Microscale Assay Research Articles

ASSESSMENT OF α-AMYLASE INHIBITION ACTIVITY BY AN OPTIMIZED AND VALIDATED IN VITRO MICROSCALE METHOD

Metabolic disorders, including hyperglycemia, characterize type-2 diabetes. One of the treatment methods used for postprandial hyperglycemia includes using potential therapeutic agents to inhibit α-amylase activity. This study utilized fractional design and the simplex method to optimize in vitro microscale assay inhibition conditions using Miller’s reaction. In addition, the effect of substrate concentration on enzyme activity was analyzed. Enzyme concentration of 0.15 U mL-1 and pre- and post-incubation times of 7.2 and 5.5 min, respectively, in water bath (15.6 min) equipment, were set up for optimized condition for the enzyme activity. Analytical validation was performed based on different international guidelines. Km was found to be 0.38 mg mL-1. Linearity was obtained at the acarbose concentration of 1.5 µg mL-1 and 5 µg mL-1. The IC50 for the positive control was found to be 0.6 µg mL-1. The relative standard deviation and Z value were found to be <4% and >0.93, respectively. Additionally, the optimized assay was applied to extracts from five different plants. Two plant extracts (Zanthoxylum fagara and Chrysactinia mexicana) inhibited α-amylase activity. The optimized and validated method was accurate, precise, and linear

A reconfigurable microscale assay enables insights into cancer-associated fibroblast modulation of immune cell recruitment.

Innate immune cell infiltration into neoplastic tissue is the first line of defense against cancer and can play a deterministic role in tumor progression. Here, we describe a series of assays, using a reconfigurable microscale assay platform (i.e. Stacks), which allows the study of immune cell infiltration in vitro with spatiotemporal manipulations. We assembled Stacks assays to investigate tumor-monocyte interactions, re-education of activated macrophages, and neutrophil infiltration. For the first time in vitro, the Stacks infiltration assays reveal that primary tumor-associated fibroblasts from specific patients differ from that associated with the benign region of the prostate in their ability to limit neutrophil infiltration as well as facilitate monocyte adhesion and anti-inflammatory monocyte polarization. These results show that fibroblasts play a regulatory role in immune cell infiltration and that Stacks has the potential to predict individual patients' cancer-immune response.

Biotransformation of cladribine by a nanostabilized extremophilic biocatalyst

Cladribine (2-chloro-2′-deoxy-β-d-adenosine) is a 2′-deoxyadenosine analogue, approved by the FDA for the treatment of hairy cell leukemia and more recently has been proved for therapeutic against many autoimmune diseases as multiple sclerosis. The biosynthesis of this compound using Thermomonospora alba CECT 3324 as biocatalyst is herein reported. This thermophilic microorganism was successfully entrapped in polyacrylamide gel supplemented with nanoclays such as bentonite. The immobilized biocatalyst (T. alba-Ac-Bent 1.00 %), was able to biosynthesize cladribine with a conversion of 89 % in 1 h of reaction and retains its activity for more than 270 reuses without significantly activity loss, showing better operational stability and mechanical properties than the natural matrix. A microscale assay using the developed system, could allow the production of at least 181 mg of cladribine in successive bioprocesses.

Evaluation of a Combined System Based on an Upflow Anaerobic Sludge Blanket Reactor (UASB) and Shallow Polishing Pond (SPP) for Textile Effluent Treatment

Abstract Color removal from textile effluents was evaluated using a laboratory-combined process based on an upflow anaerobic sludge blanket (UASB) reactor followed by a shallow polishing pond (SPP). The anaerobic reactor was fed with a real textile effluent, diluted 10-times in a 350 mg/L solution of pre-treated residual yeast extract from a brewery industry as nutrient source. The parameters color, COD, N-NH3 and toxicity were monitored throughout 45 days of operation. According to the results, decolorization and COD removal were highest in the anaerobic step, whereas the effluent was polished in the SPP unit. The overall efficiency of the complete UASB-SPP system for COD and color were 88 and 62%, respectively. Moreover, the N-NH3 generated by the residual yeast extract ammonification was below 5 mg/L for the final effluent. Finally, no toxicity was detected after the treatment steps, as shown by the Vibrio fischeri microscale assay.

Microanalysis Using Acoustically Actuated Droplets Pinned Onto a Thread

An acoustic wave-assisted microscale assay platform is demonstrated. Liquid droplets containing samples and reagents are dispensed and pinned onto a hydrophobic thread, and actuated by sound waves. The transport of droplets is achieved by modulating sound emitted by two sources. The resulting sound intensity gradient pushes the droplets in one direction. The available distance of droplet actuation is related to droplet size. The moving droplets merge at the end of their actuation range, and the merged droplet travels further due to its greater size. Fluorometric analysis of the merged droplet is accomplished by positioning an excitation light source above a defined interrogation zone along the thread. The emitted light is analyzed by a miniature spectrometer positioned orthogonally to the excitation light beam. The sound sources and detection system are controlled by universal electronic modules (Raspberry Pi, Arduino). The system is operated automatically with the aid of a custom-developed graphical user interface. Here, we demonstrate its application in enzyme assays (trypsin, β-galactosidase). In-droplet enzymatic reaction progress curves are readily obtained. The actuated droplets can be put in motion and stopped without a significant delay. Only microliter-range volumes of sample and reagent are consumed in every analysis, while sub-milliliter volumes of chemical waste are produced.

Design, solid-phase synthesis and evaluation of enterobactin analogs for iron delivery into the human pathogen Campylobacter jejuni

The human enteropathogen Campylobacter jejuni, like many bacteria, employs siderophores such as enterobactin for cellular uptake of ferric iron. This transport process has been shown to be essential for virulence and presents an attractive opportunity for further study of the permissiveness of this pathway to small-molecule intervention and as inspiration for the development of synthetic carriers that may effectively transport cargo into Gram-negative bacteria. In this work, we have developed a facile and robust microscale assay to measure growth recovery of C. jejuni NCTC 11168 in liquid culture as a result of ferric iron uptake. In parallel, we have established the solid-phase synthesis of catecholamide compounds modeled on enterobactin fragments. Applying these methodological developments, we show that small synthetic iron chelators of minimal dimensions provide ferric iron to C. jejuni with equal or greater efficiency than enterobactin.

Saturation Binding of Nicotine to Synthetic Neuromelanin Demonstrated by Fluorescence Spectroscopy

Neuromelanin (NM) has long been considered as an aging pigment, perhaps an unavoidable and undesirable byproduct of dopaminergic neural transmission. However, NM is carefully packaged into double membrane-bound structures within cells of the substantia nigra and other neural tissues, suggesting a beneficial function to maintaining these stores. It is well established that NM is able to concentrate toxic xenobiotics within pigmented cells due to its unique chemical environment. In doing so, such agents may confer susceptibility to Parkinson's disease (PD) as illustrated by model PD-inducing neurotoxins such as methyl-phenyl-pyridinium ion. It is possible that high-affinity binding interactions toward NM may contribute to the adverse effects of PD-inducing toxins, as well as neuroprotective agents. Here we aim to develop a generalized assay capable of elucidating the binding constants of chemical agents to synthetic and natural neuromelanins. Toward this end, a model neuromelanin synthesized from dopamine and cysteine was prepared according to published procedure. Using a UV/Visible spectroscopic assay, we show that dopamine, 6-hydroxy dopamine, and nicotine bind to the synthetic neuromelanin, while caffeine did not. More importantly, nicotine was further found to induce a fluorescence signal in the presence of NM which was used to establish a binding constant estimated at 0.65mM. Dopamine appears to enhance this signal, also in a saturable manner, with an estimated Kd of 0.05mM in our isolated chemical system. In summary, the micro-scale fluorescence assay described herein will allow us to overcome many of the problems inherent in the study of chemical interaction with NM through traditional spectroscopic means. Using a single standardized signal, it should now be possible to rank a number of PD-related toxins based on NM-binding affinity and shed further light on this important problem.

Development of organophosphate hydrolase activity in a bacterial homolog of human cholinesterase.

We applied a combination of rational design and directed evolution (DE) to Bacillus subtilis p-nitrobenzyl esterase (pNBE) with the goal of enhancing organophosphorus acid anhydride hydrolase (OPAAH) activity. DE started with a designed variant, pNBE A107H, carrying a histidine homologous with human butyrylcholinesterase G117H to find complementary mutations that further enhance its OPAAH activity. Five sites were selected (G105, G106, A107, A190, and A400) within a 6.7 Å radius of the nucleophilic serine Oγ. All 95 variants were screened for esterase activity with a set of five substrates: pNP-acetate, pNP-butyrate, acetylthiocholine, butyrylthiocholine, or benzoylthiocholine. A microscale assay for OPAAH activity was developed for screening DE libraries. Reductions in esterase activity were generally concomitant with enhancements in OPAAH activity. One variant, A107K, showed an unexpected 7-fold increase in its kcat/Km for benzoylthiocholine, demonstrating that it is also possible to enhance the cholinesterase activity of pNBE. Moreover, DE resulted in at least three variants with modestly enhanced OPAAH activity compared to wild type pNBE. A107H/A190C showed a 50-fold increase in paraoxonase activity and underwent a slow time- and temperature-dependent change affecting the hydrolysis of OPAA and ester substrates. Structural analysis suggests that pNBE may represent a precursor leading to human cholinesterase and carboxylesterase 1 through extension of two vestigial specificity loops; a preliminary attempt to transfer the Ω-loop of BChE into pNBE is described. Unlike butyrylcholinesterase and pNBE, introducing a G143H mutation (equivalent to G117H) did not confer detectable OP hydrolase activity on human carboxylesterase 1 (hCE1). We discuss the use of pNBE as a surrogate scaffold for the mammalian esterases, and the importance of the oxyanion-hole residues for enhancing the OPAAH activity of selected serine hydrolases.

A fluorescence-based colorimetric droplet platform for biosensor application to the detection of α-fetoprotein

Droplet methods have been successfully applied in DNA hybridization analysis and protein-protein interaction. Existing assay methods implemented in droplet platforms are severely limited by expensive and high-maintenance equipment. As a convenient detection method, colorimetry provides a new path for microscale assay since it can enhance assay efficiency and simplify the detection procedure. Here, a microscale immunoassay for α-fetoprotein (AFP) was developed for the first time by the incorporation of colorimetry and droplet platform. Ru(bpy)(2)(mcbpy-O-Su-ester)(PF(6))(2) complex (Ru) was coupled with the monoclonal antibody (Ab) of AFP to form a stable red Ru-Ab complex both as a quencher for green CdTe quantum dots (QDs) and as a capture probe for AFP. In the absence of AFP, the mixed droplet showed a red color. With the increase of AFP concentration, the color change of the droplet was from red to green as a result of the competition of AFP with QDs for Ru-Ab. The biosensor exhibited not only good sensitivity and specificity for AFP with a detection limit of 0.06 ng ml(-1), but also satisfactory performance in diluted human sera with a detection limit of 0.4 ng ml(-1). Notably, a visual droplet platform for screening cancer biomarkers by the naked eye based on this principle is anticipated.

An arrayed high-content chemotaxis assay for patient diagnosis

Chemotaxis assays are essential tools for the study of gradient sensing and directed cell migration, and have the potential to aid in the diagnosis and characterization of patients with immune disorders. Current methods are limited in their ability to meet the more demanding requirements for clinical applications. Because patient samples have a short lifespan and sometimes a limited volume (e.g. pediatrics), the operational requirements for an efficient chemotaxis assay are increased in the clinical setting. Here we describe a microscale assay platform for gradient generation that overcomes these limitations. Passive fluidic methods are leveraged to provide a reliable microfluidic gradient generation device, operable in only three pipetting steps. In addition, arrayed imaging and advanced cell tracking algorithms enabled a 50-fold increase in throughput over current methods. These methods were employed to aid in the diagnostic evaluation of an infant who presented with severe, recurrent bacterial infections. Analysis of the infant's neutrophils revealed impaired cell polarization and chemotaxis in a gradient of the chemoattractant fMLP. The patient was subsequently diagnosed with an inhibitory mutation in the Rho GTPase, Rac2. The approach also enabled a microenvironmental screen of human primary neutrophil chemotaxis on fibronectin, fibrinogen and laminin with results suggesting that fibronectin, although commonly used, may not be the most appropriate matrix protein for chemotaxis assays. Together, these findings demonstrate the use of arrayed micro-devices to aid in the diagnosis of a primary immunodeficiency disorder, and illustrate the capability for increased throughput microenvironmental studies and screening targeted to specific human diseases.

How useful are the “other” semipermeable membrane devices (SPMDs); the mini-unit (15.2 cm long)?

Mini (15.2 cm) semipermeable membrane devices (SPMDs) were used successfully in 169 streams from six metropolitan areas of the US to sequester hydrophobic organic compounds (HOCs) that are indicative of urbanization. A microscale assay the P450RGS, which responds to compounds that bind to the aryl hydrocarbon receptor (AhR), and the Fluoroscan, a chemical screen for polycyclic aromatic hydrocarbons (PAHs), were performed on each mini SPMD extract. Results show both tests were sensitive enough to respond in streams with low urbanization and responded exponentially in a predictable way to a gradient of urbanization. Mini SPMDs had sufficient sampling rates to detect HOCs using gas chromatography with mass spectrometric detection (GC/MS) in streams with low levels of urbanization. The total number of HOCs in streams had a linear response to a gradient of urbanization, where 73 of 140 targeted compounds were detected. A diverse group of compounds was found in urban streams including, PAHs, insecticides, herbicides, musk fragrances, waste water treatment compounds and flame retardants. Pentachloroanisole (PCA), a breakdown product of pentachlorophenol (wood preservative), was the most ubiquitous HOC, and was detected in 71% of streams. An evaluation of mini SPMD performance showed they can detect concentrations in water below toxicity benchmarks for many HOCs with the exception of 2,3,7,8 tetrachlorodibenzo- p-dioxin. A comparison of mini SPMDs with full sized (91.4 cm) SPMDs showed they have several distinct advantages. The most notable advantages are their low cost, small size, and reduced chance of vandalism. The greatest limitation is the inability to detect compounds at low concentrations (pg/L). Mini SPMDs perform quite well in a wide array of environmental settings and applications and should be considered as an option in environmental studies.

A new microrheometric approach reveals individual and cooperative roles for TGF‐β1 and IL‐1β in fibroblast‐mediated stiffening of collagen gels

The stiffness of the extracellular matrix can profoundly influence cell and tissue behaviors. Thus there is an emerging emphasis on understanding how matrix mechanical environments are established, regulated, and modified. Here we develop a microrheometric assay to measure the mechanical properties of a model extracellular matrix (type I collagen gel) and use it to explore cytokine-induced, cell-mediated changes in matrix mechanical properties. The microrheometric assay uses micron-scale ferrimagnetic beads embedded within collagen gels during fibrillogenesis. The beads are magnetized, then subjected to a twisting field, with the aggregate rotation of the beads measured by a magnetometer. The degree of bead rotation reflects the stiffness of the surrounding matrix. We show that the microscale assay provides stiffness measures for collagen gels comparable to those obtained with standard macroscale rheometry. To demonstrate the utility of the assay for biological discovery, we measure stiffness changes in fibroblast-populated collagen gels exposed to three concentrations of six cytokines over 2 to 14 days. Among the cytokines tested, transforming growth factor-beta1 and interleukin-1beta enhanced matrix stiffness, and together exerted cooperative effects on cellular modulation of matrix mechanics. The microrheometry approach developed here should accelerate the discovery of biological pathways orchestrating cellular modulation of matrix mechanics.

Rapid micro-scale assay for homocysteine by liquid chromatography-tandem mass spectrometry

Objectives: Increased total homocysteine (tHcy) level is an independent risk factor for atherosclerosis and cardiovascular disease. Here, we describe a rapid tHcy micro-scale assay. Methods: We developed an easy sample preparation and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the measurement of tHcy in human serum and plasma, respectively. Results: The tHcy assay was linear up to 61.6 μmol/L, the detection limit was 1.0 μmol/L, the lower limit of quantification was 1.8 μmol/L, the imprecision in the range of 9.30–25.9 μmol/L was less than 5.9% and the analytical recovery was 94.7 ± 6.3%. Conclusions: Our novel tHcy LC-MS/MS assay is a quick, precise and robust method for tHcy determination in routine diagnostics.

A microscale assay for the identification of TGF-β antagonists based on functional coupling of the heterodimeric TGF-β receptor to STAT6-driven promoter activation

Inadequate function of Transforming Growth Factor- (TGF-) β is involved in numerous disease states including immune disorders and cancer, rendering this polypeptide and its receptor an attractive target for pharmaceutical interference. We have developed a novel microscale functional test system suited for the investigation of ligand-induced receptor activation and the automatable evaluation of potential agonists and antagonists. Hybrid receptors were constructed from the cytoplasmic domain of the human interleukin-4 (IL-4) receptor α-chain and extracellular domains of a TGF-β type I and type II receptor, respectively. These chimeras were stably introduced into the factor-dependent murine cell line Ba/F3 along with an IL-4-inducible luciferase reporter gene construct, yielding a reporter cell line which responds to productive ligand?receptor interactions by specific luciferase activity in a dose-dependent fashion. A model experiment employing inhibitory peptides demonstrates that the devised reporter cell provides a rational readout for TGF-β activity on target cells and its impairment by specific antagonists.

Nonradioactive Phosphopeptide Assay by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry: Application to Calcium/Calmodulin-Dependent Protein Kinase II

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) was used to quantify the phosphopeptide produced by calcium/calmodulin-dependent protein kinase II (CaMK II). MALDI-TOF measurements were performed in a linear and positive ion mode with delayed extraction excited at various laser powers and at different sampling positions, i.e., different loci of laser illumination. We find that the ratio of the peak area of the substrate (S) to that of its monophosphorylated form (SP) for a given mixture is constant, independent of the laser powers and/or of the sample loci illuminated by the laser. We also find that the fraction of phosphorylation determined by MALDI-TOF, orfMALDI-TOF, is proportionally smaller than that determined by HPLC, orfHPLC; the ratiofMALDI-TOF/fHPLCwas 0.797 ± 0.0229 (99% confidence limit,n= 7) for a 30-mer peptide substrate used in this study. A low mass gate, which turns off the detector temporarily, improved the ratiofMALDI-TOF/fHPLCto 0.917 ± 0.0184 (99% confidence limit,n= 7). Our interpretation of this result is that the reduction of the phosphopeptide peak in the MALDI-TOF measurement is likely to be caused by a temporal loss of detector function rather than by a lower efficiency of ionization for the phosphopeptide compared with its parent species. In these measurements the experimental errors, up to the 50% phosphorylation state, were less than 5%. After an adjustment made based on thefMALDI-TOF/fHPLCratio of 0.917, MALDI-TOF gave an accurate measurement for the kinetics of the CaMK II phosphorylation reaction. Since only a small volume of the reaction mixture, typically containing 3 to 50 pmol of substrate, is required for the MALDI-TOF measurement, this method can be adapted to a nonradioactive microscale assay for CaMK II and also for other protein kinases.

Analysis of pre-ovulatory changes in cervical mucus hydration and sperm penetrability.

Changes in cervical mucus occur during the proliferative phase of the menstrual cycle and are known to correlate with receptivity to sperm and to the endocrine milieu. Prior studies, however, have often lacked biological incisiveness and technical objectivity and precision. This study analyzed daily changes in mucus water content (hydration) prior to the LH surge (LH+0) in normal women, in relation to daily levels of serum LH, FSH, estradiol and progesterone, and to daily tests of sperm penetration of the mucus. Cervical mucus was studied for 12 cycles in 10 ovulating women. Three to ten mucus specimens were collected per cycle, over the days LH-8 to LH+0. Each specimen was subjected to measurement of both water content (hydration) and penetration by spermatozoa from fresh specimens of normal human semen. For the latter, a new microscale assay was developed and applied, which was amenable to very small volumes of mucus. The new technique determines objective measures of both the numbers of penetrating sperm (motile and non-motile) and the distance penetrated by the forward most vanguard sperm. In these experiments, variations in semen quality were controlled by performing a companion penetration assay in an artificial 1.5% polyacrylamide gel. The patterns of change in mucus hydration varied quantitatively among women, with preovulatory baseline levels ranging from 93.8-96.5%. All normal cycles (as defined by endocrine profiles) displayed a significant increase in hydration over a one-day period occurring 3-4 days before the LH peak. The magnitude of this shift varied among women between 2 and 3% (absolute hydration), a distinction well within the precision of the hydration assay. This quantum increase in hydration was more pronounced than the corresponding increase in serum estradiol on the same day. The change in mucus hydration, and the associated increase in sperm penetrability, were more consistent among cycles than the changes in reproductive hormones. There was a strong but non-linear correlation between mucus hydration and sperm penetrability. Once the value of hydration rose above approximately 97.5%, there was a substantial increase in penetrability. This 'cut-off point' in sperm penetrability was in the middle of the range of hydration values (across women) which preceded the quantum jump in hydration-which, itself, preceded the surge of LH. Hydration began to increase approximately 2 days before measurable increases in sperm penetration of the mucus in vitro. These results demonstrate that mucus hydration may be a valuable marker of the approach to ovulation and delineation of the fertile period. They also provide new methods for assessing sperm penetration into both large peri-ovulatory and very small samples of collected mucus.

Protein analyses and reagents: Microscale assay of calcium-binding activity of proteins and peptides using a nitrocellulose membrane

A simple and reliable method for the measurement of calcium binding to proteins and peptides was developed. It is composed of two procedures—filtration through a nitrocellulose membrane filter and estimation of 45Ca retained on the membrane. The routine assay was completed within a few minutes, and only microgram amounts of samples were necessary. This method permitted the quantitative determination of the calcium-binding activity of proteins and peptides including one with a molecular weight of as low as 4000. This method also permitted the detection of low-affinity interactions ( K d ⋍ 10 −3 m), possibly because the nitrocellulose membrane did not show nonspecific binding of calcium and because a washing step was not employed in the routine assay. Ultrafiltration membranes when used in the apparatus gave no useful data.

An improved micro-scale liquid-chromatographic assay for piperacillin in plasma and urine.

An accurate, sensitive, and specific liquid-chromatographic method is described for measuring piperacillin in plasma and urine. Plasma samples deproteinized with two volumes of acetonitrile containing 1.2 mg of the internal standard, p-nitrobenzene sulfonamide, per liter are centrifuged. The clear supernate is evaporated under nitrogen, and the residue is reconstituted in 50 microL of the mobile phase (32/68 by vol acetonitrile/water, adjusted to pH 2.5 with 85% phosphoric acid), of which 10 microL is injected onto a reversed-phase (C-18) column. Urine samples are diluted 10-fold with distilled water, an equal volume of acetonitrile containing 3 mg of the internal standard per liter is added, and 20 microL is chromatographed. Stability studies indicate that storage conditions are critical for both plasma and urine. Piperacillin in plasma is stable at -70 degrees C for at least six weeks, but 100% of it is degraded during the same time at -20 degrees C. Piperacillin in urine is also stable at -70 degrees C for six weeks, but 20% is degraded during six weeks at -20 degrees C.

An enzymic, reaction-rate assay for serum creatinine with a centrifugal analyzer.

We describe a procedure for specific, rapid, kinetic determination of creatinine, in which a manual coupled-enzyme micro-scale assay is adapted to a centrifugal analyzer. The creatinine reaction is ultimately linked to NADH utilization, which is measured by the absorbance change at 340 nm. This procedure requires 15 microL of serum and the standard curve is linear to a creatinine concentration of 200 mg/L. A four-point kinetic algorithm allows the dynamic range of the assay to be extended without sacrificing sensitivity, and makes a separate serum blank unnecessary. The within-run precision (CV) for samples with a creatinine concentration of 11 and 52 mg/L was 5.6 and 2.4%, respectively; day-to-day CV for a creatinine concentration of 11 mg/L was 7.7% (n = 21). We compared this procedure with a kinetic Jaffé procedure, with excellent agreement (r = 0.996; y = 0.96x + 2.4 mg/L). Bilirubin, non-esterified fatty acids, and ketone bodies do not affect creatinine determinations by this method; thus the method is especially useful for monitoring the renal function of diabetics.

Fluorescence analysis of picoliter samples

The fluorescence intensity of picoliter-volume samples was quantitated by taking samples and standards into a single siliconized capillary, fixing the capillary under the objective of a microscope-fluorometer, and defining an effective “fluorescence chamber” within the capillary by placing an imaging diaphragm in the emission path. Samples were then moved, by pneumatic control with an air syringe, within the capillary to this “fluorescence chamber.” A diaphragm in the excitation path limited the volume of sample excited. Fluorescence from all samples was thus directly determined under identical optical conditions. The co-efficient of variation of replicate measurements was 3%; carry-over from sample to sample within the capillary was less than 2%. A 3-pl sample containing 0.3 amol of sodium fluorescein (about 200,000 molecules) could be discriminated from the background; fluorescence intensity was linear with concentration for three to four orders of magnitude. Fluorescence intensities of NADH and an ammonia- o-phthalaldehyde-thiol adduct were also determined. Using this “fluorescence chamber” allowed straightforward scaling down of a fluorescence assay for urea in 20-pl samples, lowering the limit of detection to 10 fmol, three orders of magnitude below a previously reported microscale assay. This technique is applicable to many fluorescence assays used in studies of cell physiology, and should allow routine measurement of metabolites in individual cells or of enzymes in individual subcellular organelles.