A Sol−Gel-Derived Acetylcholinesterase Microarray for Nanovolume Small-Molecule Screening

Abstract

A fluorimetric acetylcholinesterase (AChE) assay was developed and characterized both in solution and with the enzyme entrapped in sol-gel-derived silica. The assay is based on a disulfide-thiol interchange reaction between the intramolecularly quenched dimeric dye BODIPY FL l-cystine and thiocholine generated by the AChE-catalyzed hydrolysis of acetylthiocholine (ATCh), which results in a brightly fluorescent monomeric product owing to the cleavage of the disulfide-coupled form of the dye. The new assay was validated by comparison with the Ellman assay performed under parallel conditions and was used in both kinetic and end point assays. The assay was extended to the fabrication of functional AChE microarrays using contact pin-printing of sol-gel-derived silica. A total of 392 sol-gel formulations were screened for gelation times and 192 of these were further evaluated for array fabrication on four different surfaces using a factor analysis approach. Of these, 66 sol-gel/surface combinations produced robust microarrays, while 26 sol-gel/surface combinations were identified that could produce highly active AChE microarrays. The Z' factor for the on-array assay using an optimal sol-gel/surface combination, which considers both signal variability and difference in signals between positive and negative controls, was determined to be 0.60, which is above the minimum level required for applicability to screening. By overprinting nanoliter volumes of solutions containing the dye, ATCh, and potential inhibitors, these microarrays could be used to screen two libraries of small molecules, one composed of newly synthesized alkaloids and another consisting of ∼1000 known bioactive compounds, both as discrete compounds and mixtures thereof, for activity against AChE. IC(50) values were obtained on microarrays for compounds showing significant inhibitory activity, demonstrating the utility of arrays for quantitative inhibition assays.

A Guided Materials Screening Approach for Developing Quantitative Sol-gel Derived Protein Microarrays

“…5 To validate the assays, quantitative inhibition data were obtained using known and unknown AChE inhibitors, with results performed in duplicate and used to produce duplicate plots ( Figure 6A) and inhibition curves (Figures 6B and 6C). Spots were first overprinted with mixtures of known biologically active small-molecule inhibitors then with dye and substrate, and control mixtures containing either known inhibitors or no inhibitor were included.…”

“…Since the development of microarray technology to assess gene expression 1 in the mid-1990s, microarrays have found use in the development of high-throughput assays to identify protein-protein and protein-small molecule interactions, and to find new materials with unique properties. [2][3][4][5] More recently, microarrays have been developed wherein specific materials are used to immobilize functional proteins, producing a three-dimensional microarray element within which proteins are entrapped, allowing for facile measurement of enzymatic activity and inhibition on the microarray itself using a suitable fluorescence assay that is coupled to substrate turnover. [5][6][7][8][9][10] Importantly, such microarrays can be designed to include all necessary components to screen samples and controls together in a highly paralleled fashion.…”

“…[2][3][4][5] More recently, microarrays have been developed wherein specific materials are used to immobilize functional proteins, producing a three-dimensional microarray element within which proteins are entrapped, allowing for facile measurement of enzymatic activity and inhibition on the microarray itself using a suitable fluorescence assay that is coupled to substrate turnover. [5][6][7][8][9][10] Importantly, such microarrays can be designed to include all necessary components to screen samples and controls together in a highly paralleled fashion. 5,8 Early examples of protein microarrays were typically prepared using standard methods for protein immobilization onto solid supports, such as covalent attachment, 11 affinity capture 3 and physical adsorption.…”

“…[5][6][7][8][9][10] Importantly, such microarrays can be designed to include all necessary components to screen samples and controls together in a highly paralleled fashion. 5,8 Early examples of protein microarrays were typically prepared using standard methods for protein immobilization onto solid supports, such as covalent attachment, 11 affinity capture 3 and physical adsorption. 12 While these methods of bio-immobilization allow for increased sample concentration and accelerated reaction kinetics required for assay miniaturization, they each suffer drawbacks.…”

Immobilization of Proteins in Silica Gel: Biochemical and Biophysical Properties

“…Several additives can be added to the sol-gel in different phases of the preparation, resulting in "tailored materials" suitable for a number of proteins and detection systems [50]. Table 1 summarizes some applications of silica gel-encapsulated proteins reported in the literature [183,194,[200][201][202][203][204][205][206][207][208][209][210][211][212][213][214][215][216][217]. Notable examples include cases in which more proteins are co-encapsulated to immobilize short metabolic pathways [194].…”