![]() BP photophores have unique photochemical properties: upon n-π∗ excitation at 365 nm, a biradicaloid triplet state is formed reversibly, which can abstract a hydrogen atom from accessible C-H bonds the radicals subsequently recombine, creating a stable covalent C-C bond. The widespread applications of benzophenone (BP) photochemistry in biological chemistry, bioorganic chemistry, and material science have been prominent in both academic and industrial research. We consider the microfluidics literature from 1997 to present and close with a perspective on future approaches to protein immobilization. We also describe multifunctional surface coatings that can perform tasks that were, until recently, relegated to multiple functional coatings. Spatially encoded protein immobilization using DNA hybridization for multiplexed assays and reversible protein immobilization surfaces for repeatable assay are introduced as immobilization methods. Recent "smart immobilization" methods including the use of light, electrochemical, thermal, and chemical stimuli to attach and detach proteins on demand with precise spatial control are highlighted. We overview immobilization methods and chemistries, and discuss studies exemplar of key approaches-here with a specific emphasis on immunoassays and enzymatic reactors. In this review, we present trade-offs considerations for common immobilization surface materials. Immobilization methods and chemistries vary significantly depending on immobilization surface, protein properties, and specific assay goals. In most cases, protein immobilization densities are maximized, while native activity and conformation are maintained. For heterogeneous systems, controlled immobilization of reactants is essential for reliable, sensitive detection of analytes. For example, specific advances have been made in reagent consumption, throughput, integration of multiple assay steps, assay automation, and multiplexing capability. ![]() Microfluidic systems have shown unequivocal performance improvements over conventional bench-top assays across a range of performance metrics. For this study DNA oligonucleotides were chosen as a model system of biomolecular probes, and fluorescence detection of DNA microarrays served as method of detection. Of all photolinkers and substrates tested, PTD as photolinker and COC as substrate showed the highest photolinking efficiencies and fastest reaction times. We compared the overall photolinking efficiency of all photolinkers with respect to the polymer substrate they are applied to, and we found considerable differences for certain photolinker/substrate combinations. The influence of substrate material is discussed, and three different polymers served as representative substrates: poly(methyl methacrylate) (PMMA), polystyrene (PS), and a cycloolefin copolymer (COC). The influence of these variables are investigated for four prominent photolinkers: ketyl-reactive benzophenone (BP) and anthraquinone (AQ), nitrene-reactive nitrophenyl azide (NPA), and carbene-reactive phenyl-(trifluoromethyl)diazirine (PTD). This study addresses the selection of photolinker and the adjustment of reaction conditions, such as the concentration of biomolecule applied, and irradiation time. The use of photolinkers (photoactivatable heterobifunctional crosslinkers) is a popular method to attach biomolecules to polymer surfaces.
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