Development of xMAP Assay for Detection of Six Protein Toxins

xMAP technology was used for simultaneous identification of six protein toxins (staphylococcal enterotoxins A and B, cholera toxin, ricin, botulinum toxin A, and heat labile toxin of E. coli). Monoclonal antibody-conjugated xMAP microspheres and biotinilated monoclonal antibodies were used to detect the toxins in a sandwich immunoassay format. The detection limits were found to be 0.01 ng/mL for staphylococcal enterotoxin A, cholera toxin, botulinum toxin A, and ricin in model buffer (PBS-BSA) and 0.1 ng/mL for staphylococcal enterotoxin B and LT. In a complex matrix, such as cow milk, the limits of detection for staphylococcal enterotoxins A and B, cholera toxin, botulinum toxin A, and ricin increased 2- to 5-fold, while for LT the detection limit increased 30-fold in comparison with the same analysis in PBS-BSA. In the both PBS-BSA and milk samples, the xMAP test system was 3-200 times (depending on the toxin) more sensitive than ELISA systems with the same pairs of monoclonal antibodies used. The time required for a simultaneous analysis of six toxins using the xMAP system did not exceed the time required for ELISA to analyze one toxin. In the future, the assay may be used in clinical diagnostics and for food and environmental monitoring.     

An approach for analysis of protein toxins based on thin films of lipid mixtures in an optical biosensor

Biological membrane-like lipid films were deposited on the sensing surface in an optical biosensor instrument. The membranes were mixtures of biologically occurring lipids. Eight surfaces were prepared, some of which contained various glycolipids as minor components. One was supplemented with membrane proteins. The binding of six protein toxins (cholera toxin, cholera toxin B subunit, diphtheria toxin, ricin, ricin B subunit, staphylococcal enterotoxin B) and of bovine serum albumin at pH 7.4 and pH 5.2 to each of the sensor surfaces was studied. Each of the seven proteins gave a distinct binding pattern. The assay is rapid and simple, with no need for reagents. The lipid sensor surface is readily regenerated after binding and very stable. The concept with mixed lipid layers and assays at different pHs gives numerous combinations and could be applicable for developing a sensor for protein toxins.     

Ultraviolet native fluorescence detection in capillary electrophoresis using a metal vapor NeCu laser

Ultraviolet (UV) excitation for laser-induced native fluorescence (LINF) detection in capillary electrophoresis (CE) offers impressive performance figures of merit when assaying peptides containing tyrosine or tryptophan residues, catecholamines, indolamines, and a number of other classes of analytes with appreciable fluorescence when excited by UV radiation. One of the largest drawbacks of native fluorescence detection schemes in CE-LINF systems has been the expense and the complexity of the lasers required for excitation in the deep UV wavelength range of 200-300 nm. An improved "turnkey" NeCu laser operating at 248.6 nm interfaced to a sheath flow-based CE system obtains a performance similar to that of large frame frequency-doubled Ar ion lasers. The detection limits for serotonin and dopamine (27 nM and 8 microM, respectively, for approximately 3-nL injection) are similar to those obtained using a frequency-doubled Ar ion laser at 257 nm (21 nM and 8 microM, respectively). An example of the detection of serotonin-related analytes from a single-cell electropherogram demonstrates the performance of such a system for mass-limited measurements.     

Multiplexed toxin analysis using four colors of quantum dot fluororeagents

Quantum dots (QDs) have the potential to simplify the performance of multiplexed analysis. In this work, we prepared bioinorganic conjugates made with highly luminescent semiconductor nanocrystals (CdSe-ZnS core-shell QDs) and antibodies to perform multiplexed fluoroimmunoassays. Sandwich immunoassays for the detection of cholera toxin, ricin, shiga-like toxin 1, and staphylococcal enterotoxin B were performed simultaneously in single wells of a microtiter plate. Initially the assay performance for the detection of each toxin was examined. We then demonstrated the simultaneous detection of the four toxins from a single sample probed with a mixture of all four QD-antibody reagents. Using a simple linear equation-based algorithm, it was possible to deconvolute the signal from mixed toxin samples, which allowed quantitation of all four toxins simultaneously.     

Gangliosides as receptors for biological toxins: development of sensitive fluoroimmunoassays using ganglioside-bearing liposomes

Gangliosides, glycosphingolipids present in the membranes of neuronal and other cells, are natural receptors for a number of bacterial toxins and viruses whose sensitive detection is of interest in clinical medicine as well as in biological warfare or terrorism incidents. Liposomes containing gangliosides mimic cells that are invaded by bacterial toxins and can be used as sensitive probes for detecting these toxins. We discuss detection of three bacterial toxins-tetanus, botulinum, and cholera toxins using ganglioside-bearing liposomes. Tetanus and botulinum toxins selectively bind gangliosides of the G1b series, namely, GT1b, GD1b, and GQ1b; and cholera toxin binds GM1 very specifically. Unilamellar liposomes containing GT1b or GM1 as one of the constituent lipids were prepared by extrusion through polycarbonate membranes. To impart signal generation capability to these liposomes, fluorophore-labeled lipids were incorporated in the bilayer of liposomes. The fluorescent liposomes, containing both a marker (rhodamine) and a receptor (GT1b or GM1) in the bilayer, were used in sandwich fluoroimmunoassays for tetanus, botulinum, and cholera toxins and as low as 1 nM of each toxin could be detected. The apparent dissociation constants of liposome-toxin complexes were in 10(-8) M range, indicating strong binding. This is the first report on detection of tetanus and botulinum toxins based on specific recognition by gangliosides. The fluorescent liposomes are attractive as immunoreagents for another reason as well--they provide enormous signal amplification for each binding event as each liposome contains up to 22,000 rhodamine molecules. The present approach using receptors incorporated in bilayers of liposomes offers a unique solution to employ water-insoluble receptors, such as glycolipids and membrane proteins, for sensitive detection of toxins and other clinically important biomolecules.     

Experimental evaluation of the separation efficiency in capillary electrophoresis using open tubular and gel-filled columns.

The band dispersion phenomena in capillary zone electrophoresis (CZE) using untreated and surface-treated open tubular and gel-filled capillaries were experimentally evaluated, with emphasis on small capillary diameters (10-100 microns). Laser-induced fluorescence detection was used for high-sensitivity detection of the isoindoles originated from model amino acids. The plots of plate height vs electric field strength were generated for different column radii and compared with a theoretical model for CZE. In addition to the diffusion-controlled band dispersion in the relatively low electric field range, adsorptive interactions between a solute and the capillary wall may play a certain role in band-broadening. The sorption-desorption kinetics become important with increasing electric field strength. Thermal effects appear to contribute little to band-broadening in relatively small capillaries (less than 50-microns i.d.) within normal operating voltages (less than 30 kV), but could become significant in capillaries with larger bores (greater than 75-microns i.d.). With gel-filled capillaries of small diameters (less than 50-microns i.d.), diffusion processes can be minimized. In addition, thermal effects do not appear critical in such columns at reasonable voltages.     

Reversed-phase capillary liquid chromatography coupled on-line to capillary electrophoresis immunoassays

Capillary reversed-phase liquid chromatography (RPLC) was coupled on-line to competitive capillary electrophoresis immunoassay (CEIA) to improve concentration sensitivity of the competitive CEIA and to provide a means for detecting multiple species that cross-react with antibody. A competitive CEIA for glucagon was used for demonstration of this technique. Five-microliter samples were injected onto a 4-cm-long by 50-micron-i.d. RPLC column. Sample was desorbed by gradient elution, mixed on-line with fluorescently labeled glucagon and anti-glucagon, incubated in a continuous-flow reaction capillary, and analyzed by capillary electrophoresis with flow-gated injection and laser-induced fluorescence detection. Electrophoretic analysis of the reactor stream was performed every 1.5 s, allowing nearly continuous monitoring of the RPLC separation. Preconcentration achieved by RPLC allowed improvement in the detection limit from 760 to 20 pM. Addition of the RPLC column also allowed multiple cross-reactive species to be differentiated by first separating them chromatographically and then detecting them with the immunoassay. The technique was used to measure glucagon secretion from single islets of Langerhans and to differentiate cross-reactive forms of glucagon with one assay.     

Capillary waveguide optrodes: an approach to optical sensing in medical diagnostics

Glass capillaries with a chemically sensitive coating on the inner surface are used as optical sensors for medical diagnostics. A capillary simultaneously serves as a sample compartment, a sensor element, and an inhomogeneous optical waveguide. Various detection schemes based on absorption, fluorescence intensity, or fluorescence lifetime are described. In absorption-based capillary waveguide optrodes the absorption in the sensor layer is analyte dependent; hence light transmission along the inhomogeneous waveguiding structure formed by the capillary wall and the sensing layer is a function of the analyte concentration. Similarly, in fluorescence-based capillary optrodes the fluorescence intensity or the fluorescence lifetime of an indicator dye fixed in the sensing layer is analyte dependent; thus the specific property of fluorescent light excited in the sensing layer and thereafter guided along the inhomogeneous waveguiding structure is a function of the analyte concentration. Both schemes are experimentally demonstrated, one with carbon dioxide as the analyte and the other one with oxygen. The device combines optical sensors with the standard glass capillaries usually applied to gather blood drops from fingertips, to yield a versatile diagnostic instrument, integrating the sample compartment, the optical sensor, and the light-collecting optics into a single piece. This ensures enhanced sensor performance as well as improved handling compared with other sensors.     

Attenuated total internal reflectance infrared microspectroscopy as a detection technique for capillary electrophoresis

A novel detector for capillary electrophoresis (CE) using single-bounce attenuated total internal reflectance (ATR) Fourier transform infrared (FT-IR) microspectroscopy is presented. The terminus of the CE capillary is placed approximately 1 microm from the internal reflectance crystal at the focus of an ATR infrared microscope. Using pressure driven flow injection, concentration and volume detection limits have been determined for 25- and 10-microm-i.d. silica capillaries. Upon injection of 820 pL of succinylcholine chloride in a 10-microm capillary, a concentration detection limit of approximately 0.5 parts per thousand (ppt), or 410 pg, is found. The injection volume detection limit using a 108 ppt solution is 2.0 pL (216 pg). Sample separations using a programmed series of pressure, voltage, and again pressure on 25-, 50-, and 75-microm-i.d. capillaries are shown. CE separations of citrate and nitrate, as well as succinylcholine chloride with sodium salicylate using acetone as a neutral marker, are demonstrated. Several advantages of this CE-FT-IR technique include: (1) minimization of postcolumn broadening as a result of a small detector volume; (2) the ability to signal average spectra of the same aliquot, thereby improving the signal-to-noise in a stopped-flow environment; and (3) simplicity of design.     

Continuous cell introduction for the analysis of individual cells by capillary electrophoresis

Instrumentation for high-throughput analysis of single cells by capillary electrophoresis is described. A flow-based interface that uses electroosmotic flow (EOF) provides continuous injection of intact cells through an introduction capillary into a cell lysis junction and migration of the resulting cell lysate through a separation capillary for analysis. Specifically, two capillaries were coupled together with 5-mm-long Teflon tubing to create a approximately 5-microm gap, and the junction was immersed in a buffer reservoir. High voltage was applied across both capillaries so that cells were continuously pumped into the first capillary by EOF. Individual cells were lysed on-column at the junction without detergents, presumably owing to mechanical disruption caused by a dramatic change in flow properties at the gap. After each cell was lysed at the junction, the major proteins hemoglobin and carbonic anhydrase were separated by capillary electrophoresis and the resultant analyte zones were detected by laser-induced native fluorescence using 275-nm excitation. The detection limits of hemoglobin and carbonic anhydrase were 37 and 1.6 amol, respectively, which correlate well with the literature. The instrumentation was evaluated with intact red blood cells. The averaged time for complete analysis (i.e., continuous injection, lysis, separation, and detection) of one human erythrocyte was less than 4 min with this capillary-based setup. Moreover, this instrumentation simplifies the introduction of individual, intact cells without the use of a microscope.     

Instrumentation for medium-throughput two-dimensional capillary electrophoresis with laser-induced fluorescence detection

In two-dimensional capillary electrophoresis, a sample undergoes separation in the first dimension capillary by sieving electrophoresis. Fractions are periodically transferred across an interface into a second dimension capillary, where components are further resolved by micellar electrokinetic capillary electrophoresis. Previous instruments employed one pair of capillaries to analyze a single sample. We now report a multiplexed system that allows separation of five samples in parallel. Samples are injected into five first-dimension capillaries, fractions are transferred across an interface to 5 second-dimension capillaries, and analyte is detected by laser-induced fluorescence in a five-capillary sheath-flow cuvette. The instrument produces detection limits of 940 +/- 350 yoctomoles for 3-(2-furoyl)quinoline-2-carboxaldehyde labeled trypsin inhibitor in one-dimensional separation; detection limits degrade by a factor of 3.8 for two-dimensional separations. Two-dimensional capillary electrophoresis expression fingerprints were obtained from homogenates prepared from a lung cancer (A549) cell line, on the basis of capillary sieving electrophoresis (CSE) and micellar electrophoresis capillary chromatography (MECC). An average of 131 spots is resolved with signal-to-noise greater than 10. A Gaussian surface was fit to a set of 20 spots in each electropherogram. The mean spot width, expressed as standard deviation of the Gaussian function, was 2.3 +/- 0.7 transfers in the CSE dimension and 0.46 +/- 0.25 s in the MECC dimension. The standard deviation in spot position was 1.8 +/- 1.2 transfers in the CSE dimension and 0.88 +/- 0.55 s in the MECC dimension. Spot capacity was 300.     

Two-dimensional direct-reading fluorescence spectrograph for DNA sequencing by capillary array electrophoresis

We report a compact, two-dimensional direct-reading fluorescence spectrograph and demonstrate its application to DNA sequencing by capillary array electrophoresis. The detection cuvette is based on sheath flow, wherein the capillaries terminate in a two-dimensional array in a fluid-filled chamber that is pressurized with buffer. A thin metal plate is located downstream from the capillaries. This barrier plate has an array of holes that precisely matches the location of the capillaries. Buffer flows through the holes, drawing analyte from the capillaries in a well-defined array of thin filaments. Fluorescence is excited in the upper chamber with an elliptically shaped laser beam. The bottom chamber is sealed with a glass window and drained from the side. Fluorescence is detected by imaging the illuminated sample streams through the holes in the barrier plate. A prism is used to disperse fluorescence from each sample across a CCD camera so that the emission spectrum is monitored simultaneously from each capillary. The instrument is demonstrated in a 32-capillary configuration but can be scaled to several thousand capillaries.     

Microchip laser-induced fluorescence detection of proteins at submicrogram per milliliter levels mediated by dynamic labeling under pseudonative conditions

We have previously demonstrated on-column dynamic labeling of protein-SDS complexes on capillaries and microchips for laser-induced fluorescence (LIF) detection using both a commercially available fluor and a protein separation buffer. Upon binding to hydrophobic moieties (of the analyte or separation buffer), the fluor undergoes a conformational change allowing fluorescence detection at 590 nm following excitation with 488-nm light. Our original work showed on-chip limits of detection (LOD) comparable with those using UV detection (1 x 10(-5) M) on capillaries-falling significantly short of the detection limits expected for LIF. This was largely a function of the physicochemical characteristics of the separation buffer components, which provided significant background fluorescence. Having defined the contributing factors involved, a new separation buffer was produced which reduced the background fluorescence and, consequently, increased the available dye for binding to protein-SDS complexes, improving the sensitivity in both capillaries and microchips by at least 2 orders of magnitude. The outcome is a rapid, sensitive method for protein sizing and quantitation applicable to both capillary and microchip separations with a LOD of 500 ng/mL for bovine serum albumin. Interestingly, sensitivity on microdevices was improved by inclusion of the dye in the sample matrix, while addition of dye to samples in conventional CE resulted in a drastic reduction in sensitivity and resolution. This can be explained by the differences in the injection schemes used in the two systems. The linear range for protein quantitation covered at least 2 orders of magnitude in microchip applications. On-chip analysis of human sera allowed abnormalities, specifically the presence of elevated levels of gamma-globulins, to be determined.     

Integrating waveguide biosensor

A capillary biosensor is demonstrated which uses the waveguiding properties of the capillary to integrate the signal over an increased surface area without simultaneously increasing the background noise from the detector. This biosensor achieves limits of detection of 30-50 pg/mL in immunoassays using a diode laser for excitation and a PMT for detection. This is approximately 2 orders of magnitude greater sensitivity than was achieved using the same immunoassay reagents in a fiber optic biosensor or a planar array biosensor. Two different approaches to using the capillaries as immunosensors are described, either of which could be adapted for multianalyte sensing.     

High-efficiency nanoscale liquid chromatography coupled on-line with mass spectrometry using nanoelectrospray ionization for proteomics

We describe high-efficiency (peak capacities of approximately 10(3)) nanoscale (using column inner diameters down to 15 microm) liquid chromatography (nanoLC)/low flow rate electrospray (nanoESI) mass spectrometry (MS) for the sensitive analysis of complex global cellular protein enzymatic digests (i.e., proteomics). Using a liquid slurry packing method with carefully selected packing solvents, 87-cm-length capillaries having inner diameters of 14.9-74.5 microm were successfully packed with 3-microm C18-bonded porous (300-A pores) silica particles at a pressure of 18,000 psi. With a mobile-phase delivery pressure of 10,000 psi, these packed capillaries provided mobile-phase flow rates as low as approximately 20 nL/min at LC linear velocities of approximately 0.2 cm/s, which is near optimal for separation efficiency. To maintain chromatographic efficiency, unions with internal channel diameters as small as 10 microm were specially produced for connecting packed capillaries to replaceable nanoESI emitters having orifice diameters of 2-10 microm (depending on the packed capillary dimensions). Coupled on-line with a hybrid-quadrupole time-of-flight MS through the nanoESI interface, the nanoLC separations provided peak capacities of approximately 10(3) for proteome proteolytic polypeptide mixtures when a positive feedback switching valve was used for quantitatively introducing samples. Over a relatively large range of sample loadings (e.g., 5-100 ng, and 50-500 ng of cellular proteolytic peptides for 14.9- and 29.7-microm-i.d. packed capillaries, respectively), the nanoLC/nanoESI MS response for low-abundance components of the complex mixtures was found to increase linearly with sample loading. The nanoLC/nanoESI-MS sensitivity also increased linearly with decreasing flow rate (or approximately inversely proportional to the square of the capillary inner diameter) in the flow range of 20-400 nL/min. Thus, except at the lower loadings, decreasing the separation capillary inner diameter has an effect equivalent to increasing sample loading, which is important for sample-limited proteomic applications. No significant effects on recovery of eluting polypeptides were observed using porous C18 particles with surface pores of 300-A versus nonporous particles. Tandem MS analyses were also demonstrated using the high-efficiency nanoLC separations. Chromatographic elution time, MS response intensity, and mass measurement accuracy was examined between runs with a single column (with a single nanoESI emitter), between different columns (same and different inner diameters with different nanoESI emitters), and for different samples (various concentrations of cellular proteolytic peptides) and demonstrated robust and reproducible sensitive analyses for complex proteomic samples.     

Fluorescence-labeled peptide pI markers for capillary isoelectric focusing

Nineteen fluorescent pH standards or pI markers ranging pH 3.64-10.12 were developed for use in capillary isoelectric focusing using laser-induced fluorescence detection. Tetra- to tridecapeptides containing one cysteine residue were designed to focus sharply at their respective isoelectric points by including amino acids that contain charged side chains, the pKa values of which are close to the corresponding pI values. An iodoacetylated derivative of tetramethylrhodamine was coupled to the thiol group of cysteine to yield fluorescent pI markers. The pI values of the labeled peptides were precisely determined after isoelectric focusing on polyacrylamide gel slabs by direct measurement of the pH of the focused bands. The markers were subjected to capillary isoelectric focusing for 10-15 min in coated capillaries under conditions of low electroosmosis and were detected by means of a scanning laser-induced fluorescence detector down to a level of subpicomolar range. The markers permitted the calibration of a wide-range pH gradient formed in a capillary by fluorescence detection for the first time and should facilitate the development of highly sensitive analytical methods based on a combination of capillary isoelectric focusing and laser-induced fluorescence detection.     

Angular distribution of fluorescence from dye-filled capillaries

We studied the angular distribution of fluorescence from a small, lossy capillary filled with a laser-dye solution. We found that the fluorescence is isotropic for the liquid core and that, far from the liquid-solid phase transition, this isotropy shows no temperature dependence. This result, an extension of studies with solid cylinders, is at variance with theoretical expectations for solids as well as with previous reports by other investigators but is explained by the motion of the molecules in the liquid. Therefore the optimal viewing angle for capillary zone electrophoresis experiments is near 90° because the elastic scattering of the incident laser light is at or near a minimum for these small capillaries. This reduces contamination of the fluorescence signal as a result of stray laser light in the optical system.     

Tantalum-filament pulling device for fabrication of submicrometer fused-silica tips

A simple and low-cost pulling device for fused-silica capillaries was developed. By using a tantalum heating filament and the self-tension in a bent capillary, tips and constricted regions with outer diameters of approximately 1 microm and inner diameters of a few hundred nanometers could be reproducibly pulled from 50-microm-i.d., 375-microm-o.d. capillaries. The tips can be used in different applications such as microinjection, micromanipulation, and single-channel patch-clamp, injection ends for CE or as electrospray tips. Constricted capillaries with optimized dimensions to minimize cylindrical lensing effects and to match the size of a diffraction-limited laser focus can be used as optical detection windows in CE and micro-HPLC. Fused silica has several advantages over other glasses such as high melting temperature and superior optical and mechanical properties.     

Electrophoretic separation of proteins on a microchip with noncovalent, postcolumn labeling

Proteins were separated by microchip capillary electrophoresis and labeled on-chip by postcolumn addition of a fluorogenic dye, NanoOrange, for detection by laser-induced fluorescence. NanoOrange binds noncovalently with hydrophobic protein regions to form highly fluorescent complexes. Kinetic measurements of complex formation on the microchips suggest that the reaction rate is near the diffusion limit under the conditions used for protein separation. Little or no band broadening is caused by the postcolumn labeling step. Lower limits of detection for model proteins, alpha-lactalbumin, beta-lactoglobulin A, and beta-lactoglobulin B, were <0.5 pg (approximately 30 amol) of injected sample. The relative fluorescence and reaction rates are compared with those of a number of other fluorogenic dyes used for protein labeling.     

Laser ablation construction of on-column reagent addition devices for capillary electrophoresis

A simple and reproducible technique for constructing perfectly aligned gaps in fused-silica capillaries has been developed for postcolumn reagent addition with capillary electrophoresis. This technique uses laser ablation with the second harmonic of a Nd:YAG laser (532 nm) at 13.5 mJ/pulse and a repetition rate of 15 Hz to create these gaps. A capillary is glued to a microscope slide and positioned at the focal point of a cylindrical lens using the focused beam from a laser pointer as a reference. Gaps of 14.0 +/- 2.2 microm (n = 33) at the bore of the capillary are produced with a success rate of 94% by ablation with 400 pulses. This simple method of gap construction requires no micromanipulation under a microscope, hydrofluoric acid etching, or use of column fittings. These structures have been used for reagent addition for postcolumn derivatization with laser-induced fluorescence detection and have been tested for the separation of proteins and amino acids. Detection limits of 6 x 10(-7) and 1 x 10(-8) M have been obtained for glycine and tranferrin, respectively. Separation efficiencies obtained using these gap reactors range from 38,000 to 213,000 theoretical plates.