Article pubs.acs.org/ac
Twist on Protein Microarrays: Layering Wax-Patterned Nitrocellulose to Create Customizable and Separable Arrays of Multiplexed Affinity Columns Victoria de Lange and János Vörös* Laboratory of Biosensors and Bioelectronics, Institute for Biomedical Engineering, University and ETH Zurich, Gloriastrasse 35, 8092 Zurich, Switzerland S Supporting Information *
ABSTRACT: We developed the simple and inexpensive FoRe microarray to simultaneously test several 1 μL samples for multiple proteins. By combining forward and reverse phase microarrays into an innovative three-dimensional format, the FoRe array exploits the advantages and eliminates several drawbacks of the traditional approaches (i.e., large sample volumes, protein loss, and cross-reactivity between detection antibodies). Samples are pipetted into an array of separable, multiplexed affinity columns. Several nitrocellulose membranes, each functionalized with a different capture antibody, are stacked to create a customizable affinity column. The nitrocellulose is patterned with wax to form 25 isolated microspots on each layer, allowing us to analyze multiple samples in parallel. After running the immunoassay, the stacks are quickly disassembled, revealing 2D microarrays of different fractions from multiple samples. By combining the stack-and-separate technique with wax patterning, we keep the arrays low cost and easily tailored to a variety of applications. We successfully performed 3D multiplexing using a model system with mouse and rabbit IgG. Binding proved to be independent of the position in the stack, and the limit of detection for a mouse IgG sandwich assay was 7.3 pM in BSA and 15 pM in human plasma. The FoRe microarray makes it possible to identify protein expression patterns across several minute volume samples; for example, it could be used to analyze cell lysate in drug response studies or pricks of blood from small animal studies.
P
tested for the presence of one protein (Figure 1B). The FoRe array involves layers of captures sites with parallel sample analysis on each layer (i.e., multiple protein samples tested for multiple targets) (Figure 1C). When detecting proteins, both the forward and reverse phase arrays present problems. Labels (e.g., enzymes, fluorescent dyes, quantum dots, or metallic nanoparticles) are commonly used to visualize the presence of the target protein.7 In a forward phase array, the target is either directly labeled or detected with a second, labeled antibody (i.e., sandwich assay). The fragile and complex nature of proteins discourages direct labeling.1,6,7 The sandwich assay has higher specificity and sensitivity but suffers from additional cross reactivity between detection antibodies.1,6 As a result, multiplexing in the system is limited to detecting 30−50 antigens in parallel.1,3 Additionally, relatively large sample volumes, i.e., up to several hundred microliters, are needed to submerge the test sites. Clinical sample volumes, e.g., biopsy material or blood from an infant heel prick, can be
