Beyond In Situ Hybridization; Novel Multiplex Immunoassay Applications of HybriWell™ Microslide Chambers

IN HybriWell, Product Applications, Seals and chambers, Uncategorized

One of the best aspects of working with Grace Bio-Labs and the in-situ hybridization and chambering devices is the opportunity to learn some novel applications which are developed by the users of these tools.  Sometimes our customers can teach us a few things about applications for our products!As recently as this summer, a very interesting paper was published in Analyst, the journal of the Royal Society of Chemistry, by two groups in Michigan utilizing HybriWells™.  Here we see inventive Mechanical Engineers and Biological Scientists in collaboration resulting in entirely new applications for these products.

The HybriWell™ is a water-tight, flexible, enclosed and ported microscope slide chambering device configured in a variety of formats.  Traditionally, they are utilized in proteomic or genomic methods requiring in situ hybridization within very small volume sample sizes.  They are popular with these users by virtue of their ability to seal small volumes of liquids under a range of extreme temperatures and then subsequently be cleanly removed.  In the case of Al-Ameen, et al’s publication, instead of using the HybriWell™ for in situ hybridization, they created hydrogel micro-particle casts within the chamber.  These micro-particles were then used in a multiplexed immunoassay system to detect angiogenic markers VEGF, FGF and PDGF in cancer cell lines.

This group was seeking  an alternate solution to multiplexed immunoassays from traditional flow-bead cytometric or spatially based solid-phase protein microarrays.   Flow-bead applications are problematic due to immuno-affinity cross reactivity in liquid phase applications along with prohibitively expensive and complex instrumentation.  Requiring a modest number of analytes to query, they also sought a more flexible content application than high density solid-phase microarrays.  The solution was to fabricate shape-coded hydrogel microparticles, which could be impregnated with immuno-affinity reagents (capture antibodies) and assayed with serum samples in liquid phase.  The shape-coded hydorgel micorparticles are fluorescently detected using image analysis to identify and count representative coded-particle presence and signal intensity.

In order to fabricate specifically shaped < 1 µm hydrogel microparticles, acryl-labeled antibodies were mixed in pre-polymer solutions and place inside the HybriWells™.  These chambers were uniquely effective in this application due to the precise dimensions of the HybriWell™ chamber which was required for casting of the microparticle shapes.   Microparticles were cast by covering with a mask and curing via UV exposure.  This created three shapes of microparticles (rectangular, circular and triangular) corresponding to three capture analytes (VEGF, PDGF and FGF).  These three microparticles were utilized in an immunoassay to query MDA-MB-231 metatastic breast cancer cell lines.  Results of this multiplex assay were comparable to results obtained with single-plex ELISA assay with respect to sensitivity and reproducibility.

Full text can be viewed here:

Analyst, 2015,140, 4530-4539  DOI: 10.1039/C5AN00358J