Diseño estructural de Sala

7.1 Introduction

Multiplexed bead arrays are a powerful tool in the development of sensitive, high- throughput, on-chip immunoassays(Ferguson et al. 2000; Goodey et al. 2001; Fan et al. 2006; Ng et al. 2008; Xu et al. 2008; Barbee and Huang 2008; Lee et al. 2009). In this chapter, we demonstrate this concept with two different chip designs. In the first approach, a chiplet containing a microwell array patterned on a silicon wafer (Illumina Inc., San Diego, CA) is integrated into a standalone pouch-based reagent delivery system. In the second approach, avoiding packaging that increases device complexity and may adversely impact device reliability (Qiu et al. 2009; Xu et al. 2008; Li et al. 2005; Ng et al. 2008), an embossed microwell array is directly integrated into a plastic chip. The relative merits of each approach are discussed.

In both approaches, two types of functionalized polymeric monodisperse microbeads from a master library containing both bead types are dispensed onto the microwell array. Once the wells are populated with beads, the identification and location of each bead in the array is recorded by means of a decoding process, whereby each bead type is identified by its fluorescent intensity with a CCD camera. The biosensing capability of the array is tested using beads coated with antibodies to Interleukin-8 (IL-8, 8 kDa) and beads coated with antibodies to Vascular Endothelial Growth Factor (VEGF, 42 kDa) to demonstrate the specific detection of IL-8. The transparent chip materials enabled in situ imaging of the beads to quantify the amount of target captured at each bead while exhibiting low background fluorescence. In an alternative deliberate loading

approach (not requiring decoding) discussed in Thompson et al. 2010, individual magnetic beads can also be controllably placed in predetermined microwells using a custom-made magnetic probe.

7.2 Pouch-Based Immunoassay with Integrated Etched Silicon Bead Array

The uniqueness of the pouch-based cassette we designed for this assay stems in part from our recognition that a device for use at home by an individual, at the point-of- care (e.g. in a doctor's or dentist's office), or in the field (e.g. to test a water supply for different types of bacterial contamination) must be fully contained with on-board storage of all reagents and have a shelf life of several months to a year. The pouch-based cassette consists of two inexpensive parts (~$2 per chip without mass production) that are fabricated by CNC machining. The upper part contains the reagent pouches (~50-100 µL

in volume) and valves and is formed by laminating a flexible membrane to a plastic (polyethylene) substrate. The pouches store the various buffers and wash solutions. The lower part of the cassette is made of polycarbonate and contains the flow conduits, reaction chambers, microbead array (chiplet; Figure 7.1), and needles to facilitate hydraulic connections with the upper part. Prior to use, the two parts are mated using alignment pins to ensure a proper connection. The needles penetrate a thin piece of double-sided adhesive aluminum foil that seals the bottom of the polyethylene substrate and form quick, leak-free connections between the two parts. The transparent cassette materials enable in situ imaging of the chiplet with a CCD camera to determine the registry of target-specific beads according to their fluorescent coding, and to detect the amount of specific target captured at each bead.

Figure 7.2 shows a cassette designed for a simple immunoassay that integrates the microbead array. Figure 7.2b shows the bottom side of a mated cassette and illustrates how the chiplet interfaces with the fluidic channel and detection chamber. Upon mating the top and bottom pieces, the pouches are depressed in a predetermined sequence to squeeze their liquid contents into the conduits. In addition to their role as storage chambers, the pouches act as micropumps and facilitate the transport of the sample from one reaction chamber to another. When the chemistry is relatively simple such as immunoassays for the detection of antibodies and antigens, the pouches and valves are actuated manually with a rigid actuator and pins. In more complicated cases such as the processing of nucleic acids, individually controlled solenoid actuators may be required to actuate individual pouches. The pouch system also enables two connected pouches—one

plastic substrate silicon chiplet 2.4 mm 2.0 mm Illumina chiplet contains ~100,000 wells 3 µm empty wells

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Figure 7.1: The Illumina silicon chiplet containing a hexagon microbead array. (a)

Micrograph of the microarray of etched wells in the chiplet; after loading, some of the wells are populated with functionalized beads. (b) Relative size of the chiplet. (c) Schematic of the chiplet interfacing with the detection chamber milled in a plastic substrate by CNC machining.

initially empty and one full—to work in tandem for mixing and incubation. As the full pouch is compressed, the empty pouch fills up; the process is then repeated emptying the full pouch into the empty receiving pouch. The liquid is propelled back and forth between alternating empty and full pouches. This reciprocating flow action is used to enhance mass transfer and improve the reaction kinetics between target analytes, labels, and immobilized ligands. Alternating flow provides an advantage over commonly used commercially available microarrays, where interaction kinetics are governed mostly by diffusion and can take many hours.

7.2.1 Experiments

To demonstrate the utility of the cassette with the microbead array, we performed a bead-based fluorescence sandwich immunoassay. The steps associated with the assay are depicted in Figure 7.3 (which is a modification of a figure presented in Blicharz et al.

membrane valves

finger-actuated pouches for reagents and mixing

Storage