Comparison of photoactivatable crosslinkers for in-gel immunoassays.

While fluorescence readout is a key detection modality for hydrogel-based immunoassays, background fluorescence due to autofluorescence or non-specific antibody interactions impairs the lower limit of detection of fluorescence immunoassays. Chemical modifications to the hydrogel structure impact autofluorescence and non-specific interactions. Benzophenone is a common photoactivatable molecule, and benzophenone methacrylamide (BPMA) has been used for cross-linking protein in polyacrylamide (PA) hydrogels. However, previous studies have suggested that the aromatic structure of benzophenone can contribute to increased autofluorescence and non-specific hydrophobic interactions with unbound fluorescent probes. Here, we synthesize diazirine methacrylamide (DZMA) as an alternative photoactivatable molecule to crosslink into PA hydrogels for in-gel protein capture for in-gel immunoassays. We hypothesize that the less hydrophobic structure of diazirine (based on previously reported predicted and experimental log P values) exhibits both reduced autofluorescence and non-specific hydrophobic interactions. We find that while equal concentrations of DZMA and BPMA result in lower protein target photocapture in the diazirine configuration, increasing the DZMA concentration up to 12 mM improves in-gel protein capture to be on par with previously reported and characterized 3 mM BPMA hydrogels. Furthermore, despite the higher concentration of diazirine, we observe negligible autofluorescence signal and a 50% reduction in immunoassay fluorescence background signal in diazirine gels compared to BPMA gels resulting in comparable signal-to-noise ratios (SNR) of the probed protein target. Finally, we test the utility of DZMA for single-cell immunoblotting in an open microfluidic device and find that protein migrates ∼1.3× faster in DZMA hydrogels than in BPMA hydrogels. However, in DZMA hydrogels we detect only 15% of the protein signal compared to BPMA hydrogels suggesting that the diazirine chemistry results in greater protein losses following electrophoretic separations. We establish that while diazirine has lower background fluorescence signal, which may potentially improve immunoassay performance, the lower capture efficiency of diazirine reduces its utility in open microfluidic systems susceptible to sample losses.

Ferguson analysis of protein electromigration during single-cell electrophoresis in an open microfluidic device.

In an open microfluidic device, we investigate protein polyacrylamide gel electrophoresis (PAGE) separation performance on single-cell lysate. Single-cell protein electrophoresis is performed in a thin layer of polyacrylamide (PA) gel into which microwells are molded. Individual cells are isolated in these open microwells, then lysed on-chip with a dual lysis and electrophoresis sodium dodecyl sulfate (SDS) buffer. We scrutinize the effect of sieving gel composition on electromigration of protein targets, using a wide range of cellular protein standards (36 kDa to 289 kDa). We find that as PA concentration increases, protein electromigration deviates from the empirical log-linear relationship predicted between migration distance and molecular mass. We perform Ferguson analysis to calculate retardation coefficients and free solution mobilities of nine cellular protein standards and observe that the largest-molecular-mass protein, mTOR (289 kDa), does not behave as predicted by established linear-fit models for SDS-denatured proteins, indicating that mTOR is beyond the linear range of this assay. Lastly, we performed in-gel immunoprobing on the single-cell electrophoretic separations and observed that smaller pore-size gels (higher gel concentration) reduce protein diffusion out of the gel, which does not notably impact the measured immunoprobed protein expression. Compared to larger pore-size gels, the smaller pore-size gels lead to higher local concentrations of the target protein in each protein band, resulting in an increase in the signal-to-noise ratio (SNR) for each protein. Understanding the separation and immunoprobing performance at different gel concentrations improves assay design and optimization for target proteins.