94.8 1.8 99.7 3.4

ACC (%) 96.6 CV (%) 8.8 ACC (%) 97.4 CV (%) 9.1 ACC (%) 99.4 CV (%) 5.6

96.6 4.9 98.2 7.2 96.4 6.6

99.7 2.9 98.9 2.0 99.1 2.5

96.0 2.1 98.7 3.7 99.8 3.1

linear multichannel channel 1 channel 2 channel 3 channel 4 channel 5 channel 6 channel 7 channel 8

ACC (%) CV (%) ACC (%) CV (%) ACC (%) CV (%) ACC (%) CV (%) ACC (%) CV (%) ACC (%) CV (%) ACC (%) CV (%) ACC (%) CV (%)

reconfigured pipette channel 1 channel 2 channel 3


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Table 2. Results of equivalence testing for volume dispensing experiments for all multichannel pipette configurations with respect to a calibrated single-channel pipette.

device single linear 1 linear 2 linear 3 linear 4 linear 5 linear 6 linear 7 linear 8 triangle 1 triangle 2 triangle 3

dispensed volume (µL) 2.5 5.0 7.5 10.0 TRUE TRUE TRUE TRUE TRUE TRUE TRUE FALSE TRUE TRUE TRUE TRUE FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE FALSE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE


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For TOC only.


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(2)

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(3)

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(7)

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(8)

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(10) Well Bottom Elevation; 6-2012; The Society for Laboratory Automation and Screening: St. Charles, IL, October 2011. (11) Allen, D.; Zier, R. H.; Kalmakis, G. P.; Torti, V. A.; Nelson, G. E. Uniformly Expandable Multi-Channel Pipettor. U.S. Patent 6,235,244, May 22, 2001. (12) O’Connell, K. P.; Vitale, N. R. Liquid End Assembly For A Handheld Multichannel Pipette With Adjustable Nozzle Spacing. U.S. Patent Application 2009/0104079, April 23, 2009. (13) Hnasko, R. M.; Stanker, L. H.; McGarvey, J. A.; Lin, A. V.; Navarrete, M. A.; Hnasko, T. S.; Chennuru, S.; Pavuluri, P. R.; Lin, A.; Salvador, A.; Carter, J. M.; Clotilde, L. M.; Yu, H.; Carbonell, M. L.; Ching, K. H.; He, X.; Patfield, S. A.;


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