Digital Microfluidics for Immunoprecipitation

Abstract

Immunoprecipitation (IP) is a common method for isolating a targeted protein from a complex sample such as blood, serum, or cell lysate. In particular, IP is often used as the primary means of target purification for the analysis by mass spectrometry of novel biologically derived pharmaceuticals, with particular utility for the identification of molecules bound to a protein target. Unfortunately, IP is a labor-intensive technique, is difficult to perform in parallel, and has limited options for automation. Furthermore, the technique is typically limited to large sample volumes, making the application of IP cleanup to precious samples nearly impossible. In recognition of these challenges, we introduce a method for performing microscale IP using magnetic particles and digital microfluidics (DMF-IP). The new method allows for 80% recovery of model proteins from approximately microliter volumes of serum in a sample-to-answer run time of approximately 25 min. Uniquely, analytes are eluted from these small samples in a format compatible with direct analysis by mass spectrometry. To extend the technique to be useful for large samples, we also developed a macro-to-microscale interface called preconcentration using liquid intake by paper (P-CLIP). This technique allows for efficient analysis of samples >100× larger than are typically processed on microfluidic devices. As described herein, DMF-IP and P-CLIP-DMF-IP are rapid, automated, and multiplexed methods that have the potential to reduce the time and effort required for IP sample preparations with applications in the fields of pharmacy, biomarker discovery, and protein biology.

Pre-concentration by liquid intake by paper (P-CLIP): a new technique for large volumes and digital microfluidics

“…As a reference, we also tested triangles cut from Kimwipe tissues, which we have used previously to wick away unwanted waste on a DMF device 28 or as a wick when P-CLIP was applied for proteomic sample concentration for mass spectrometry. 27 These wicks also had fast imbibition times (10 s) but were tedious to cut and assemble. Thus, two-ply 10 mm × 10 mm wicks formed from SureWick material were used for all subsequent experiments.…”

“…20 While surface-binding on magnetic microparticles can be a versatile pre-concentration technique, a disadvantage is the risk of clogging when used with microchannel systems. 21 "Open"-format microfluidic systems such as those powered by digital microfluidics (DMF) eliminate the problem of clogging and have proven particularly useful for handling magnetic micro-particles 22 for the analysis of small molecules, 23,24 proteins, [25][26][27][28][29][30][31] and nucleic acids. 32,33 In the most common DMF device format, discrete droplets of liquid are sandwiched between two plates: the bottom plate comprises an array of electrodes that is covered by an insulating dielectric layer and a hydrophobic layer, and the top plate comprises a ground electrode that is covered with a hydrophobic layer.…”

“…In this two-plate format, droplets cannot be manipulated in the z-axis but are unrestricted by any walls or barriers in the xy-plane. 34 Despite its proven utility in handling magnetic microparticles, [22][23][24][25][26][27][28][29][30][31][32][33] DMF is limited in its ability to effect preconcentrations of significant magnitude. Specifically, the difference between the smallest and largest volumes that can be (practically) manipulated on most DMF devices is often quite smalle.g., in the devices used here, this range runs from ∼0.9 μL (a "unit" droplet covering one electrode) to ∼9 μL (a droplet covering 10 electrodes; volumes larger than this are impractical to use).…”

“…We recently introduced one variant of P-CLIP via a proof-of-concept demonstration of its utility for immunoprecipitating proteomic samples prior to elution, collection and analysis off-chip by mass spectrometry. 27 Here, we comprehensively characterize the performance of P-CLIP implemented in several different formats, and apply it to an on-chip immunoassay (with chemiluminescent detection) for Plasmodium falciparum L-lactate dehydrogenase (PfLDH), a biomarker for malaria. 35 These results suggest that P-CLIP may be useful for integrating sample pre-concentration for diagnostic assays for infectious disease.…”

“…More generally, the new technique should be incorporated in any DMF assay relying on functionalized magnetic microparticles. [22][23][24][25][26][27][28][29][30][31][32][33]…”