MULTIPLEX PINWHEEL ASSAY: MICRO-SCALE OPTICAL AND LABEL-FREE QUANTITATION OF DNA WITH HIGH THROUGHPUT AND LOW COST

Departments of
1Chemistry, 2Mechanical

Jingyi Li1, 4 and James P. Landers1, 2, 3, 4 and Aerospace Engineering, 3Pathology, 4Center For Microsystems For The Life Sciences, University of Virginia, Charlottesville, VA 22904 USA

ABSTRACT Pinwheel assay refers to a new technique for DNA quantitation and cell counting by monitoring the aggregation of superparamagnetic beads in a rotation magnetic field. In this paper, a multiplexed format of the pinwheel assay is developed with a bidirectional magnetic field, enabling simultaneous quantitation of 32 samples in 20 min. The new setup can detect DNA down to 10 pg/L. Moreover, it improves automation, minimizes hands-on operation and significantly enhances the throughput. KEYWORDS: multiplex, pinwheel, DNA, quantitation, superparamagnetic beads INTRODUCTION At MicroTAS 2010, we presented the pinwheel assay, where monitoring DNA-induced aggregation of superparamagnetic beads in a rotating magnetic field (RMF) allowed for DNA quantitation with limit of detection ~1 pg/L, and enumeration of cells based on their DNA content [1]. One of the limitations was the need to accurately position the beads at the center of RMF to obtain an image with quantitative information. With multiple wells, this required finicky manual manipulation, and was subject to poor efficiency and reproducibility. Here, we report an improved design of the pinwheel assay that solves this problem, allowing for sample multiplexing, with improved throughput, analysis speed, precision, and accuracy. EXPERIMENTAL The novel solution that allows for multiplex analysis is three-fold: 1) the use of a bidirectional motor (clockwise and counterclockwise) to replace the unidirectional magnetic stirrer (Figure 1A); 2) the use of a circular multiwell accommodating 32 samples; and 3) the ability for a single image to provide quantitation in all 32 wells. Laser-etched circular PMMA chips with thirty-two 25-L microwells allowed beads to simultaneously experience the same magnetic environment, while the bidirectional RMF (bRMF) ensured the beads remained evenly distributed throughout the wells and capable of pinwheeling (Figure 1B). Furthermore, only a single snapshot of all 32 microwells was required for quantitative analysis,

(A)

(B)

Figure 1. (A) Experimental Setup. A magnet on a bidirectional motor generates the new RMF from the bottom of pinwheel chip, and images of beads are obtained from the top with a digital camera. (B) Microchip Design. An array of thirty-two wells was cut through a PMMA sheet with CO2 laser and bonded to another PMMA sheet with proper heat and pressure. The pinwheel chip was aligned with the magnet so that all the microwells were exposed to the same RMF environment. The direction of rotation was reversed every 20 seconds, which is the time that the beads migrated from one side of microwell to the other. instead of previous one snapshot per microwell. RESULTS AND DISCUSSION Bead aggregation in bRMF with DNA is shown in Figure 2. The direction of bRMF was reversed every 20 seconds, which allows beads migrate from one side of the microwell to the other. Images were acquired every 5 seconds, and aggregates were observed at 25, 45, and 65 seconds (Figure 2). No aggregation occurred without DNA (Figure 3A). To

validate synchronized bead aggregation in multiple microwells, three identical DNA samples were mixed with beads in adjacent microwells. After 5-minute exposure in the bRMF, aggregates formed in all three microwells (Figure 3B),

Figure 2. The process of aggregation in bRMF was monitored with a digital camera. The RMF was turned on at t = 0 after 0.8 ng/L -DNA was mixed with beads, and images were taken every 5 seconds. The beads were distributed near the center of microwell at every 5 seconds after reversing rotating direction (i.e. 25, 45, and 65 sec), and aggregation was observed at these time points.

Figure 3. (A) Detecting DNA in bRMF. Aggregates formed with 0.8 ng/L -DNA after 2 min of exposure in RMF, whereas the beads remained dispersed without DNA. (B) Simultaneously detecting DNA in multiple samples. Three identical -DNA samples (0.8 ng/L) were mixed with beads in adjacent microwells, and aggregates formed in all samples simultaneously in 5 minutes.

(B)

140 120 100 80 60 40 20 0 0 7.8125 15.625 31.25 62.5 [DNA] (pg/L)

Figure 4. DNA Quantitation in 27 samples. (A) Nine serially diluted -DNA samples were mixed with beads in triplicate. Aggregates formed after being exposed to bRMF for 5 min. The extent of aggregation increases as DNA samples become more concentrated, as shown in the circled microwells and the corresponding cropped images on the right. (B) Standard curve for DNA quantitation generated from these samples (n = 3, and error bars denote the standard deviation). Dark area values were acquired from image analysis, normalized against a negative control, and correlated with [DNA]. indicating the ability of the new setup to multiplex DNA detection. For DNA quantitation, multiple measurements are often required to obtain accurate results. Nine serially-diluted DNA samples were mixed with beads in triplicate (total 27 samples) and analyzed simultaneously. As DNA concentration increased, the beads transformed into tighter, visually-distinct aggregated states within 5 minutes (Figure 4A). Isodata algorithm [2] was applied to the images, which generated a quantitative correlation between DNA concentration and the extent of aggregation (% dark area) as a standard curve (Figure 4B). DNA was detected down to 10 pg/L, which is comparable to a singleplex pinwheel and conventional fluorescence spectroscopy. The reduction in time for the 27

Dark Area (%)

measurements (~20 minutes) compared to singleplex pinwheel (~50 minutes) represents a significant decrease in analysis time. CONCLUSION In summary, a new experimental setup, a new magnetization strategy and a new chip design for the pinwheel assay constitutes the innovation here. This improves automation, minimizes hands-on operation and significantly enhances the throughput. Current effort focuses on integration of the multiplex pinwheel assay with serial dilution to develop a microfluidic device for rapid and inexpensive quantitation of DNA and cells with sample-in-answer-out capability. REFERENCES [1] J. Li, D.C. Leslie, D.M. Haverstick, K.A. Kelly, N.S. Barker, and J.P. Landers, Pinwheel Assay: A Visual and LabelFree Method for DNA Quantitation, Proc. Micro Total Analysis Systems Conferences, 61-63 (2010). [2] T.W. Ridler, S. Calvard, Picture thresholding using an iterative selection method, IEEE Trans. System, Man and Cybernetics, SMC-8, 630-632 (1978).