A filter paper-based assay for laboratory evolution of hydrolases and dehydrogenases

Industrially important enzyme classes such as hydrolases and dehydrogenases are often not amenable to laboratory evolution methods due to a lack of sensitive and reliable high-throughput screening (HTS) systems. We developed a conceptually novel and technically simple high-throughput screening system based on detection of volatile aldehydes with the sensitive reagent Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole). The aldehyde detection takes place on a filter-paper that is pre-soaked with Purpald and covers the microtiter plate. The filter paper-based Purpald assay separates aldehyde detection from biocatalytical conversion and thereby avoids interferences from biological materials with assay components. This screening principle allows, to our knowledge, for the first time to determine the synthetic activity of hydrolases such as lipases and esterases in organic solvents in a 96-well whole-cell format. Its simplicity and cost-effectiveness make the reported HTS system suitable as fast pre-screen in laboratory evolution experiments and for semi-quantitative assays of improved mutants.     

Fluorescence polarization is a useful technology for reagent reduction in assay miniaturization

The use of fluorescence polarization (FP) has increased significantly in the development of sensitive and robust assays for high throughput screening of chemical compound libraries during the past few years. In this study, we show that FP is a useful assay miniaturization technology for reagent reduction during high throughput screening. We developed and optimized several FP assays for binding to estrogen receptor alpha and two protein kinases with an assay volume of 100 microl. Without any re-optimization, a consistent signal window was maintained in 384- or 1536-well format when the assay volume varied from 2.5-100 microl at constant concentrations of all assay components. In contrast, the signal window decreased with decreasing assay volume at constant reagent concentration in the protein kinase C scintillation proximity assay (SPA) and prompt fluorescence assay. In addition, the effect of evaporation on the signal window was minimal for the FP assays. Our study suggests that FP is superior to SPA and prompt fluorescence in terms of reagent reduction in the miniaturized assay format.     

A digital microfluidic method for multiplexed cell-based apoptosis assays

Digital microfluidics (DMF), a fluid-handling technique in which picolitre-microlitre droplets are manipulated electrostatically on an array of electrodes, has recently become popular for applications in chemistry and biology. DMF devices are reconfigurable, have no moving parts, and are compatible with conventional high-throughput screening infrastructure (e.g., multiwell plate readers). For these and other reasons, digital microfluidics has been touted as being a potentially useful new tool for applications in multiplexed screening. Here, we introduce the first digital microfluidic platform used to implement parallel-scale cell-based assays. A fluorogenic apoptosis assay for caspase-3 activity was chosen as a model system because of the popularity of apoptosis as a target for anti-cancer drug discovery research. Dose-response profiles of caspase-3 activity as a function of staurosporine concentration were generated using both the digital microfluidic method and conventional techniques (i.e., pipetting, aspiration, and 96-well plates.) As expected, the digital microfluidic method had a 33-fold reduction in reagent consumption relative to the conventional technique. Although both types of methods used the same detector (a benchtop multiwell plate reader), the data generated by the digital microfluidic method had lower detection limits and greater dynamic range because apoptotic cells were much less likely to de-laminate when exposed to droplet manipulation by DMF relative to pipetting/aspiration in multiwell plates. We propose that the techniques described here represent an important milestone in the development of digital microfluidics as a useful tool for parallel cell-based screening and other applications.     

A miniaturized glucocorticoid receptor translocation assay using enzymatic fragment complementation evaluated with qHTS

Nuclear translocation is an important step in glucocorticoid receptor (GR) signaling and assays that measure this process allow the identification of nuclear receptor ligands independent of subsequent functional effects. To facilitate the identification of GR-translocation agonists, an enzyme fragment complementation (EFC) cell-based assay was scaled to a 1536-well plate format to evaluate 9,920 compounds using a quantitative high throughput screening (qHTS) strategy where compounds are assayed at multiple concentrations. In contrast to conventional assays of nuclear translocation the qHTS assay described here was enabled on a standard luminescence microplate reader precluding the requirement for imaging methods. The assay uses beta-galactosidase alpha complementation to indirectly detect GR-translocation in CHO-K1 cells. 1536-well assay miniaturization included the elimination of a media aspiration step, and the optimized assay displayed a Z' of 0.55. qHTS yielded EC(50) values for all 9,920 compounds and allowed us to retrospectively examine the dataset as a single concentration-based screen to estimate the number of false positives and negatives at typical activity thresholds. For example, at a 9 microM screening concentration, the assay showed an accuracy that is comparable to typical cell-based assays as judged by the occurrence of false positives that we determined to be 1.3% or 0.3%, for a 3sigma or 6sigma threshold, respectively. This corresponds to a confirmation rate of approximately 30% or approximately 50%, respectively. The assay was consistent with glucocorticoid pharmacology as scaffolds with close similarity to dexamethasone were identified as active, while, for example, steroids that act as ligands to other nuclear receptors such as the estrogen receptor were found to be inactive.     

Surface modification for enhancing antibody binding on polymer-based microfluidic device for enzyme-linked immunosorbent assay

A novel surface treatment method using poly(ethyleneimine) (PEI), an amine-bearing polymer, was developed to enhance antibody binding on the poly(methyl methacrylate) (PMMA) microfluidic immunoassay device. By treating the PMMA surface of the microchannel on the microfluidic device with PEI, 10 times more active antibodies can be bound to the microchannel surface as compared to those without treatment or treated with the small amine-bearing molecule, hexamethylenediamine (HMD). Consequently, PEI surface modification greatly improved the immunoassay performance of the microfluidic device, making it more sensitive and reliable in the detection of IgG. The improvement can be attributed to the spacer effect as well as the functional amine groups provided by the polymeric PEI molecules. Due to the smaller dimensions (140x125 microm) of the microchannel, the time required for antibody diffusion and adsorption onto the microchannel surface was reduced to only several minutes, which was 10 times faster than the similar process carried out in 96-well plates. The microchip also had a wider detection dynamic range, from 5 to 1000 ng/mL, as compared to that of the microtiter plate (from 2 to 100 ng/mL). With the PEI surface modification, PMMA-based microchips can be effectively used for enzyme linked immunosorbent assays (ELISA) with a similar detection limit, but much less reagent consumption and shorter assay time as compared to the conventional 96-well plate.     

Transitioning enantioselective indicator displacement assays for alpha-amino acids to protocols amenable to high-throughput screening

Enantioselective indicator displacement assays (eIDAs) for alpha-amino acids were conducted in a 96-well plate format to demonstrate the viability of the technique for the high-throughput screening (HTS) of enantiomeric excess (ee) values. Chiral receptors [Cu(II)(1)](2+) and [Cu(II)(2)](2+) with the indicator chrome azurol S were implemented for the eIDAs. Enantiomeric excess calibration curves were made using both receptors and then used to analyze true test samples. These results were compared to those previously obtained with a conventional UV-vis spectrophotometer, and they showed little to no loss of accuracy, while the speed of analysis was increased. A sample of valine of unknown ee was synthesized through an asymmetric reaction to produce a realistic reaction sample, which was analyzed using receptor [Cu(II)(1)](2+). The experimentally determined ee using our eIDA was compared to that obtained by chiral HPLC and (1)H NMR chiral shift reagent analysis. This gave errors of 4.7% and 12.0%, respectively. In addition to the use of ee calibration curves, an artificial neural network (ANN) was used to determine the % L-amino acid of the test samples and of the sample of valine of unknown ee from the asymmetric reaction. This method obtained errors of 5.9% and 2.2% compared to chiral HPLC and (1)H NMR chiral shift reagent analysis, respectively. The technique using calibration curves for the determination of ee on a 96-well plate allows one to determine 96 ee values in under a minute, enabling its use for HTS of asymmetric reactions with acceptable accuracy.     

An integrated enzyme-linked immunosorbent assay system with an organic light-emitting diode and a charge-coupled device for fluorescence detection

A fluorescence detection system for a microfluidic device using an organic light-emitting diode (OLED) as the excitation light source and a charge-coupled device (CCD) as the photo detector was developed. The OLED was fabricated on a glass plate by photolithography and a vacuum deposition technique. The OLED produced a green luminescence with a peak emission at 512 nm and a half bandwidth of 55 nm. The maximum external quantum efficiency of the OLED was 7.2%. The emission intensity of the OLED at 10 mA/cm(2) was 13 μW (1.7 mW/cm(2)). The fluorescence detection system consisted of the OLED device, two band-pass filters, a five microchannel poly(dimethylsiloxane) (PDMS) microfluidic device and a linear CCD. The fluorescence detection system was successfully used in a flow-based enzyme-linked immunosorbent assay on a PDMS microfluidic device for the rapid determination of immunoglobulin A (IgA), a marker for human stress. The detection limit (S/N=3) for IgA was 16.5 ng/mL, and the sensitivity was sufficient for evaluating stress. Compared with the conventional 96-well microtiter plate assay, the analysis time and the amounts of reagent and sample solutions could all be reduced.     

In situ DMSO hydration measurements of HTS compound libraries

Compounds used in high throughput screening (HTS) are typically dissolved in DMSO. These solutions are stored automation-friendly racks of wells or tubes. DMSO is hygroscopic and quickly absorbs water from the atmosphere. When present in DMSO compound solutions, water can accelerate degradation and precipitation. Understanding DMSO hydration in an HTS compound library can improve storage and screening methods by managing the impact of water on compound stability. A non-destructive, acoustic method compatible with HTS has been developed to measure water content in DMSO solutions. Performance of this acoustic method was compared with an optical technique and found to be in good agreement. The accuracy and precision of acoustic measurements was shown to be under 3% over the tested range of DMSO solutions (0% to 35% water by volume) and insensitive to the presence of HTS compounds at typical storage concentrations. Time course studies of hydration for wells in 384-well and 1536-well microplates were performed. Well geometry, fluid volume, well position and atmospheric conditions were all factors in hydration rate. High rates of hydration were seen in lower-volume fills, higher-density multi-well plates and when there was a large differential between the humidity of the lab and the water content of the DMSO. For example, a 1536-well microplate filled with 2microL of 100% DMSO exposed for one hour to a laboratory environment with approximately 40% relative humidity will absorb over 6% water by volume. Understanding DMSO hydration rates as well as the ability to reverse library hydration are important steps towards managing stability and availability of compound libraries.     

Droplet-based microfluidic platforms for the encapsulation and screening of Mammalian cells and multicellular organisms

High-throughput, cell-based assays require small sample volumes to reduce assay costs and to allow for rapid sample manipulation. However, further miniaturization of conventional microtiter plate technology is problematic due to evaporation and capillary action. To overcome these limitations, we describe droplet-based microfluidic platforms in which cells are grown in aqueous microcompartments separated by an inert perfluorocarbon carrier oil. Synthesis of biocompatible surfactants and identification of gas-permeable storage systems allowed human cells, and even a multicellular organism (C. elegans), to survive and proliferate within the microcompartments for several days. Microcompartments containing single cells could be reinjected into a microfluidic device after incubation to measure expression of a reporter gene. This should open the way for high-throughput, cell-based screening that can use >1000-fold smaller assay volumes and has approximately 500x higher throughput than conventional microtiter plate assays.     

Evaluation of 384-well formatted sample preparation technologies for regulated bioanalysis

The capabilities and limitations of 384-well formatted sample preparation technologies applied to regulated bioanalysis were evaluated by developing two assays for the simultaneous quantitation of lopinavir and ritonavir, the active ingredients of Kaletra. One method used liquid-liquid extraction (LLE), and the other used solid-phase extraction (SPE). The steps and apparatuses employed by the two methods covered most of those used for bioanalysis. Briefly, the previously validated 96-well formatted assays were adapted to the 384-format with minor modifications. Because the wells of a 384-well plate are clustered together, cross-contamination between adjacent wells was evaluated critically, along with sensitivity, assay throughput, and ruggedness. Samples (35 microL) containing plasma samples (15 microL), internal standard (10 microL), and sodium carbonate (0.5 M, 10 microL to basify the sample) were placed in a 384-well microtiter plate that may contain saquinavir or amprenavir as contamination markers. For LLE preparation, the samples were placed in a deep 384-well plate (300-microL well volume) and extracted with 150 microL of ethyl acetate. Approximately 50 microL of the extracts were removed from each well after phase separation for analysis. For SPE preparation, the fortified samples were transferred to a 384-formatted SPE plate (C18, 5 mg packing). The extracts were eluted from the plate with basified 2-propanol. The LLE or SPE extracts were dried and reconstituted for column-switching high-performance liquid chromatography with tandem mass spectrometric detection (HPLC/MS/MS). The lower limit of quantitation and the assay range were the same as the 96-well formatted assay. If combined with appropriate automation, sample preparation in the 384-well format would be up to five times more efficient than the 96-well format.     

Miniaturization of a panel of high-throughput yeast-cell-based nuclear receptor assays in 384- and 1536-well microplates

A panel of luminescent Saccharomyces cerevisiae cell-based nuclear receptor assays, consisting of human estrogen receptors α and β, androgen receptor, and aryl hydrocarbon receptor, was miniaturized from the standard 96-well microplate format to high-throughput 384- and 1536-well microplate formats. In these assays, firefly luciferase lacking the peroxisome targeting sequence was used as a reporter and D-luciferin substrate was pre-mixed with the yeast cells before the incubation step, eliminating cell lysis and substrate addition steps, and allowing multiple readings at any desired time point. All of the assays were highly functional in the 384-well format, and most functioned well in the 1536-well format. The detection limit of the estrogen receptor α assay was even lower in the miniaturized microplate formats than in the original 96-well format. The panel of yeast-cell-based nuclear receptor assays can be used for high-throughput chemical testing and environmental monitoring of potential endocrine-disrupting activity of compounds and samples.     

A fluorescent high-throughput assay for double-stranded DNA: the RediPlate PicoGreen assay

The fluorescent PicoGreen reagent for detection and quantitation of double-stranded DNA has been adapted for high-throughput screening: the RediPlate PicoGreen double-stranded DNA assay format. In the RediPlate PicoGreen assay format, the PicoGreen reagent is predistributed and co-dried into either 96- or 384-well microplates with the excipient trehalose. The user resuspends the dried reagents upon adding DNA, and measures the resulting fluorescence after a five minute incubation. Replicate fluorescence measurements on nominally identical wells have less than a 5% coefficient of variation. The assay is linear from 5 to 500 ng/ml DNA in a 200 micro l volume. The RediPlate PicoGreen assay format retains the advantages of the original PicoGreen reagent - sensitivity, speed, and specificity - but in a high-throughput format.     

Adhesive microarrays for multipurpose diagnostic tools

We are reporting here a new technology for the straightforward production of integrated microarrays. The approach is based on the use of adhesive supports enabling (i) the immobilization of biomolecules as microarrays (up to 2500 spots per cm(2)) and (ii) the easy assembly of these microarrays with complex 3D structures such as 96-well bottomless microplates or polymer and glass microfluidic networks. The analytical performances of the system were demonstrated for sandwich protein detection (C-reactive protein) and hybridization assays, both in classical 96-well microplate format and microfluidic environment.     

High-density microfluidic arrays for cell cytotoxicity analysis

In this paper, we report on the development of a multilayer elastomeric microfluidic array platform for the high-throughput cell cytotoxicity screening of mammalian cell lines. Microfluidic channels in the platform for cell seeding are orthogonal to channels for toxin exposure, and within each channel intersection is a circular chamber with cell-trapping sieves. Integrated, pneumatically-actuated elastomeric valves within the device isolate the microchannel array within the device into parallel rows and columns for cell seeding and toxin exposure. As a demonstration of the multiplexing capability of the platform, a microfluidic array containing 576 chambers was used to screen three cell types (BALB/3T3, HeLa, and bovine endothelial cells) against a panel of five toxins (digitonin, saponin, CoCl(2), NiCl(2), acrolein). Evaluation of on-chip cell morphology and viability was carried out using fluorescence microscopy, with outcomes comparable to microtiter plate cytotoxicity assays. Using this scalable platform, cell seeding and toxin exposure can be carried out within a single microfluidic device in a multiplexed format, enabling high-density parallel cytotoxicity screening while minimizing reagent consumption.     

Automated high content screening for phosphoinositide 3 kinase inhibition using an AKT 1 redistribution assay

High Content Screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modelling systems. In this work, we established a medium to high throughput HCS assay in the 384-well format to measure cellular type I phosphoinositide 3 kinase (PI3K) activity. Type I PI3K is involved in several intracellular pathways such as cell survival, growth and differentiation as well as immunological responses. As a cellular model system we used Chinese Hamster Ovary (CHO) cells that had been stably transfected with human insulin receptor (hIR) and an AKT1-enhanced green fluorescent protein (EGFP) fusion construct. Upon stimulation of the hIR with insulin-like growth factor-1 (IGF-1), PI3K was activated to phosphorylate phosphatidylinositol (PtdIns)-4,5-bisphosphate at the 3-position, resulting in the recruitment of AKT1-EGFP to the plasma membrane. The AKT1-EGFP redistribution assay was robust and displayed little day-to-day variability, the quantification of the fluorescence intensity associated with plasma membrane spots delivered good Z' statistics. A novel format of compound dose-response testing was employed using serial dilutions of test compounds across consecutive microtiter plates (MTPs). The dose response testing of a PI3K inhibitor series provided reproducible IC50 values. The profiling of the redistribution assay with isoform-selective inhibitors indicates that PI3Kalpha is the main isoform activated in the CHO host cells after IGF-1 stimulation. Toxic compound side effects could be determined using automated image analysis. We conclude that the AKT1-EGFP redistribution assay represents a solid medium/high throughput screening (MTS/HTS) format to determine the cellular activity of PI3K inhibitors under conditions of growth factor stimulation.     

Peptide protein binding assay using ImageFlashPlates or Imaging Beads

Imaging devices used for the measurement of radioligand-receptor binding assays are typically based on charge-coupled device (CCD) cameras, which are more sensitive for red-shifted scintillation. In the past, red-shifted scintillants had only been integrated into microspheres, referred to as scintillation proximity assay (SPA) Imaging Beads. More recently, ImageFlashPlates have been developed that emit light at 615 nm when exposed to beta-radiation. In this article, we report the establishment of peptide-protein binding assays using either streptavidin-coated ImageFlashPlates or Imaging Beads in a low volume 384-well format. In these assays, we employed a biotinylated peptide X and a [33P]-phosphorylated protein Y as the binding partner. The FlashPlates required a washing step, the bead-filled microtiter plates (MTPs) needed a centrifugation step for optimal performance in the scintillation measurements. Both the peptide X-loaded FlashPlates and the beads displayed saturable binding of [33P]-phosphorylated protein Y with a similar scintillation efficiency. A KD value of about 30 nmol/l was measured using the bead-based assay. Due to the washing step in the FlashPlate experiment, approximately two-thirds of the [33P]-phosphorylated protein Y were withdrawn from equilibrium binding. This resulted in correspondingly lower scintillation signals for the FlashPlate experiment. For this reason, the FlashPlate produced a Z' value of 0.64 that was lower than the Z' value of 0.87 for the beads. Using a reference inhibitor in a competition assay produced similar IC50 values for the bead-based assay as for the FlashPlate. Depending on the local automation environment either the centrifugation step for the beads or the washing step for the FlashPlates may be considered more or less of a challenge. Low volume 384-well high-throughput screening (HTS) applicable assay formats are achievable using either the ImageFlashPlates or the Imaging Beads.     

New fluorogenic probes for oxygen and carbene transfer: a sensitive assay for single bead-supported catalysts.

A high-throughput screening assay for atom transfer catalysis has been developed. This assay is based on two probes, developed herein, which generate highly fluorescent products upon carbene or oxygen atom transfer. The emission wavelength of probes 1 and 5 shift significantly (up to 90 nm) upon epoxidation, allowing detection of product at 3% conversion. Probe 7 is not fluorescent, while fluorescence emission by carbene insertion/rearrangement product 8 allows detection at less than 1% conversion. Such sensitivity allows for examination of single-bead reactions in a high throughput array format (1536 wells per plate), and provides a broad detection window ranging from single to high turnover numbers. Thousands of metal complexes are evaluated in a single screening experiment. Preliminary screening of a diverse ligand library with probe 7 in the presence of Rh(II) uncovered new catalysts capable of cyclopropanation and C-H insertion.     

A digital microfluidic approach to heterogeneous immunoassays

A digital microfluidic (DMF) device was applied to a heterogeneous sandwich immunoassay. The digital approach to microfluidics manipulates samples and reagents in the form of discrete droplets, as opposed to the streams of fluid used in microchannels. Since droplets are manipulated on relatively generic 2-D arrays of electrodes, DMF devices are straightforward to use, and are reconfigurable for any desired combination of droplet operations. This flexibility makes them suitable for a wide range of applications, especially those requiring long, multistep protocols such as immunoassays. Here, we developed an immunoassay on a DMF device using Human IgG as a model analyte. To capture the analyte, an anti-IgG antibody was physisorbed on the hydrophobic surface of a DMF device, and DMF actuation was used for all washing and incubation steps. The bound analyte was detected using FITC-labeled anti-IgG, and fluorescence after the final wash was measured in a fluorescence plate reader. A non-ionic polymer surfactant, Pluronic F-127, was added to sample and detection antibody solutions to control non-specific binding and aid in movement via DMF. Sample and reagent volumes were reduced by nearly three orders of magnitude relative to conventional multiwell plate methods. Since droplets are in constant motion, the antibody–antigen binding kinetics is not limited by diffusion, and total analysis times were reduced to less than 2.5 h per assay. A multiplexed device comprising several DMF platforms wired in series further increased the throughput of the technique. A dynamic range of approximately one order of magnitude was achieved, with reproducibility similar to the assay when performed in a 96-well plate. In bovine serum samples spiked with human IgG, the target molecule was successfully detected in the presence of a 100-fold excess of bovine IgG. It was concluded that the digital microfluidic format is capable of carrying out qualitative and quantitative sandwich immunoassays with a dramatic reduction in reagent usage and analysis time compared to macroscale methods.