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In this reported work, we achieved high-throughput, highly sensitive fluorescent analysis using an enzyme-linked immunosorbent assay (ELISA) that employed a metallic nanoparticle (NP)-immobilized 96-well plate. The immobilization of metallic NPs on a 96-well plate effectively amplified fluorescent signals of the assay. The silver (Ag) NP-immobilized plate showed the best fluorescent enhancement effect of all plates immobilized by metal NPs. Our results demonstrate the potential of applying Ag NPs to enhance the efficiency of direct and indirect ELISA by the labeling of targets.

Abstract We present a real-time, high-throughput, and cost-effective method of detecting apoptosis in vitro using a previously developed reagent that detects caspase activation by fluorescence. Current methods of assessing apoptosis fail to account for the dimension of time, and thus are limited in data yielded per sample. This reagent allows real-time detection of apoptosis, but until now has been restricted to a costly automated detection system. Here, we describe apoptosis detection with the Essen Bioscience IncuCyte® Caspase-3/7 Reagent using a multimode microplate reader, a common instrument in biological laboratories, which may be used prior to or in lieu of the automated system. This modified microplate reader apoptosis assay was validated against the established automated system, and was shown to detect a strong dose-response relationship (automated system r2 = 0.9968, microplate reader r2 = 0.9924). We also propose a quick and reliable method of quantifying cell density by Hoechst 33342 nuclear staining in microplates (r2 = 0.8812 between Hoechst signal and cell density). We assert that the dimension of time should not be overlooked, and that the method presented here is an accessible strategy for many researchers due to low startup cost and precise detection of apoptosis in real time. Highlights • Popular methods of apoptosis detection yield a single reading per sample • Accounting for kinetics of the apoptotic response provides more robust and precise data outputs • Current real time methods of apoptosis detection are either expensive or have low sample capacity • A high-throughput microplate reader based assay is shown here to accurately detect apoptosis in real time [ABSTRACT FROM AUTHOR]

Accurate methods that can continuously detect low concentrations of hydrogen peroxide (H2O2) have a huge application potential in biological, pharmaceutical, clinical and environmental analysis. Luminescent probes and nanomaterials are used for fabrication of sensors for H2O2 that can be applied for these purposes. In contrast to previous reviews focusing on the chemical design of molecular probes for H2O2, this mini-review highlights the latest luminescent nanoparticular materials and new luminescent optical sensors for H2O2 in terms of the nanomaterial composition and luminescent receptor used in the sensors. The nanomaterial section is subdivided into schemes based on gold nanoparticles, polymeric nanoparticles with embedded enzymes, probes showing aggregation-induced emission enhancement, quantum dots, lanthanide-based nanoparticles and carbon based nanomaterials, respectively. Moreover, the sensors are ordered according to the type of luminescent receptor used within the sensor membranes. Among them are lanthanide complexes, metal-ligand complexes, oxidic nanoparticles and organic dyes. Further, the optical sensors are confined to those that are capable to monitor the concentration of H2O2 in a sample over time or are reusable. Optical sensors responding to gaseous H2O2 are not covered. All nanomaterials and sensors are characterized with respect to the analytical reaction towards H2O2, limit of detection (LOD), analytical range, electrolyte, pH and response time/incubation time. Applications to real samples are given. Finally, we assess the suitability of the nanomaterials to be used in membrane-based sensors and discuss future trends and perspectives of these sensors in biomedical research.

There has been growing interest in fluorescence-based microplate methods to measure enzyme activities due to the sensitivity of fluorimetric detection and the potential for simultaneous and rapid assaying of multiple enzyme activities in the same soil suspension. However, micro-scale methods could introduce considerable operator error such as: 1) the requirement to put soil samples into a suspension; 2) the very small amounts of soil placed in each microplate well; 3) pipetting error because μL volumes are required; and 4) the need for standard curve calibration with every sample to account for quenching. For valid data comparison and interpretation, there is clearly a need to have a strict and agreed-upon enzyme assay protocol to standardize the microplate-based method. Therefore, the objectives were to: 1) determine the reproducibility and comparability of the standard p -nitrophenol bench-scale and 4-methylumbelliferone microplate enzyme assays measured by five laboratories for β-glucosidase (EC 3.2.1.21) and acid phosphomonoesterase (EC 3.1.3.2) on the same soil samples; and 2) determine the degree and the sources of variability associated with the assays within and among the laboratories. The results showed that overall variability was highest for replication on the microplate (n = 4), whereas suspension replication had low CVs. This suggests an important source of variation is from pipetting not variability from soil suspensions. A major effort was made to control for methodological differences by using air-dried soils (therefore more stable over varying storage periods) and operator consistency for each task across the labs (e.g. preheated reagents, microplate reader sensitivity set to the highest standard, readings taken within an hour of reaction termination, and controls for substrate autohydrolysis). As a result, the differences among labs were much smaller than differences due to soil type for the microplate method, indicating operator error can be minimized by following the same strict protocol. At the molar level, enzyme activity rates measured across the five labs were not the same between bench and MUF microplate methods (although they were within an order of magnitude), but were quite similar in terms of ranking of soil management treatments and soil types ( Table 2 ). Correlations between bench and microplate assays were strong for both enzymes, although slightly stronger for acid phosphomonoesterase (r = 0.93) than β-glucosidase (r = 0.81). Additionally, for both acid phosphomonoesterase and β-glucosidase, correlation r values were mostly similar for MUF microplate and PNP bench method correlation with EL-FAME biomarkers, suggesting both methods were measuring activity originating from the same microbial groups. We conclude that different labs using the same MUF microplate protocol tested, gives reasonably similar absolute activity values, variability, and ranking of treatments (highest to lowest). We propose that the MUF microplate method described in this study be considered as a standard protocol for assaying soil enzyme activities, providing that the buffer pH for the incubation be adjusted to the optimal pH according to the enzyme of interest. [ABSTRACT FROM AUTHOR]

A functional modification of the surface of a 96-well microplate coupled with a thin layer deposition technique is demonstrated for enhanced fluorescence-based sandwich immunoassays. The plasma polymerization technique enabling the deposition of organic thin films was employed for the modification of the well surface of a microplate. A silver layer and a plasma-polymerized film were consecutively deposited on the microplate as a metal mirror and the optical interference layer, respectively. When Cy3-labeled antibody was applied to the wells of the resulting multilayered microplate without any immobilization step, greatly enhanced fluorescence was observed compared with that obtained with the unmodified one. The same effect could be also exhibited for an immunoassay targeting antigen directly adsorbed on the multilayered microplate. Furthermore, a sandwich immunoassay for the detection of interleukin 2 (IL-2)was performed with the multilayered microplates, resulting in specific and 88-fold--enhanced fluorescence detection. [ABSTRACT FROM AUTHOR]

The assessment of new therapeutic strategies to cure surfactant-associated lung disorders would greatly benefit from assay systems allowing routine evaluations of surfactant functions. We present a method to measure surfactant adsorption kinetics into interfacial air-liquid interfaces based on fluorescence microplate readers. The principle of measurement is simple, robust, and reproducible: Wells of a microtiter plate contain an aqueous solution of a light-absorbing agent. Fluorescence is excited and collected from the top of the wells so that fluorescently labeled surfactant injected into the bulk can be detected only once adsorbed into the air-liquid interface. Mass transfer from the bulk to the interface is achieved by orbital shaking implemented in the plate reader instrument. The method has been tested and validated by using phospholipids or surfactants of different origins, by using albumin as surfactant inhibitor, and by comparison of results with Wilhelmy balance measurements. The method is suited for implementation in high-throughput screening routines for conditions affecting, or improving, surfactant film formation. In contrast to surface tension measurements, our method gives a direct readout of the amount of surfactant adsorbing into the interface, including the functionally important amount of material firmly associating with the interfacial film.

Traditional assays for secreted proteins include methods such as Western blot and enzyme-linked immunosorbent assay (ELISA) detection of the protein in the cell culture medium. We describe a method for the detection of a secreted protein based on fluorescent measurement of an mCherry fusion reporter. This microplate reader-based mCherry fluorescence detection method has a wide dynamic range of 4.5 orders of magnitude and a sensitivity that allows detection of 1 to 2 fmol fusion protein. Comparison with the Western blot detection method indicated greater linearity, wider dynamic range, and a similar lower detection threshold for the microplate-based fluorescent detection assay of secreted fusion proteins. An mCherry fusion protein of matrix metalloproteinase-9 (MMP-9), a secreted glycoprotein, was created and expressed by transfection of human embryonic kidney (HEK) 293 cells. The cell culture medium was assayed for the presence of the fluorescent signal up to 32 h after transfection. The secreted MMP-9–mCherry fusion protein was detected 6 h after transfection with a linear increase in signal intensity over time. Treatment with chloroquine, a drug known to inhibit the secretion of many proteins, abolished the MMP-9–mCherry secretion, demonstrating the utility of this method in a biological experiment. [ABSTRACT FROM AUTHOR]

Hydrogels are an increasingly important class of medical device materials that enable diverse and unique function, but can also be subject to significant biofouling and contamination. Although it is challenging to accurately quantify protein biofouling in hydrogels, spectroscopic detection of fluorescently labeled proteins is one method with the potential to provide direct, sensitive quantitation in transparent materials. Therefore, it is important to understand how fluorophores can affect protein-material interactions in hydrogels. This work uses an independent method, native ultraviolet fluorescence (native UV) of proteins, in conjunction with labeled protein fluorescence and the bicinchoninic acid assay (BCA), to assess the effect of fluorescent labels on protein sorption in polymer hydrogels. Bovine serum albumin (BSA) and lysozyme (LY) were labeled with two common but structurally different fluorophores and used as model biofouling proteins in three contact lens hydrogel materials. Native UV was used to directly measure both labeled and unlabeled protein sorption, while orthogonal measurements were performed with extrinsic fluorescence and BCA assay to compare with the native UV results. Sorption of labeled proteins was found to be <2-fold higher than unlabeled proteins on most protein-material combinations, while differences of up to 10-fold were observed for labeled BSA in more hydrophobic hydrogels. Fluorescence recovery after photobleaching (FRAP) also showed that the fluorescent label chemistry can significantly affect surface adsorption of sorbed proteins on the internal surfaces of hydrogels. This study reveals the complex nature of fluorophore-protein-material interactions and shows the potential of native UV for investigating unlabeled protein biofouling in hydrogels. [ABSTRACT FROM AUTHOR]

Abstract: The first optical sensing microplate is described for repetitive high-throughput screening of hydrogen peroxide (H2O2) in solutions of physiological pH. The fluorescent probe HP Green which shows a photoinduced electron transfer (PET) was incorporated in a polyurethane polymer matrix (type D4) on the bottom of microplate wells. Enzymatic oxidation of H2O2 Green in the presence of horseradish peroxidase (HRP) enables the determination of H2O2 concentrations up to 300μM with a LOD as low as 8.0μM. The calibration plots show high correlation coefficients (R 2 =0.996). The new sensing microplate (V-bottom) enables parallel and rapid (96 samples in 20min) detection of H2O2 with a standard fluorescence microplate reader. The response time is in the range of 10–20min, and the response is reversible. Finally, the H2O2 sensing microplate has successfully been applied to the determination of H2O2 in colored cell nutrition medium as real biological sample with a strong matrix effect. [Copyright &y& Elsevier]

Abstract: Measurement of proteasome activity is fast becoming a commonly used assay in many laboratories. The most common method to measure proteasome activity involves measuring the release of fluorescent tags from peptide substrates in black microplates. Comparisons of black plates used for measuring fluorescence with different properties show that the microplate properties significantly affect the measured activities of the proteasome. The microplate that gave the highest reading of trypsin-like activity of the purified 20S proteasome gave the lowest reading of chymotrypsin-like activity of the 20S proteasome. Plates with medium binding surfaces from two different companies showed an approximately 2-fold difference in caspase-like activity for purified 20S proteasomes. Even standard curves generated using free 7-amino-4-methylcoumarin (AMC) were affected by the microplate used. As such, significantly different proteasome activities, as measured in nmol AMC released/mg/min, were obtained for purified 20S proteasomes as well as crude heart and liver samples when using different microplates. The naturally occurring molecule betulinic acid activated the chymotrypsin-like proteasome activity in three different plates but did not affect the proteasome activity in the nonbinding surface microplate. These findings suggest that the type of proteasome activity being measured and sample type are important when selecting a microplate. [Copyright &y& Elsevier]

The optical design for an LEDs-based microplate reader that can perform fluorescence intensity (top and bottom), absorbance, luminescence and time-resolved fluorescence measurements is described. The microplate reader is the first microplate reader in the marketplace that incorporates LEDs as excitation light sources. Absorbance measurements over the 0-3.5 optical density range for caffeine solution are presented. Additionally, fluorescence intensity readings collected at 535 and 625 nm from a green and a red RediPlate™ are reported. Furthermore, fluorescence decay lifetime measurements obtained for Eu (europium) and Sm (samarium) standard solutions using 370 nm excitation are presented. The microplate reader detection limits for the fluorescence intensity top, fluorescence intensity bottom, fluorescence polarization and time-resolved fluorescence modes are 1.5 fmol 100 μL-1 fluorescein (384-well plate), 25 fmol 100 μL-1 fluorescein (384-well plate), 5 mP at 10 nM fluorescein (black 384-well plate) and 30 amol 100 μL-1 europium solution (white 384-well plate), respectively. [ABSTRACT FROM AUTHOR]

Abstract: Microplating that (i) does not necessitate complex or precise machinery to dispense small liquid volumes, (ii) enables fluorescent optical diagnosis, and (iii) permits simple analyte mixing mechanically is desirable. We advance here a novel approach that employs the formation of a liquid bridge held in place by capillary forces between glass rod tubes located parallel to each other. Experimental investigations made on liquid filling characteristics show conformance to theoretical notions. Analytical development showed the presence of regions of minimal uncertainty in the cross-sectional area of the liquid body arising from variations in the contact angle which permit consistent fluorescence measurements. Cyclical translation of the rods relative to each other, which cause rupture and reattachment of the liquid bridge, was found to engender good mixing. Strong linear trends were found in fluorescence signals relative to EGFP fluorophore concentration using standard and optical fiber (which offer targeted) excitation illumination. The open nature of liquid handling in the approach reported here and the positive results obtained portend the ability for development as integrated lab-on-a-chip devices. [Copyright &y& Elsevier]

Abstract: We report on a systematic study on the reactivity of the pyrylium dye Py-1 toward various classes of amines (primary aliphatic, primary aromatic, secondary, tertiary) and its use in environmental sensing of amines. While most primary aliphatic amines react almost quantitatively with Py-1 within 10min, sterically hindered primary aliphatic amines and primary aromatic amines react more slowly. Secondary and tertiary amines just induce a decomposition of the chromophore of Py-1 but do not yield a fluorescent product. This makes Py-1 a suitable receptor and transducer in an optical sensing microplate for rapid screening of primary aliphatic amines in water and soil samples. Py-1 is embedded into a polymeric cocktail, which is deposited on the bottom of wells in microtiter plates to yield a high-throughput fluorescence sensing tool. On reaction with primary aliphatic amines, a significant fluorescence increase (λ exc =485nm/λ em =620nm) of the sensor spots is detected in a standard microplate reader after 10min incubation at 25°C. The linear calibration plots of eight primary aliphatic amines are in the range of 0.350–70.0μgmL−1. The limits of detection (LODs) are from 0.119 to 0.589μgmL−1 and the limits of quantitation (LOQs) are from 0.399 to 1.965μgmL−1. We further show that the sensing plate is suitable for sensing mixtures of primary amines and that the total content of amines (TAC) found is a good measure for the sum of the contents of the individual amines present. Finally, the sensing plate was successfully applied to the quantitative analysis of primary aliphatic amines in 3 samples of water and soil, respectively. [Copyright &y& Elsevier]

ABSTRACT A 96-well microplate-based HPLC endpoint assay is described for the determination of NADPH-cytochrome P450 reductase (CPR) activity. Novel sampling of NADPH into microplates was optimized. Separation was performed on a Zorbax Eclipse XDB-C18 analytical 4.6 × 150 mm, 5 µm column. To validate the method, recombinant human NADPH-P450 reductase and microsomes with cytochrome P450 CYP1A1 were used. The mobile phase consisted of 80% acetonitrile and 20% water at a flow-rate of 0.8 mL/min. The CPR activity was quantified using NADPH fluorescence at λEx = 340 nm and λEm = 450 nm. Enzymatic activity was directly proportional to the decrease in NADPH fluorescence. This analytical process enables a highly sensitive endpoint determination for reductase activity in vitro and monitoring of the consumption of NADPH in enzymatic reactions. The method avoids the use of substrates and of organic solvents that may affect CPR and cytochrome P450 activity. In the reaction, molecular oxygen served as a proton source. The method can substitute spectrophotometric detection methods for its accuracy, high reproducibility (~100%) and sensitivity. The lower limit of detection, shown using the Agilent 1200 aparatus, is in the 250 nmol range. In addition, using this method it is possible to set up reactions in a high-throughput format. Copyright © 2011 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: Microplates for use in resource-limited laboratories should ideally not require processes that involve substantial large-scale production in order to be viable. We describe and demonstrate here an approach of using a silicone sheet with holes, conveniently cut out precisely using an inexpensive cutting plotter to correspond with regions where liquid is to be dispensed, and attaching it to a transparency to create very thin well arrays. With this, the contact angle hysteresis behavior of liquid could be harnessed to produce taller drop shapes so that the fiber probe used could read in the emitted light more effectively. Experimentation conducted revealed fluorescence measurements that were significantly more sensitive than standard microplates, notwithstanding that smaller volumes of liquid were needed. This was achieved using both the fiber optic and imaging evaluation modes. The two methods investigated, one with a lid placed and one without, showed the latter to produce marginally more sensitive readings as opposed to improved immunity from the environment with the former. These favorable measurement characteristics were found to be achievable with an estimated production cost of AU $0.40 and fabrication times of 3.5min (96 wells) and 6.5min (384 wells) per plate. [Copyright &y& Elsevier]

The presence of bubbles in liquid samples residing in microplate wells causes inaccuracies in fluorescence measurements. In addition, pipetting errors, if not adequately managed, can result in misleading data and wrong interpretations of assay results, particularly in the context of high-throughput screening. In this work, the authors describe an adapted design to the capillary wells microplate approach that permits side viewing. They demonstrate a prototype that detects bubbles and pipetting errors during actual assay runs to ensure accuracy in screening. [ABSTRACT FROM PUBLISHER]

The ability to rapidly genotype a large number of individuals is the key to any successful marker-assisted plant breeding program. One of the primary bottlenecks in high-throughput screening is the preparation of DNA samples, particularly the quantification and normalization of samples for downstream processing. A rapid and simple Sybr Green I-based quantification procedure that can he performed in a 96-well format is outlined. In this procedure, a dual standard curve method is used to allow better resolution of dilute samples and to reduce fluorescence value variation between samplings. A method to quickly normalize samples, and the importance of normalization, is also explored. We demonstrate that successful fragment amplification of a Theobroma grandiflorum (Willd ex Spreng) Schum. population is increased from 70% to 98% when each DNA extract is quantified and normalized as opposed to quantifying only a subset and normalizing all the samples based on tee average of that subset. Improved micro-satellite amplification was also observed among individuals in the monocot genus Phaedranassa Herb. ssp. Additionally, when our normalization method is applied to a Persea americana Mill. population, 97% of the samples normalized to 4 ng·µL-1 amplify at least three of six microsatellite regions, whereas only 30% of the samples below 4 ng·µL-1 (i.e., samples that could not be normalized) amplify at least three regions. We describe an undemanding method to quantify and normalize a large number of samples, which can be done manually or can be automated. [ABSTRACT FROM AUTHOR]

Abstract: This note describes the development of a method to rapidly quantify the metal-binding ability of low-volume DOM samples based on their fluorescence properties. The method uses 96-well microplates to screen the fluorescence quenching observed when increasing concentrations of various metals are added to the pH-buffered DOM sample. Only 1.6 mL of DOM sample is required to quantify the binding affinity for each metal and the result is obtained in a few minutes. This study presents results for a surface soil DOM sample for which binding was assessed for Cu2+, Fe2+, Ni2+ at pH 5 and 9. [Copyright &y& Elsevier]

Successful high-throughput screening requires methods for determining the precision of liquid dispensing. We compared absorbance and fluorescence methods. We found that plate-to-plate variability was large enough to mask the difference in optical methods. When a dual-dye method was used, we found that absorbance and fluorescence gave similar results in 96- and 384-well plates. In 1536-well plates, the two methods gave differing results, but centrifugation reduced the difference, suggesting that entrapped air bubbles or unsettled meniscus shapes artificially raised the coefficients of variation of fluorescence reads. [Copyright &y& Elsevier]

A fluorescence-based microplate assay was developed to quantify cell death based upon the measurement of glucose-6-phosphate dehydrogenase (G6PD) activity. G6PD is a cytosolic enzyme and leaks from cells when plasma membrane integrity is compromised. In this assay, cell death is measured by correlating the activity of extracellular G6PD to the reduction of resazurin to the fluorescent product, resorufin, via a coupled-enzyme reaction. The coupled-enzyme reaction permits rapid signal amplification from small amounts of G6PD, an advantage over assays based on resazurin alone. This assay is rapid, nontoxic, and amenable to high-throughput screening. The assay has a Z′ factor of 0.78. [Copyright &y& Elsevier]

We have successfully configured a new ultrasensitive fluorescent phosphate assay that detects free phosphate in solution through the formation of the fluorescent product resorufin. The phosphate assay relies on coupling phosphate generation to purine nucleoside phosphorylase, xanthine oxidase, and horseradish peroxidase. The response is excellent in the nanomolar range, being linear between 50 nM and 5 μM phosphate. This method is more sensitive (more than 10-fold) than other reported methods and is amenable to miniaturization. In particular, we have demonstrated the utility of this new method in a format suitable for ultra-high-throughput screening. [Copyright &y& Elsevier]

Abstract: The immense cost effectiveness of using transparencies as analyte handling implements in microplate instrumentation offers the possibility of application even in resource-limited laboratories. In this work, a standard microplate was adapted to serve as the permanent base for disposable scribed transparencies. The approach is shown to ameliorate evaporation, which can affect assay accuracy when analytes need to be incubated for some time. It also offers assurance against fluorescence measurement errors due to the cross-talk of samples from adjacent wells. [Copyright &y& Elsevier]