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

Microarrays have found use in the development of high-throughput assays for new materials and discovery of small-molecule drug leads. Herein we describe a ...

McMaster University 

Monodisperse silica core-shell particles with a high surface area and large pore size were rapidly prepared via a multilayer-by-multilayer (ML-b-ML) process, with 6-7 layers of silica nanoparticles deposited per coating cycle onto an oppositely charged polyelectrolyte in 0.1 M NH(4)NO(3) at pH 2.7. The resultant porous shells show much greater porosity compared to particles obtained by the traditional layer-by-layer process.

Rapid fabrication of core-shell silica particles using a multilayer-by-multilayer approach.

Structure-switching signaling aptamers (ss-aptamers) are single-stranded DNA molecules that are generated through in vitro selection and have the ability to switch between a duplex composed of a quencher-labeled DNA strand (QDNA) hybridized adjacent to a fluorophore label on the aptamer, and an aptamer-target complex wherein the QDNA strand is released, generating a fluorescence signal. While such species have recently emerged as promising biological recognition and signaling elements, very little has been done to evaluate their potential for solid-phase assays. In this study, we demonstrate that high surface area, sol-gel-derived macroporous silica films are suitable platforms for high-density affinity-based immobilization of functional ss-aptamer molecules, allowing for binding of both large and small target analytes with robust signal development. These films are formed using a poly(ethylene glycol) (PEG)-doped sodium silicate material, and we show that it is possible to control the pore size distribution and surface area of the silica film by varying the amount of PEG. Materials with the highest surface area are shown to be able to immobilize up to 6-fold more ss-aptamer than planar glass surfaces, providing greater detection sensitivity and somewhat improved detection limits as compared to immobilization on conventional glass. The solid-phase assay is performed using two different structure-switching signaling aptamers with high selectivity for adenosine 5'-triphosphate and platelet-derived growth factor, respectively, demonstrating that this immobilization scheme should be suitable for a variety of target ligands.

Surface immobilization of structure-switching DNA aptamers on macroporous sol-gel-derived films for solid-phase biosensing applications.

A method is described for identifying bioactive compounds in complex mixtures based on the use of capillary-scale monolithic enzyme-reactor columns for rapid screening of enzyme activity. A two-channel nanoLC system was used to continuously infuse substrate coupled with automated injections of substrate/small molecule mixtures, optionally containing the chromogenic Ellman reagent, through sol-gel derived acetylcholinesterase (AChE) doped monolithic columns. This is the first report of AChE encapsulated in monolithic silica for use as an immobilized enzyme reactor (IMER), and the first use of such IMERs for mixture screening. AChE IMER columns were optimized to allow rapid functional screening of compound mixtures based on changes in the product absorbance or the ratio of mass spectrometric peaks for product and substrate ions in the eluent. The assay had robust performance and produced a Z' factor of 0.77 in the presence of 2% (v/v) DMSO. A series of 52 mixtures consisting of 1040 compounds from the Canadian Compound Collection of bioactives was screened and two known inhibitors, physostigmine and 9-aminoacridine, were identified from active mixtures by manual deconvolution. The activity of the compounds was confirmed using the enzyme reactor format, which allowed determination of both IC(50) and K(I) values. Screening results were found to correlate well with a recently published fluorescence-based microarray screening assay for AChE inhibitors.

Continuous flow immobilized enzyme reactor-tandem mass spectrometry for screening of AChE inhibitors in complex mixtures.

High surface area, sol-gel derived macroporous silica films doped with gold nanoparticles (AuNP) are used as a platform for high-density affinity-based immobilization of functional structure-switching DNA aptamer molecules onto Michelson interferometer long-period grating (LPG) fiber sensors, allowing for label-free detection of small molecular weight analytes such as adenosine triphosphate (ATP). The high surface area afforded by the sol-gel derived material allowed high loading of DNA aptamers, while the inclusion of gold nanoparticles within the silica film provided a high refractive index (RI) overlay, which is required to enhance the sensitivity of the LPG sensor according to our numerical simulations. By using a structure-switching aptamer construct that could release an oligonucleotide upon binding of ATP, the effective change in RI was both enhanced and inverted (i.e., binding of ATP caused a net reduction in molecular weight and refractive index), resulting in a system that prevented signals originating from nonspecific binding. This is the first report on the coupling of aptamers to LPG fiber sensors and the first use of high RI AuNP/silica films as supports to immobilize biomolecules onto the LPG sensor surface. The dual functionality of such films to both improve binding density and LPG sensor cladding refractive index results in a substantial enhancement in the sensitivity of such sensors for small molecule detection.

Enhancing sensitivity and selectivity of long-period grating sensors using structure-switching aptamers bound to gold-doped macroporous silica coatings.

We demonstrate a novel approach for rapid, selective, and sensitive detection of heavy metals using a solid-phase bioactive lab-on-paper sensor that is inkjet printed with sol-gel entrapped reagents to allow colorimetric visualization of the enzymatic activity of Î²-galactosidase (B-GAL). The bioactive paper assay is able to detect a range of heavy metals, either alone or as mixtures, in as little as 10 min, with detection limits as follows: Hg(II) = 0.001 ppm; Ag(I) = 0.002 ppm, Cu(II) = 0.020 ppm; Cd(II) = 0.020 ppm; Pb(II) = 0.140 ppm; Cr(VI) = 0.150 ppm; Ni(II) = 0.230 ppm. The paper-based assay was immune to interferences from nontoxic metal ions such as Na(+) or K(+), could be used to detect heavy metals that were spiked into tap water or lake water, and provided quantitative data that was in agreement with values obtained by atomic absorption. With the incorporation of standard chromogenic metal sensing reagents into a multiplexed bioactive paper sensor, it was possible to identify specific metals in mixtures, albeit with much lower detection limits than were obtained with the enzymatic assay. The paper-based sensor should be valuable for rapid, on-site screening of trace levels of heavy metals in resource limited areas and developing countries.

Î²-Galactosidase-based colorimetric paper sensor for determination of heavy metals.

The stability of a paper-immobilized antibody was investigated over a range of temperatures (40-140 Â°C) and relative humidities (RH, 30-90%) using both unmodified filter paper and the same paper impregnated with polyamide-epichlorohydrin (PAE) as supports. Antibody stability decreased with increasing temperature, as expected, but also decreased with increasing RH. At 40 Â°C, the half-life was more than 10 days, with little dependence on RH. However, at 80 Â°C, the half-life varied from ~3 days at low RH to less than half an hour at 90% RH, demonstrating that hydration of the antibody promotes unfolding. Antibody stability was not influenced by the PAE paper surface treatment. This work shows that antibodies are good candidates for development of bioactive paper as they have sufficient stability at high temperature to withstand printing and other roll-to-roll processing steps, and sufficient low temperature stability to allow long-term storage of bioactive paper materials.

Effects of temperature and relative humidity on the stability of paper-immobilized antibodies.

Rapid, sensitive, on-site detection of bacteria without a need for sophisticated equipment or skilled personnel is extremely important in clinical settings and rapid response scenarios, as well as in resource-limited settings. Here, we report a novel approach for selective and ultra-sensitive multiplexed detection of Escherichia coli (non-pathogenic or pathogenic) using a lab-on-paper test strip (bioactive paper) based on intracellular enzyme (Î²-galactosidase (B-GAL) or Î²-glucuronidase (GUS)) activity. The test strip is composed of a paper support (0.5 Ã— 8 cm), onto which either 5-bromo-4-chloro-3-indolyl-Î²-D: -glucuronide sodium salt (XG), chlorophenol red Î²-galactopyranoside (CPRG) or both and FeCl(3) were entrapped using sol-gel-derived silica inks in different zones via an ink-jet printing technique. The sample was lysed and assayed via lateral flow through the FeCl(3) zone to the substrate area to initiate rapid enzyme hydrolysis of the substrate, causing a change from colorless-to-blue (XG hydrolyzed by GUS, indication of nonpathogenic E. coli) and/or yellow to red-magenta (CPRG hydrolyzed by B-GAL, indication of total coliforms). Using immunomagnetic nanoparticles for selective preconcentration, the limit of detection was ~5 colony-forming units (cfu) per milliliter for E. coli O157:H7 and ~20 cfu/mL for E. coli BL21, within 30 min without cell culturing. Thus, these paper test strips could be suitable for detection of viable total coliforms and pathogens in bathing water samples. Moreover, inclusion of a culturing step allows detection of less than 1 cfu in 100 mL within 8 h, making the paper tests strips relevant for detection of multiple pathogens and total coliform bacteria in beverage and food samples.

Multiplexed paper test strip for quantitative bacterial detection.

Structure-switching, fluorescence-signaling DNA and RNA aptamers have been reported as highly versatile molecular recognition elements for biosensor development. While structure-switching DNA aptamers have been utilized for solid-phase sensing, equivalent RNA aptamers have yet to be successfully utilized in solid-phase sensors due to their lack of chemical stability and susceptibility to nuclease attack. In this study, we examined entrapment into sol-gel derived organic-inorganic composite materials as a platform for immobilization of structure-switching fluorescence-signaling RNA aptamer reporters, using both the synthetic theophylline- and naturally occurring thiamine pyrophosphate-binding RNA aptamers as test cases. Structure-switching versions of both aptamers were entrapped into a series of sol-gel derived composites, ranging from highly polar silica to hydrophobic methylsilsesquioxane-based materials, and the target-binding and signaling capabilities of these immobilized aptamers were assessed relative to solution. Both immobilized aptamers demonstrated sensitivity and selectivity similar to that of free aptamers when entrapped in a composite material derived from 40% (v/v) methyltrimethoxysilane/tetramethoxysilane. Importantly, this material also conferred protection from nuclease degradation and imparted long-term chemical stability to the RNA reporter systems. Given the versatility of sol-gel entrapment for development of biosensors, microarrays, bioaffinity columns, and other devices, this entrapment method should provide a useful platform for numerous solid-phase RNA aptamer-based devices.

Stabilizing structure-switching signaling RNA aptamers by entrapment in sol-gel derived materials for solid-phase assays.

This paper reports the development of a method to control the flow rate of fluids within paper-based microfluidic analytical devices. We demonstrate that by simply sandwiching paper channels between two flexible films, it is possible to accelerate the flow of water through paper by over 10-fold. The dynamics of this process are such that the height of the liquid is dependent on time to the power of 1/3. This dependence was validated using three different flexible films (with markedly different contact angles) and three different fluids (water and two silicon oils with different viscosities). These covered channels provide a low-cost method for controlling the flow rate of fluid in paper channels, and can be added following printing of reagents to control fluid flow in selected fluidic channels. Using this method, we redesigned a previously published bidirectional lateral flow pesticide sensor to allow more rapid detection of pesticides while eliminating the need to run the assay in two stages. The sensor is fabricated with sol-gel entrapped reagents (indoxyl acetate in a substrate zone and acetylcholinesterase, AChE, in a sensing zone) present in an uncovered "slow" flow channel, with a second, covered "fast" channel used to transport pesticide samples to the sensing region through a simple paper-flap valve. In this manner, pesticides reach the sensing region first to allow preincubation, followed by delivery of the substrate to generate a colorimetric signal. This format results in a uni-directional device that detects the presence of pesticides two times faster than the original bidirectional sensors.

Creating fast flow channels in paper fluidic devices to control timing of sequential reactions.

Aminoglycoside phosphotransferase 3'IIIa (APH3'IIIa) is a bacterial enzyme involved in antibiotic resistance through phosphorylation of aminoglycosides, which can potentially be overcome by co-administration of an APH3'IIIa inhibitor with the antibiotic. Current assay methods for discovery of APH3'IIIa inhibitors suffer from low specificity and high false positive/negative hit rates. Here, we describe a method for screening APH3'IIIa inhibitors based on direct detection of kanamycin A phosphorylation using MALDI-MS/MS, which is more rapid than conventional assays and does not require secondary assays or sample cleanup. The MALDI-MS/MS assay operates at an ionic strength of 45 mM and co-factors can be utilized at near-physiological levels for optimal enzyme activity. Detection via MALDI-MS/MS allowed for improved reproducibility when compared to ESI-MS/MS. Furthermore, the use of MS/MS provided better signal-to-noise ratios relative to MS alone on the MALDI instrument. The assay was validated via generation of Z'-factors, with values of 0.78 and 0.56 in the absence and presence of 0.2% DMSO, respectively. The assay was used to screen a kinase directed library of >200 compounds, assayed as 21 mixtures of 10 compounds each. Five novel synthetic inhibitors were identified following mixture deconvolution. Inhibition constants were obtained for the aforementioned inhibitors using the MALDI-MS/MS assay, revealing several low to mid micromolar "hits", and highlighting the quantitative nature of the assay.

A matrix-assisted laser desorption/ionization tandem mass spectrometry method for direct screening of small molecule mixtures against an aminoglycoside kinase.

Efficient DNA repair mechanisms frequently limit the effectiveness of chemotherapeutic agents that act through DNA damaging mechanisms. Consequently, proteins involved in DNA repair have increasingly become attractive targets of high-throughput screening initiatives to identify modulators of these pathways. Disruption of the XRCC4-Ligase IV interaction provides a novel means to efficiently halt repair of mammalian DNA double strand break repair, however the extreme affinity of these proteins presents a major obstacle for drug discovery. A better understanding of the interaction surfaces is needed to provide a more specific target for inhibitor studies. To clearly define key interface(s) of Ligase IV necessary for interaction with XRCC4, we developed a competitive displacement assay using ESI-MS/MS and determined the minimal inhibitory fragment of the XRCC4-interacting region (XIR) capable of disrupting a complex of XRCC4/XIR. Disruption of a single helix (helix 2) within the helix-loop-helix clamp of Ligase IV was sufficient to displace XIR from a pre-formed complex. Dose-dependent response curves for the disruption of the complex by either helix 2 or helix-loop-helix fragments revealed that potency of inhibition was greater for the larger helix-loop-helix peptide. Our results suggest a susceptibility to inhibition at the interface of helix 2 and future studies would benefit from targeting this surface of Ligase IV to identify modulators that disrupt its interaction with XRCC4. Furthermore, helix 1 and loop regions of the helix-loop-helix clamp provide secondary target surfaces to identify adjuvant compounds that could be used in combination to more efficiently inhibit XRCC4/Ligase IV complex formation and DNA repair. Â© Proteins 2013;. Â© 2013 Wiley Periodicals, Inc.

John D. Brennan

Department of Chemistry and Chemical Biology

McMaster University

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