CENTRIFUGAL FLUIDIC SYSTEM FOR ENHANCED MIXING AND REDUCING INCUBATION TIMES DURING PROTEIN MICROARRAY

PROCESSING Z. Noroozi1*, H. Kido1, 2, R. Peytavi3, R. Sasaki4, A. Jasinskas4, P. Felgner4 and M. J. Madou1, 5

(1) Department of Mechanical and Aerospace Engineering, Henri Samueli School of Engineering, University of California, Irvine, 92697, USA

(2) RotoPrep Inc., 2913 El Camino Real, #242, Tustin, CA 92782, USA (3) Centre de Recherche en Infectiologie, Université Laval, Québec, Canada, PQ G1V4G2

(4) Department of Medicine, Division of Infectious Diseases, University of California, Irvine, 92697, USA (5) World Class University (WCU), UNIST, South- Korea

ABSTRACT A novel centrifugal fluidic system for fast semi-automated microarray processing was developed. It works by using centrifugal acceleration acting upon a liquid within a rotating disc to generate and store pneumatic energy that can be released by reduction of centrifugal acceleration, resulting in a reversal of direction of flow of the liquid. Through an alternating sequence of high and low centrifugal acceleration, the system reciprocates the flow of liquid within a fluidic system to maximize chemical encounters between molecules. This results in a reduction in processing time and reagent consumption by one order of magnitude. KEYWORDS: Centrifugal, Microfluidics, Mixing, Microarray

INTRODUCTION The use of protein microarray for development of vaccines and detection of infectious diseases has emerged as a

popular method because of its high throughput and sensitivity [1]. Enzyme-Liked Immunosorbent Assay (ELISA) is one of the most widely used methods for screening of proteins. In a typical ELISA process, all the proteins of interest are produced in vitro and then spotted onto a solid phase, such as a nitrocellulose membrane, to make protein microarrays. These microarrays can then be exposed to the sera of patients that have developed immunity to the specific disease, in the search for the antigens that elicit the best immune responses. These are manifested by the specific attachment of patient antibodies (found in the sera) to the immobilized antigens. The antibodies can then be detected by the use of second antibodies tagged with enzymes, radioisotopes, or fluorescent molecules to visualize the antibody/antigen complexes [2]. In the search for more effective drugs or vaccines for diseases, potentially thousands of molecules must be analyzed. The process of manually exposing the antigen microarrays to sera, washing, incubation with the second tagged antibody, and incubating with substrate is labor intensive and time-consuming. Herein we present a semi-automated centrifugal microfluidic platform for faster protein microarray processing. The system is based on the novel technique published earlier by these authors [3] for reciprocating fluid samples in the radial direction on a Compact Disc (CD) to enhance mixing in a miniaturized bioassay chamber. It works by using centrifugal acceleration acting upon a liquid within a rotating disc to generate and store pneumatic energy in situ for later use to reverse the direction of flow of the liquid as centrifugal acceleration is reduced (by reducing angular frequency). Through a sequence of increasing and decreasing centrifugal acceleration (Fig.1), the system makes it possible to reciprocate the flow of liquid within a fluidic system to affect mixing as well as to maximize chemical encounters and specific interactions between molecules in the liquid phase and solid phase. This results in a reduction in time and reagent consumption necessary to perform a bioassay by 90%. THEORY

The CD based fluidic structure as shown in Fig. 1 consists of a loading chamber for introducing the fluid to the system, an upper chamber for holding liquid as it reciprocates in the radial direction, a pressure chamber for storing pneumatic pressure, a waste chamber, a reaction chamber where solid phase reaction between immobilized proteins and the molecules suspended in the fluid takes place, and several microchannels for transferring liquid between the chambers. Liquid is loaded into the loading reservoir and the disc is placed on a rotating platform and spun at a high acceleration rate to a high angular velocity. The induced centrifugal force [4] drives the liquid downstream through the upper and reaction chambers resulting in compression of the air trapped in pressure chamber [3]. The high acceleration rate prevents the liquid from priming the siphon valve at this point [5]. Next the angular velocity is reduced gradually and the flow reverses direction and moves towards the upper chamber as the pneumatic pressure is released. The minimum velocity at which the liquid will reach the crest of the siphon before falling over depends on the properties of the liquid and is measured experimentally. This sequence of increasing and decreasing angular velocity is repeated for the desired

978-0-9798064-3-8/µTAS 2010/$20©2010 CBMS 79 14th International Conference onMiniaturized Systems for Chemistry and Life Sciences

3 - 7 October 2010, Groningen, The Netherlands

number of cycles. The angular velocity is then lowered rapidly to a small value to force the liquid to move up and pass the crest of the siphon and prime it. The capillary valve draws the liquid almost completely into the waste chamber and the system is ready for introduction of the next fluid. The spin program was set according to the angular velocity profile presented in Fig. 2. EXPERIMENTAL

The fluidic structure was designed using SolidWorks 2007 (SolidWorks Corporation, Concord, MA) and fabricated by layered assembly of polycarbonate plastic parts and pressure sensitive adhesives. Deep features were machined in 1.1 mm polycarbonate sheets (McMaster-Carr, USA) using a QuickCircuit 5000 CNC router machine (T-Tech Corporation, USA). Microchannels and the reaction chamber were patterned in a 100-µm pressure sensitive double adhesive (FLEXcon, USA) using a Graphtec CE-2000 vinyl cutter (Graphtec America Inc., USA). All loading and ventilation holes were drilled in a clear DVD (0.6 mm thick) using QuickCircuit. The bottom layer casted with 6 x 6 mm nitrocellulose membranes (GraceBio Labs, OR, USA) was a clear CD (1.2 mm thick). All layers were then aligned and bonded together. The experimental setup as described previously [5] included a mechanical spin stand assembly (including a servomotor and a controller) for rotating the disc, a high speed camera, a strobe light, a fiber optic sensor, and a personal computer running Windows Vista Operating System. MS Visual Basic 2008 was used to develop the software to control the motor through a user interface. Microarrays of 5 x 5 Immunoglobulin (IgG) spots with concentrations varying in each row were printed onto nitrocellulose membranes cast on both discs and glass slides using an Omni Grid 100 array printer (Genomic Solutions). All reagents and buffers were prepared as described before [1] and divided appropriately for use in both conventional experiments using glass slides and centrifugal microfluidic experiments that were carried out side by side. On the glass slides, first the membranes were dehydrated with protein array blocking buffer (Whatman) for 30 min and then incubated with Alkaline Phosphatase (AP)-conjugated Goat Anti-Human IgG diluted to 1/100 in protein blocking buffer for 1 h at room temperature. After a sequence of 6 washes in 20 mM Tris-0.5 mM NaCl containing 0.05% Tween-20 buffer, the slides were developed with a 1-Step nitro blue tetrazolium/5-bromo-4-chloro-3-indoyl phosphate (NBT/BCIP) mixture. The reaction was stopped by two washes with deionized (DI) water after the dark array spots appeared and the slides were dried with brief centrifugation. Centrifugal-based assays were carried out using the consecutive application of the same reagents and buffers explained above to the microfluidic system in a number of steps. At each step, 25 µl of sample was injected into the loading chamber and the disc was mounted onto the spinstand and rotated at angular velocities varying with time according to the profile presented in Fig. 3. Incubation times for each step varied according to table 1 and Fig. 4. The developed arrays on both glass slides and CD were then scanned using HP ScanJet 9200 and the spot intensities were quantified using Perkin Elmer QuantArray software. The results were imported in to a Microsoft Excel Spreadsheet and the graphs were created for comparison.

Figure 1 - Schematic illustration of the fluidic system

Figure 3 – Flow reciprocation work cycle profile: Angular velocity vs. Time.

Figure 2 - Time-lapse images of the system in operation. (a) Liquid is added to the loading reservoir, (b) Compression of air in pressure chamber as a result of high RPM, (c) relaxation of air and pumping of liquid towards the center, (d) increase of RPM to maximum, (e) priming of the siphon as the liquid level rises above the crest of the siphon, f) the empty system.

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RESULTS AND DISCUSSION Increasing the rate of reaction in bimolecular encounters in microarray technology is of paramount interest. Molecular

diffusion, which is the primary mode of mass transport for molecules in the bulk of the flow to the molecules bound on the solid surface, is slow [6]. Research studies have demonstrated that convection or transport of molecules in bulk, increases the reaction rate for a number of reasons such as thinning of the diffusion boundary layer as well as creating turbulence and eddy diffusion and hence, intersecting the streamlines in the laminar flow. With this knowledge in mind, we designed our fluidic system to utilize the centrifugal and capillary forces as well as the pneumatic pressure to promote mixing and a faster reaction. We conducted a series of experiments to study how the reaction rate increased as a result of flow reciprocation. Our first goal was to find the incubation times, centrifugal force, and the rate of reciprocation needed to get the same results achieved in conventional microarray processing experiments performed on glass slides. Fig. 4 presents the average spot intensity of a developed microarray for different incubation times between IgG proteins with different concentrations printed on the nitrocellulose membranes and AP-labeled Goat Anti-Human IgG molecules. Table 1 shows the duration for performing other incubation and washing steps. A reciprocating cycle of 30 s equivalent to a Reynolds number (Re) of ~1.5 was used for all steps. The curves demonstrate that the incubation time of 5 min

Table 1 – Different steps of the centrifugal-based assay

generates the same (or better) spot intensity when compared to the results of 1 h incubation using conventional method. We also observed that (results not shown here) the minimum required incubation time decreases as the cycle time decreases to 10 s and the Re increases by three fold. CONCLUSION

This work presents a novel solution for the inherent problems of microarray processing on a CD using centrifugal microfluidics. By reciprocating flow between the center and perimeter of the disc with relatively small usable area (100 mm), we were able to prolong the movement of the flow. In doing so, we employed flow-through microfluidics and active mixing phenomena to develop a faster and semi-automated methodology for processing microarrays. ACKNOWLEDGEMENT

This project was supported in part by the National Science Foundation and the ARCS foundation through their generous fellowship grants. We would like to thank Grace Bio Labs for casting nitrocellulose membranes on discs. REFERENCES [1] D.H. Davis et al, Proc. Natl. Acad. Sci. USA, 102 (3), 547–552, 2005 [2] S. Sundaresh, et al, Bioinformatics, vol 22, pp. 1760-1766, 2006. [3] Z. Noroozi et al, Review of Scientific Instruments, vol. 80, pp. 075102-8, 2009. [4] M. Madou, Fundamentals of microfabrication, 2nd edn, CRC Press, Boca Raton, 2002 [5] H. Kido et al, Colloids Surf B Biointerfaces, 58:44–51, 2007. [6] B. R. Munson et al, Fundamentals of Fluid Mechanics, Fifth edition, John Wiley & Sons Inc. (2006) CONTACT *Z. Noroozi, tel: +1-949-824-4143; norooziz@uci.edu

Steps Time (s)

Blocking Buffer 60

AP & Secondary AB Varying (See Figure 4)

TTBS 30, 60, 90

TBS 30, 60

NBT/BCIP 210

DI Water 30, 60, 90

Figure 4 - Results of the reciprocating microfluidic system were compared with results of one h conventional method.

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