Nanoliter Samples Research Articles

Capillary batch injection analysis: a novel approach for analyzing nanoliter samples

A significantly miniaturized version of batch injection analysis (BIA) is described. The new technique is based on solution handling by means of a piece of fused silica capillary and is, therefore, termed as capillary batch injection analysis (CBIA). The operation of CBIA is characterized by dispensing nanoliter samples onto the surface of a micro- or minidisk electrode which is immersed in a 1 ml blank solution. The effects of various parameters such as injection volume, capillary outlet/electrode distance, dispensing rate, presence or absence of stirring and capillary/electrode dimensions on the CBIA response are studied and optimized conditions for CBIA operation are derived. Repetitive injections of 188 nl aliquots of hexacyanoferrate(II) are performed with an injection rate of 600 samples/h, with a good precision of the signal height expressed by a relative standard deviation of 1.1% ( n=31). Furthermore, CBIA measurements are combined with square-wave voltammetry by recording complete voltammograms during the injection of sub-μl samples. The enhancement of selectivity is demonstrated by the simultaneous determination of ferrocene and ferrocenemonocarboxylic acid at concentrations ranging from 2.5×10 −5 M to 2.5×10 −4 M. The square-wave voltammograms of CBIA are nearly identical to those obtained in a large-volume batch experiment.

High-resolution microcoil NMR for analysis of mass-limited, nanoliter samples.

An improved nanoliter-volume NMR probe design places the microcoil and capillary at the magic angle (57.7 degrees) with respect to the external magnetic field. Using an NMR probe which requires a total sample volume of just 200 nL, high-resolution 300-MHz 1H-NMR spectra (line width, 0.6 Hz) are presented of 10 mM alpha-bag cell peptide for an observe quantity of 45 ng (50 pmol in 5 nL). For the volume of sample inside the microcoil (the observe volume, Vobs), the 3 sigma limit of detection (LOD) is 9 ng (10 pmol, 2mM) for data obtained in 15 h. To reduce the data acquisition time, a probe with a greater Vobs is developed. As an example of a rapid, mass-limited analysis, a concentration corresponding to 400 ng of menthol dissolved in Vobs = 31 nL (82.6 mM) yields a spectrum in 9 min (LOD = 6.9 ng, 44 pmol, 1.4 mM). To illustrate improvements in concentration sensitivity, a spectrum is acquired in 45 min for 400 ng of menthol dissolved in a total sample volume of 200 nL (12.8 mM). Compared to a commercial nanoprobe for the same mass of menthol, these two examples reduce data acquisition time by at least 95%. Both model compounds demonstrate substantially improved concentration LODs compared to those obtained in previous high-resolution, microcoil NMR work. These advances illustrate the utility of enhanced sensitivity provided by NMR microcoils applied to nanoliter volumes of mass-limited samples.

A simple procedure for the chromatographic analysis of nanoliter samples

A simple method for the analysis of nanoliter droplets is proposed, which is profitable when larger samples cannot be collected as, for instance, in the case of several biological fluids and particularly in clinical chemistry. A glass capillary associated to a micromanipulator is used to collect submicroliter volumes which are partially transferred into transparent polymeric tubings with known internal diameters (120–178 μm), where the volumes sampled are measured by meniscus collimations with a collimator microscope at suitable magnification. Both ends of these tubings are preliminarily equipped with ferrules and fittings, so as to make them suitable for connection as the loop to a conventional high-pressure injection valve. The reliability of this procedure has been tested for the analysis of Na+, K+ and Ca2+ present in minute synthetic standard samples (10–200 nL) by a conventional ion-chromatographic instrumentation. Relative standard deviations in peak area measurements (5–6%) are discussed in terms of the whole approximation affecting volume measurements, which depends on both the inconstancy of the inner diameter of the polymeric tubings employed and the uncertainty characterizing meniscus collimations. The proposed procedure can be easily extended to the determination of any organic or inorganic species present in very small samples, provided that their detection can be achieved by any chromatographic approach or, more generally, by flow injection analysis.

Directly coupled CZE-NMR and CEC-NMR spectroscopy for metabolite analysis: paracetamol metabolites in human urine.

Direct coupling of NMR spectroscopic detection with both capillary zone electrophoresis (CZE) and capillary electrochromatography (CEC) was applied to the separation of metabolites of the drug paracetamol in an extract of human urine. Continuous-flow CZE-NMR and CEC-NMR allowed the detection of the major metabolites, the glucuronide and sulfate conjugates of the drug and the endogenous material hippurate. Identification of these substances was achieved by examination of individual rows of the NMR chromatogram and this also gave estimates of the detection limits. For CEC-NMR, spectra were also obtained in the stopped-flow mode including a two-dimensional TOCSY NMR experiment which afforded confirmatory evidence for paracetamol glucuronide. Characterisation of drug metabolites using NMR spectroscopy is therefore possible with nanolitre sample volumes.

Microanalysis of lung airway surface fluid by capillary electrophoresis with conductivity detection.

The thin layer of fluid that covers the surface of the epithelia lining the conducting airways plays an important role in primary pulmonary defense, and its composition may be a critical factor in the pathogenesis of several lung diseases including cystic fibrosis. Despite its physiological importance, the composition of airway surface fluid (ASF) is poorly understood due to considerable difficulties in sample collection from the 5-30 microns thick layer and subsequent analysis. We have used a novel technique for sample collection and microanalysis of ASF (nanoliter sample required) by capillary electrophoresis with conductivity detection. Limitations on the diameters of capillary required for the sample injection process and for the conductivity detector require the use of coupled separation capillaries with different external diameters. Two different methods were used to construct a butt-joint coupling for capillaries of different outer diameters. Reasonable efficiency is observed with the coupled capillaries (N = 100000 plates m-1) compared to an unbroken single capillary (N = 180000 plates m-1). The use of conductivity detection allows greater flexibility in method development and the possibility of determining a greater variety of ions than with a previous indirect-UV method. In the present study, we describe the analysis of cations (Na+, K+, Ca2+, Mg2+) and anions (Cl-, NO2-, NO3-, SO4(2-), PO4(2-), HCO3-) in rat ASF. Particular attention was paid to developing washing procedures which limited fouling of the conductivity sensor. In healthy rats, ASF was found to be hypotonic compared to plasma levels, consistent with some observations made in human airways.

Minireview: Fluorescence Correlation Spectroscopy (FCS) - A Highly Sensitive Method to Analyze Drug/Target Interactions

Fluorescence Correlation Spectroscopy (FCS), a new analytical technology, allows binding properties to be determined very accurately in biological assays at the level of single molecules. At concentrations of ≥ 10-12 M, binding constants, on/off-rates, and even reaction/enzyme kinetics can be determined in real-time, and in sample volumes as low as 10-9 μl. The FCS technology can be applied to study molecular and cellular interactions in homogeneous assays. Assay times in the range of seconds in combination with nanoliter sample volumes allow FCS to be used for high throughput screening to identify new pharmaceutical lead structures or new pharmacological targets. FCS is fully compatible with standard microtiter plate formats. However, for high throughput screening, specially designed sample carriers containing many thousand sub-microliter sample wells may be used in combination with a nanopipetting and sample retrieval system.

Biochemical comparison of plaque fluid on tooth and acrylic surfaces during a sucrose challenge

Previous studies have investigated variations in dental plaque fluid composition within a single mouth after a sucrose exposure. The purpose of this study was to determine a potential source of calcium and phosphorus in plaque by comparing the pH, calcium and phosphorus concentrations in plaque fluid obtained from an acrylic appliance with samples taken from supragingival tooth surfaces within the same individual after a sucrose challenge. Separate plaque samples from 14 individuals were collected from an acrylic appliance or tooth surfaces within same individual before and 15 min after a 20% sucrose rinse. Each plaque sample was centrifuged and nanolitre samples of plaque fluid were analysed for pH with a pH microelectrode, for total calcium concentration by atomic absorption in a graphite furnace, and for phosphorus concentration by spectrophotometry. There was an increase in the calcium and phosphorus concentration in the plaque after the sucrose challenge and a significant increase in calcium and phosphorus concentrations in the plaque taken from the teeth compared to the acrylic surfaces. The results indicate that the increased total calcium and phosphorus in plaque during a sucrose challenge is probably derived from the demineralization of enamel or extracellular demineralized components.

Separation of nanoliter samples of biological amines by a comprehensive two-dimensional microcolumn liquid chromatography system.

A two-dimensional liquid chromatography system based on the combination of a charge separation mechanism, provided by anion exchange chromatography, with a hydrophobic separation mechanism, provided by reversed phase chromatography, is presented. A 90 cm long anion exchange microcolumn is coupled to a 3 cm long reversed phase microcolumn. Both microcolumns are interfaced by two electronically controlled valves. Effluent from the anion exchange microcolumn is collected in a sample loop and then concentrated onto the head of the reversed phase microcolumn. Analyte peaks consisting of tagged amines elute from the second column and are detected by laser-induced fluorescence. The entire operation is controlled by a personal computer. The resolving power of this system is demonstrated with a two-dimensional chromatogram of the peptides obtained from a tryptic digest of porcine thyroglobulin. The sensitivity of the system is demonstrated with a two-dimensional chromatogram of the contents of a single bovine chromaffin cell.

Amperometric detection for capillary flow injection analysis

Ultrasmall-volume measurements of oxidizable compounds have been accomplished by coupling a capillary flow injection system with amperometric detection. Remarkably low (femtomole) mass detection limits result from the combination of nanoliter sample volume and the inherent sensitivity of the wall-jet detector. A substantial economy of reagent consumption and disposal accrues from the operation of the nl/min flow regime. Variables influencing the physical dispersion in the capillary flow injection system, including capillary length, sample volume or flow rate, are explored and optimized.

A simple method to determine millimolar concentrations of sodium in nanoliter samples

Measurement of sodium in the nanoliter samples obtained from in vitro studies of isolated, perfused nephron segments has traditionally been associated with time-consuming sample preparation and/or expensive instrumentation. A new instrument is described which can measure picomole quantities of sodium without the need for special processing. The instrument is based on the continuous flow devices developed by Vurek and a sodium-sensitive glass detector. The response to varying concentrations of NaCl was logarithmic as predicted from the Nernst equation. The response to 50 mM NaCl was 22.1 +/- 0.2 mV; 100 mM, 29.9 +/- 0.2 mV; 150 mM, 35.2 +/- 0.2 mV; and 200 mM, 39.3 +/- 0.6 mV (mean +/- SD). The slope of the standard curve was 27.8 mV/log unit change in sodium concentration. Potassium, calcium and ammonium, possible interfering ions, had no effect on the response to 100 mM NaCl. When the response to 100 mM NaCl was measured in buffered solutions of varying pH, it was 29.1 +/- 0.2 mV at pH 6.21, 28.9 +/- 0.4 mV at pH 7.04 and 28.7 +/- 0.4 mV at pH 8.54, indicating that pH did not alter the response to 100 mM NaCl. By treating the detector with NaOH and HCl, the response to 50, 100 and 150 mM improved to the point that the slope of the standard curve was 51.2 mV/log unit change in sodium concentration. The calculated resolution after treatment was 1.0 mM in the 50 to 100 mM range and 1.3 mM in the 100 to 150 mM range using a 10.9 nl pipet.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased performance in electrothermal atomic absorption spectrometry of lithium in renal tubular fluid by use of tantalum foil

A method for the determination of lithium by electrothermal atomic absorption spectrometry in nanolitre samples of tubular fluid of rat kidneys was developed. The signal-to-noise ratio was improved by use of tantalum foil in the atomization step. Peak height absorbance increased ten times and integrated absorbance four times. The atomization temperature could be reduced by 300 °C. The sensitivity increased from 1.7 to 0.45 pmol, which permits the determination of lithium in 10 nl samples. Addition of lithium-free tubular fluid to lithium standards showed that no matrix effects were present and that background correction was not required. At a level of 0.25 mmol l–1, the within-day and between-day relative standard deviations (RSDs) were 5 and 4%, respectively. For plasma, the within-day and between-day RSDs were also 5 and 4%, respectively, at a level of 0.25 mmol l–1. The recovery of lithium standards added to plasma was 99 ± 4%. In untreated anaesthetized rats, the tubular fluid-to-plasma lithium concentration ratio for the late proximal part of the renal tubule was 1.10 ± 0.10 (mean ± SD; n= 180) and for the early distal part of the tubule 1.08 ± 0.13 (n= 102). Since tubular fluid-to-plasma concentration ratios can be assessed in both proximal and distal tubular fluid, the proposed method provides a tool to test whether lithium is a good marker of proximal tubular sodium reabsorption.

Osmolality and Electrolyte Composition of Pleon Fluid in Porcellio Scaber (Crustacea, Isopqda, Oniscidea): Implications for Water Vapour Absorption

ABSTRACT Active water vapour absorption in Porcellio scaber is associated with the endogenous production of strongly hyperosmotic fluid in the pleoventral chamber (pleoventralraum; PV). Pre-desiccated animals show increased pleon fluid secretion within 1–2 min of transfer to suprathreshold humidities (>87 % relative humidity). The conspicuous increase in fluid volume is accompanied by a rise in osmolality from isosmotic (approximately 750mosmolkg−1) up to as much as 8.2osmol kg−1. Vapour absorption is marked by the onset of metachronal pleopodal ventilation and a subsequent decline in fluid osmolality as uptake fluid approaches equilibrium with ambient water activities. The effects of sealing of the PV by the margins of the depressed pleopods, along with the observation that animals ventilate even when PV fluid activities are somewhat below ambient, suggest that resultant pressure increases may elevate humidity within the PV and thereby augment vapour uptake. Ion-selective microelectrode measurements of Na+, K+, Ca2+ and Cl− concentrations in nanolitre samples of isolated pleon fluid identify Na+ and Cl− as the major osmolytes. Possible preadaptions favouring evolution of vapour absorption in the Oniscidea are discussed.

Measures of tear exchange under rigid contact lenses: Part I. The method

Although previous tear-exchange measurements under contact lens have been primarily qualitative in nature, Cuklanz and Hill in 1969 proposed a mathematical model to determine tear exchange based on particle movement. They first tested that model utilizing flame photometry to determine the number of particles in a given sample. The considerable sample dilution required at that time diminished the accuracy attainable. Recently available nanoliter sample freezing-point osmometry techniques, however, are introduced here to reexplore the CuklanzHill mathematical model more precisely.

Determination of low concentrations of lithium in biological samples using electrothermal atomic absorption spectrometry

A method is described for the determination of endogenous lithium concentrations in serum and urine using electrothermal atomic absorption spectrometry. The method has also been applied to the analysis of nanolitre samples of micro-puncture fluid from rat kidney tubules containing pharmacological concentrations of lithium. The graphite tubes are coated in situ with tantalum to give improved sensitivity and increased pre-atomization stability. A mini-flow of gas at a flow-rate of 30 ml min–1 is used during atomization to eliminate background interference. Background correction is not necessary. The characteristic mass is 0.98 pg of lithium. Serum samples are deproteinized with an equal volume of 10% nitric acid before analysis. Urine samples are diluted 5-fold with 5% nitric acid. The normal range of serum lithium is up to 0.39 µmol l–1 and normal urinary excretion is up to 9 µmol over a period of 24 h.

Quantitation of total carbon dioxide in nanoliter samples by flow-through fluorometry

A new ultramicrofluorometric assay for the measurement of picomole amounts of total CO2 is described. The assay is based on a sequence of two linked enzymatic reactions: HCO3 reacts with phosphoenolpyruvate forming oxaloacetate, and oxaloacetate is then converted to malate with associated oxidation of NADH to NAD+. The fluorescence of NADH is measured in a modified ultramicrofluorometer. The HCO3 content of the sample is proportional to the decrease in NADH fluorescence. The method can resolve differences as small as 5.5 pmol total CO2 in 3-nl samples containing 0-160 pmol. The assay is 5- to 10-fold more sensitive than the widely used microcalorimetric assay. The assay can also measure total CO2 in biological samples. The assay will be useful in measuring transepithelial total CO2 transport in short distal nephron segments with low rates of total CO2 transport without resorting to extremely long collection times.

Continuous-flow quantitation of Na+ and K+ in nanoliter samples using chromogenic macrocyclic ionophores.

Continuous-flow colorimetric methods for measurement of sodium and potassium contents in 1- to 30-nl aqueous samples are described. The methods employ macrocyclic ionophores (cryptahemispherands) that selectively bind sodium or potassium, altering the absorbance spectra of covalently attached chromophores. The potassium method is linear in the range 0-350 pmol and exhibits a high degree of precision (coefficient of variation of approximately 2% in the 150- to 350-pmol range). It can detect as little as 8 pmol of K+. The sodium method is linear in the range 0-2,700 pmol and also exhibits a high degree of precision (coefficient of variation of approximately 3% in the 1,700- to 2,700-pmol range). It can detect as little as 57 pmol of Na+. The instrumentation and reagents are available from commercial sources. These methods are proposed for measurement of K+ and Na+ fluxes in isolated perfused tubules.

Microcolumn separations and the analysis of single cells.

Capillary zone electrophoresis and open tubular liquid chromatography are two examples of an emerging area of analytical instrumentation known as microcolumn separations. The high resolution and small sample requirements of these methods make them suitable for the quantitative, multicomponent chemical analysis of single cells. Appropriate instrumentation for the analysis of nanoliter and subnanoliter samples is discussed. Data from the analysis of individual neurons are presented, including amino acid and neurotransmitter content.

Real-Time Electron Diffraction. Part III: Image Transfer via Fiber Optics

Improvements are described in photodiode-array real-time data recording for gas electron diffraction (GED). When the conventional glass window and lens optics in a previously reported detector configuration are replaced by fiber optic components, two significant effects arise: (1) detector gain is enhanced to the extent that it is now possible to detect nanoliter samples in combined GED-GC (gas chromatography) experiments, and (2) for the first time since the development of the real-time recording scheme, molecular mean amplitudes of vibration are within error limits of literature values. Thus the method now affords full molecular structure determinations, including bond distances and angles, and their associated mean vibrational properties.

Determination of sodium and potassium in nanoliter volumes of biological fluids by furnace atomic absorption spectrometry.

Renal tubular fluid samples are nanoliter (10/sup -9/ L) volumes containing sodium and potassium concentrations that are within the range of determination by furnace atomic absorption. Modification of nanoliter handling techniques and the use of microboats with the IL 951/655 provided a method for rapid precise analyses (relative standard deviation of 5%). Determinations of sodium and potassium were precise; however, inaccuracies occurred with anion substitution of sodium salts. NaHCO/sub 3/ solutions gave consistently higher peak height absorbance and area absorbance compared with those of NaCl: the peak area absorbance correlated linearly with the concentration of bicarbonate. Pretreatment of the microboat with boric acid eliminated this phenomenon and the associated inaccuracy. Comparison of determination of sodium in nanoliter samples by graphite furnace atomic absorption with macroanalysis by flame emission gave relative errors of less than 2.0%. Addition of sodium and potassium to tubular fluid samples yielded mean recoveries of 102.6% and 99.7%, respectively. The authors conclude that graphite furnace can be an accurate method for measurement of sodium and potassium in nanoliter volumes of biological fluids.

Micro-HPLC and transfer techniques for directly measuring drug (indomethacin) transport across the isolated perfused renal proximal straight tubule

We wished to study the active transport of indomethacin across isolated segments of renal proximal tubules. To do so, we developed a microbore HPLC technique capable of directly measuring pharmacologic concentrations of indomethacin in nanoliter sample volumes. The technique also required development of methods for transferring of samples, introduction of internal standard and identification of an oil, under which the sample could be collected without interference with the assay itself or the compounds of interest. We found that gamma-terpinene could be used as the oil, for it did not impede the assay, did not dissolve indomethacin or inulin, and indomethacin and inulin remained stable in the water phase. Transfer of sample was effected with a glass tube containing 640 nL of water plus internal standard into which the indomethacin sample pipette (110 nL) could be introduced. This transfer tube was then capped with PE-60 tubing, which could later be used for withdrawing sample into an injection syringe for HPLC assay. This method proved feasible for measuring indomethacin transport across isolated perfused proximal tubule segments and should be applicable for similar studies of other drugs.