The UC Berkeley - UCSF Graduate Program in Bioengineering, 94720 Berkeley, CA, USA.
Department of Research and Development, Thermo Fisher Scientific, Rockford, Illinois, USA.
Analyst. 2021 Oct 25;146(21):6621-6630. doi: 10.1039/d1an01309b.
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 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.
尽管荧光读出是基于水凝胶的免疫分析的关键检测模式，但由于自发荧光或非特异性抗体相互作用引起的背景荧光会降低荧光免疫分析的检测下限。水凝胶结构的化学修饰会影响自发荧光和非特异性相互作用。苯甲酮是一种常见的光活化分子，苯甲酮甲基丙烯酰胺（BPMA）已被用于聚丙酰胺（PA）水凝胶中的蛋白质交联。然而，先前的研究表明，苯甲酮的芳构结构会导致自发荧光增加和未结合的荧光探针的非特异性疏水性相互作用增加。在这里，我们合成了叠氮甲基丙烯酰胺（DZMA）作为一种替代的光活化分子，用于 PA 水凝胶中的蛋白质捕获，用于胶内免疫分析。我们假设，根据先前报道的预测和实验 log 值，氮丙啶的疏水性结构较低，表现出较低的自发荧光和非特异性疏水性相互作用。我们发现，虽然 DZMA 和 BPMA 的等浓度导致氮丙啶结构中蛋白质靶标光捕获减少，但将 DZMA 浓度增加到 12mM 可提高胶内蛋白质捕获能力，与先前报道和表征的 3mM BPMA 水凝胶相当。此外，尽管氮丙啶的浓度较高，但与 BPMA 凝胶相比，我们观察到氮丙啶凝胶中的荧光背景信号可忽略不计，免疫分析荧光背景信号降低 50%，导致探测的蛋白质靶标信号与噪声比（SNR）相当。最后，我们在开放式微流控设备中测试了 DZMA 用于单细胞免疫印迹的实用性，发现蛋白质在 DZMA 水凝胶中的迁移速度比在 BPMA 水凝胶中快约 1.3 倍。然而，在 DZMA 水凝胶中，我们检测到的蛋白质信号仅为 BPMA 水凝胶的 15%，这表明氮丙啶化学会导致电泳分离后蛋白质损失更大。我们发现，虽然氮丙啶的背景荧光信号较低，这可能潜在地改善免疫分析性能，但氮丙啶的较低捕获效率降低了其在易发生样品损失的开放式微流控系统中的实用性。
