An automated materials screening approach for the development of sol–gel derived monolithic silica enzyme reactor columns

Abstract

Fabrication of monolithic protein-doped capillary columns was reported almost 10 years ago. These columns were derived from a diglycerylsilane precursor, however this material is not commercially available, is difficult to produce in large quantities and has very short phase separation and gelation times, which leads to issues with column reproducibility. Herein, we investigate the use of sodium silicate (SS), a commercially available biofriendly sol-gel precursor, for the fabrication of bimodal meso/ macroporous protein-doped monolithic silica columns that are suitable for immobilized enzyme reactor (IMER) assays. Using an automated liquid handler and platereader, a hierarchical materials screening approach was applied to $1400 formulations, from which we have identified materials with long gelation times that can form robust bimodal meso/macroporous materials suitable for fabrication of monolithic silica columns. A subset of these materials was observed to have good chromatographic behavior (appropriate backpressure and good stability). A secondary screen around lead materials was performed to identify optimal materials for fabrication of IMER columns. These materials were tested for leaching and activity of immobilized acetylcholine esterase to identify an optimal material for IMER column fabrication. The optimal material was formed from 2% (w/v) silica which was combined with 1.25% PEG 600 at pH 6.4 in 100 mM TRIS buffer. Such columns showed reproducible IMER performance and were able to quantitatively measure the inhibition of immobilized AChE by galanthamine with an inhibition constant of 175 AE 5 nM, which is in excellent agreement with the literature value.

Enzymes in a golden cage

“…6 In general, immobilizations have been carried out in 0D architectures such nanoparticles, 7 in 1D architectures such as nanobers, 8 in the common 2D architectures of adsorption or covalent attachment to surfaces, 9 and in 3D architectures in which the enzyme is entrapped physically or chemically within a porous matrix. 10,11 The latter has been of particular interest, mainly because 3D immobilization provides high surface areas in small volumes, because the internal regions of the immobilizing material provides efficient protection against denaturing processesheat, extreme pH, destructive chemicalsand because this immobilization reduces the susceptibility of detachment from the support. 12,13 Of the three large family of materialsinorganic, (bio)organic and metalsthe largest libraries of 3D immobilization matrices methodologies belong to the rst two, examples being metal-oxide matrices such as sol-gel family of materials, 10,[13][14][15] and organic matrices such as cross-linked polymers.…”

“…10,11 The latter has been of particular interest, mainly because 3D immobilization provides high surface areas in small volumes, because the internal regions of the immobilizing material provides efficient protection against denaturing processesheat, extreme pH, destructive chemicalsand because this immobilization reduces the susceptibility of detachment from the support. 12,13 Of the three large family of materialsinorganic, (bio)organic and metalsthe largest libraries of 3D immobilization matrices methodologies belong to the rst two, examples being metal-oxide matrices such as sol-gel family of materials, 10,[13][14][15] and organic matrices such as cross-linked polymers. 16 The most common metal used in the immobilization context is gold, because of its inertness to most biological processes and its low toxicity, 17 which have led to the use of gold in diagnostics and many other therapeutic elds.…”

Sol–Gel‐Derived Biohybrid Materials Incorporating Long‐Chain DNA Aptamers

“…We selected ac utoff of 20 % transmittance,b elow which materials were considered to be macroporous. [9] Figure 1d emonstrates that transmittance (and hence morphology) can be carefully controlled by adjusting the precursor and porogen properties.M any materials comprised of SS,T MOS or 40 vol %M TMS in TMOS with variable amounts of PEG demonstrated transmittance values indicative of macroporosity and formed self-…”

“…Herein, we demonstrate that concatemeric aptamers can be entrapped into specially-designed macroporous sol-gel-derived organosilicate composites with high target-binding activity and minimal leaching, allowing for fabrication of flow-through biosensors to detect both small and large targets.Our initial goal was to identify an appropriate porous material for entrapping concatemeric aptamers.T he morphology of sol-gel materials is affected by several parameters that control gelation time and phase separation, which include types of silica precursors and porogens,reaction pH and ionic strength. [3a,c,9] Fort his reason, we adapted ap reviously reported screening approach [9] to identify suitable compositions that could retain concatemeric aptamers with minimal leaching, allow concatemer accessibility to both small molecule and protein targets,p roduce self-supporting monolithic capillary columns,a nd allow pressure-driven flow through acapillary column with low backpressure.Atotal of 140 formulations were prepared from four silica precursors-sodium silicate (SS), tetramethoxysilane (TMOS), methyltrimethoxysilane( MTMS) or 40 vol % MTMS in TMOS,which were previously used for entrapping functional aptamers (see Supporting Information for experimental detail). [10] Each precursor was combined with five concentrations (0, 1.25, 2.5, 5, 10 %w /v) of seven poly(ethylene glycol) (PEG) species varying in MW.M acroporosity was assessed by measuring the transmittance of each material at 400 nm, which decreases owing to increased light scattering as materials become more macroporous (see Figure S1 in the Supporting Information).…”

“…Our initial goal was to identify an appropriate porous material for entrapping concatemeric aptamers.T he morphology of sol-gel materials is affected by several parameters that control gelation time and phase separation, which include types of silica precursors and porogens,reaction pH and ionic strength. [3a,c,9] Fort his reason, we adapted ap reviously reported screening approach [9] to identify suitable compositions that could retain concatemeric aptamers with minimal leaching, allow concatemer accessibility to both small molecule and protein targets,p roduce self-supporting monolithic capillary columns,a nd allow pressure-driven flow through acapillary column with low backpressure.…”