The development of functional biomaterials that contain covalently attached, biologically active proteins and peptide sequences has greatly impacted fields related to tissue engineering, biosensing, and biomedical research.  Specifically, antibody surface immobilization techniques have had a significant impact on the antigen detection for clinical diagnostics.  To date, most assays rely on monolayer adsorption of antibodies to surfaces.  However, the ability to covalently bind antibodies with control over their density and clustering can provide numerous advantages with respect to high sensitivity detection. While most covalent attachment chemistries are time intensive, substrate limited, and difficult to control spatially, living radical photopolymerization (LRP) techniques overcome these issues. Methods to derivatize antibodies to create a polymerizable, yet active form are also critical.

In this work, acrylated whole antibodies were synthesized with the goal of establishing a unique polymerization method to immobilize antibodies to polymer surfaces in a manner that leads to increased accessibility and high mobility.  This approach enables covalent binding of the antibodies with independent control over their density and surface orientation, which improves detection sensitivity and response time. In this research we demonstrate three significant milestones: the ability to polymerize whole antibodies as polymer grafts using LRP chemistry, which is integral in maintaining antibody activity and selectivity, the retention of antimer-antigen selectivity in a variety of biologically relevant analyte environments, and the application of a novel photopolymerization method, which allows patterning and fabrication of microfluidic assays based on antibody-antigen detection.  These milestones have been established using a variety of antigens with varying molecular weight, biological stability and function.

Results and Discussion. Synthesis of acrylated, PEGylated, photopolymerizable proteins is a useful means of creating surfaces that have been functionalized for biomaterial integration as well as for biodetection applications.  Here, affinity purified, whole anti-glucagon (GLGN) antibody protein was acrylated using NHS:NH₂ bioconjugation chemistry, for the detection purposes presented in this text.  Degree of acrylation was verified via SDS-Page and a trinitrobenzene sulfonic acid (TNBS) assay. The degree of acrylation was determined to be around 15-50%, depending on the reaction stoichiometry used to synthesize the acrylated antibody.  Once acrylated, anti-GLGN antibody was further determined to remain biologically active as compared to unconjugated anti-GLGN antibody.  This was shown using an indirect ELISA technique to compare the detection limits and activity of acrylated antibody in capturing GLGN antigen in a biologically relevant environment.  A statistical difference in detection capabilities was not observed post-modification.

Once synthesized and determined to be biologically active, whole antibody monomer was grafted in the presence of poly(ethylene glycol) (375) monoacrylate (PEG (375)) co-monomer to prevent non-specific protien adsorption onto grafted detection patterns and to provide increased solubility and mobility for surface detection.  Time-consuming blocking and washing steps could also be prevented through tailoring graft chemistry, leading to a rapid immunoassay platform that only requires 12 minutes to complete.  By using these parameters and the increased surface density alloted by using LRP chmeistry, platform sensitivities were incrased by 3 orders of magnitude (< pM) as compared to standard ELISA protocols (~nM).  Detection signal was also significantly increased by 5X compared to standard techniques.  Further, GLGN was shown to be detectable at this level in whole blood and plasma environments, otherwise not possible using standard immunoassays, due to the short half life (~10 min.) [1] of GLGN in a biologically relevant environment.  Some evidence has also been reported suggesting that the PEG spacer (spacing the antibody from the graft backbone) has a profound impact on antigen sensing capabilities.  Current interests related to this research include varing the PEG spacer length to further improve detection using this assay as well as investigations related to assay amplification methods and chemistries that are useful in the incorporation of multiple antibodies on grafted surfaces for simultaneous antigen detection.

Figure 1:  A microfluidic detection device demonstrating positive detection of two specific antigens in parallel.  The left well is a negative control well, consisting of a PEG only graft, in the absence of antibody.

To demonstrate the utility of using LRP-based grafting combined with photolithography, a microfluidic device with antibody-grafted detection wells was constructed. This device was created to demonstrate the ability to detect multiple analytes simultaneously, also confirming that the immobilized antbodies have retained their specificity for their respective antigen.  2 mm diameter wells incorporated on a microassay were modified with grafted antibody.  After proper swelling and cleaning, the 2 mm diameter, grafted wells were utilized for detection purposes as presented in Figure 1. References.

 ADDIN EN.REFLIST 1.         Gotthardt, M.; et al. Euro. Jour. of Nucl. Med. and Molec. Imag. 2002, 29, 597-606 (2002).