Anal. Chem. 1996, 68, 697-700
Detection of Attomolar Concentrations of Alkaline Phosphatase by Capillary Electrophoresis Using Laser-Induced Fluorescence Detection Douglas B. Craig, Jerome C. Y. Wong, and Norman J. Dovichi*
Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada
Alkaline phosphatase can be assayed by monitoring the conversion of the fluorogenic substrate AttoPhos into the highly fluorescent product AttoFluor. We have used capillary electrophoresis with laser-induced fluorescence detection to monitor this reaction. The concentration limit of detection (3σ) of alkaline phosphatase is 1.5 × 10-17 M (2.1 fg/mL), which corresponds to a mass limit of detection of nine molecules (1.5 × 10-23 mol) contained within a 1-µL sample volume. Enzymatic reactions are used in the diagnosis of disease and as amplifiers for the detection of molecules in complex mixtures. These assays utilize enzymes that catalyze the conversion of a substrate into a product that can be detected with high sensitivity. The enzyme is attached to a highly specific probe for the analyte of interest, typically either an antibody or a piece of complementary oligonucleotide.1,2 The probe binds to the analyte, and unbound probe is removed. The adduct is incubated with substrate for the conjugated enzyme. The amount of product formed is proportional to the analyte present and the incubation period. Because enzymes are catalytic, they act as amplifiers and dramatically increase sensitivity. One enzyme routinely used in these applications is alkaline phosphatase. A critical step in the further development of assays of this type is the development of more sensitive systems for the detection of the enzyme. The use of capillary electrophoresis (CE) in enzyme assays has advantages over conventional assays in terms of speed, small sampling capability, and the ability to separate and quantify products and substrates that are very similar in structure. The use of a highly sensitive detection method such as post column laser-induced fluorescence (LIF) allows for extreme sensitivity. High-sensitivity detection limits for CE-LIF range from 1000 molecules to one molecule of fluorescent dye.3-8 CE-LIF has been used in the development of sensitive assays for oligosaccharide synthesizing and hydrolyzing enzymes and a high-sensitivity assay (1) deFrutos, M.; Paliwal, S. K.; Regnier, F. E. Anal. Chem. 1993, 65, 21592163. (2) Cano, R. J.; Torres, M. J.; Klem, R. E.; Palomares J. C. Biotechniques 1992, 12, 264-267. (3) Chen, D. Y.; Dovichi, N. J. J. Chromatogr. B 1994, 657, 265-269. (4) Chen D. Y.; Adelhelm, K.; Cheng X. L.; Dovichi, N. J. Analyst 1994, 119, 349-352. (5) Wu, S.; Dovichi, N. J. J. Chromatogr. 1989, 480, 141-155. (6) Zhao, J. Y.; Dovichi, N. J.; Hindsgaul, O.; Gosselin, S.; Palcic, M. M. Glycobiology 1994, 4, 239-242. (7) Timperman, A. T.; Khatib, K.; Sweedler, J. V. Anal. Chem. 1995, 67, 139144. (8) Chen, D. J.; Dovichi, N. J. Anal. Chem. 1996, 68, 690-696. 0003-2700/96/0368-0697$12.00/0
© 1996 American Chemical Society
for β-galactosidase.6,9 In this paper, we report the use of CE-LIF for the detection of alkaline phosphatase activity at attomolar (1 aM ) 10-18 M) concentrations. EXPERIMENTAL SECTION Instrumentation. Details of the instrument have been published previously.10 A 10-µm-i.d., 145-µm-o.d., 37-cm-long fused silica capillary (Polymicro Technologies, Phoenix, AZ) was used. The injection end is immersed into the running buffer or sample along with a platinum electrode that supplies positive high-voltage from a high voltage power supply (CZE1000R, Spellman, Plainview, NY). The detection end of the capillary, from which the polyimide coating has been removed by a gentle flame, is placed inside the sheath flow cuvette. The sheath flow cuvette (250- × 250-µm inner bore) is used to provide hydrodynamic focusing of the capillary flow and to reduce light scattering of the laser beam. Excitation of eluting species with the 457.9-nm line from a multiwavelength argon ion laser (Innova 90-4, Coherent) is achieved by focusing the laser beam with a 6.3×, 0.20 NA microscope objective (Melles Griot). Fluorescence is collected at 90° from the direction of excitation by a 60× 0.70 NA microscope objective (Model 60×-LWD, Universe Kogaku) and selectively transmitted through a slit and a 580DF40 bandpass filter (Omega Optical) to an R1477 photomultiplier tube (PMT) (Hamamatsu). Optimum laser power for the detection of AttoFluor is ∼5 mW at 457.9 nm. The analog PMT signal is collected at 10 Hz and digitized by a Macintosh IIsi via a NB-MIO-16XH-18 I/O board (National Instruments). The same input/output board is used to control the power supply for CE. Materials and Methods. Calf intestinal alkaline phosphatase (EC 3.1.3.1, 4900 units/mg, 140 000 g/mol) and p-nitrophenylphosphate (pNPP) were obtained from Sigma. AttoPhos is a weakly fluorescent proprietary substrate for alkaline phosphatase marketed by JBL Scientific and is converted to the highly fluorescent product AttoFluor. All other reagents were of analytical grade. Standardization Assay. The concentration of alkaline phosphatase as supplied by Sigma was determined by the standardization assay. Dephosphorylation of 10 mM pNPP in 1.0 M diethanolamine (pH 9.8) containing 0.5 mM MgCl2 at 37 °C by alkaline phosphatase was followed spectrophotometrically at 405 nm. The absorption coefficient of p-nitrophenol in this buffer is (9) Craig, D.; Arriaga, E.; Banks, P.; Zhang, Y.; Renborg, A.; Palcic, M. M.; Dovichi, N. J. Anal. Biochem. 1995, 226, 147-153. (10) Arriaga, E. A.; Zhang, Y.; Dovichi, N. J. Anal. Chim. Acta 1995, 299, 319326.
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18.45 cm-1 mmol-1. One unit of calf intestinal alkaline phosphatase is defined as the amount required to hydrolyze 1 µmol of pNPP in 1 min under the conditions described. Units were converted to concentration on the basis of a specific activity of 4900 units/mg of protein (Sigma product information). CE-LIF Assay. Alkaline phosphatase was serially diluted and incubated at 24 °C in 2.4 M diethanolamine (pH 10.0) containing 57 µM MgCl2 and 1 mM AttoPhos for 60 min. Alkaline phosphatase concentrations ranged from 4.6 × 10-16 (64 fg/ml) to 4.6 × 10-11 M (64 ng/mL). Assays were done in triplicate. Blanks contained no enzyme. Reaction mixture was electrokineticly injected (5 s, 135 V/cm) into a 37-cm-long capillary. An electric field of 400 V/cm was applied for the separation. The sheath buffer was 100 mM borate (pH 9.5) and the running buffer was 80 mM borate (pH 9.5), containing 20% v/v formamide. All peaks eluted within 10 min. Data were smoothed by 10 passes with a binomial filter. All injected samples contained 1.4 × 10-8 M fluorescein, which was used as an internal standard. Peak heights were converted to concentrations for determination of enzyme turnover numbers by comparison to the height of injections of AttoFluor standards of 1.7 × 10-8 M. AttoPhos was found to contain some AttoFluor as an impurity, which was removed by three extractions with an equal volume of CHCl3. It is important to prevent residual CHCl3 from entering the capillary; the nonpolar material appears to coat the inner wall of the capillary, distorting the electroosmotic flow. Several steps were taken to minimize contamination from exogenous enzyme. All buffers and vessel used in the assay were autoclaved prior to use in order to denature any enzyme contaminants. Solutions were prepared in a laminar-flow cleanair hood to minimize contamination from microbes. Latex gloves were worn during sample preparation to minimize transfer of enzyme from skin. All solutions were prepared in disposable plasticware and with use of disposable micropipet tips. RESULTS AND DISCUSSION The sample buffer used was 2.4 M diethanolamine (pH 10.0) containing 57 µM MgCl2, and the running buffer was 80 mM borate (pH 9.5) containing 20% v/v formamide. Although the concentrations of the standards are known, the volume of the injected sample cannot be precisely calculated due to differences in conductivity of the sample and the running buffer. However, with a total capillary volume of 29 nL, typical injection volumes are well below 1 nL. This injection volume will be consistent because injection conditions were identical for all assays. The sensitivity of the instrument for AttoFluor was determined from injections of 1.7 × 10-8 M AttoFluor, Figure 1, that contained 1.4 × 10-8 M fluorescein as an internal standard. Three peaks are detected. Peaks B and C are due to AttoFluor and fluorescein, respectively. Peak A is associated with the sample buffer and is present upon injection of buffer alone. The limit of detection (LOD) for AttoFluor, which is defined as that amount required to give a signal of 3 times the background noise, is 6.0 × 10-11 M, and sensitivity is 2.7 × 107 V mol-1 L-1. The relative standard deviation of the signal, after normalization to the internal standard, was 3% (n ) 5). To reduce the amount of AttoFluor present as an impurity in the substrate, the AttoPhos was subjected to extraction with CHCl3. Extraction cannot completely eliminate the AttoFluor impurity because AttoPhos undergoes a slow nonenzymatic breakdown during the course of the assay. To correct for this 698
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Figure 1. Electropherogram of 1.7 × 10-8 M AttoFluor standard (B) containing 1.4 × 10-8 M fluorescein (C) in 2.4 M diethanolamine (pH 10.0) containing 57 µM MgCl2. Peak A is due to the sample buffer. Injection was performed for 5 s at 135 V/cm, with separation at an electric field strength of 400 V/cm in 80 mM borate (pH 9.5) containing 20% v/v formamide. Capillary: 10-µm-i.d., 145-µm-o.d., 37-cm-long fused silica.
hydrolysis, a plot of the blank peak of AttoFluor versus time was produced. This relationship was found to be linear and was fit with the equation
AttoFluornonenzym ) AttoFluorinit + kt
(1)
where AttoFluornonenzym is the height of the peak of the nonenzymatically produced AttoFluor at a given time t from the preparation of the substrate, AttoFluorinit is the height of the AttoFluor peak at the time of preparation of the substrate, and k is the rate of signal increase due to nonenzymatic hydrolysis. Values for Attofluorinit and k were 2.7 ( 5.0 mV and 0.63 ( 0.03 mV/min, respectively. In each assay, the nonenzymatically produced AttoFluor present at the time of injection was corrected on the basis of this plot. The LOD in this assay is not determined by the background noise of the electropherogram, but rather by the error associated with the height of the nonenzymatically produced AttoFluor peak. The standard deviation of this peak is given by the equation
SD ) [tSDslope2 + SDintercept2]1/2
(2)
where SD is the standard deviation of the AttoFluor peak in the blank at time t from the preparation of the substrate and SDslope
Figure 2. Electropherograms of assay mixture: 4.6 × 10-16 M alkaline phosphatase was incubated with 1 mM AttoPhos in 2.4 M diethanolamine (pH 10.0) containing 57 µM MgCl2 for 1 h at 24 °C and electrokineticly injected into the capillary. Peaks A, B, and C are as per Figure 1. Peaks D and E are due to the presence of the substrate (lower trace). The upper trace is that of a blank and contained no enzyme. CE was performed as per Figure 1.
and SDintercept are the standard deviations of the slope and intercept of eq 1, respectively. The LOD of the assay is defined as that amount of enzyme that produces an AttoFluor peak that exceeds that of the blank by 3 times its standard deviation. Figure 2 shows an electropherogram of the injection of a 60min incubation of 4.6 × 10-16 M alkaline phosphatase with AttoPhos. Peaks A, B, and C correspond to those in Figure 1. Peaks D and E are due to the presence of the substrate. A log-log plot of the signal, corrected for dilution, was linear with alkaline phosphatase concentration over 6 orders of magnitude, 4.6 × 10-16-4.6 × 10-11 M (slope ) 1.02 ( 0.01, r2 ) 0.9996, n ) 18). At concentrations greater than 4.6 × 10-15 M, the sample required dilution prior to injection to avoid saturating the PMT. At the highest concentration of alkaline phosphatase used in this assay, ∼20% of the substrate is consumed in the 60-min incubation period. Linearity is expected to be lost at higher concentrations of enzyme due to substrate depletion. The concentration LOD under these conditions is 1.7 × 10-16 M. Since injection volumes in this instrument are typically much less than 1 nL, this limit corresponds to injection of fluorescent product generated by less than 1 enzyme molecule. However, in practical terms, the LOD in the amount of alkaline phosphatase required per analysis is set by the smallest volume required to prepare a sample. It is difficult to manipulate or inject sample volumes smaller than 1 µL. Thus, the practical mass LOD is 100 enzyme molecules (1.7 × 10-22 mol) in 1 µL. However, if the
reaction is performed within a microvessel, such as a single cell, then the nanoliter sampling capability of CE can be utilized fully. Detection of a single alkaline phosphatase molecule within the volume of a cell is within the ability of the system. The turnover number of alkaline phosphatase was calculated to be 80 000 molecules of AttoFluor produced per molecule of alkaline phosphatase per minute. The turnover number of alkaline phosphatase in 2.4 M diethanolamine (pH 10.0) containing 57 µM MgCl2 for AttoPhos at 31 °C is 100 000 molecules of AttoFluor produced per molecule of alkaline phosphatase per minute (JBL Scientific, personal communication). This is 25% greater than the value we obtain and likely reflects the difference in incubation temperatures employed, 24 vs 31 °C. Because the LOD in this assay is set by the rate of nonenzymatic hydrolysis of the substrate, reduction of this rate will result in an improved LOD. To obtain additional sensitivity, we employed a 50-µm-i.d., 145-µm-o.d. capillary using an electric field of 400 V/cm, 18 mM borate containing 10% dimethyl sulfoxide (pH 9.5) as running and sample buffer, and 20 mM borate (pH 9.5) as sheath flow buffer. AttoPhos concentration in the sample remained at 1 mM. The rate of nonenzymatic hydrolysis is lower in this buffer than in the diethanolamine buffer. The catalytic rate of alkaline phosphatase is also reduced, but to a lesser extent. A longer incubation is also expected to increase sensitivity. Under these conditions, we can detect 4.6 × 10-17 M alkaline phosphatase using a 21-h incubation period at room temperature. The LOD, based on a peak height that exceeds the blank peak by 3 times its standard deviation, is 1.5 × 10-17 M. The injection volume, which can be calculated under these conditions because the running and sample buffers are the same, was 2.4 nL. The detection limit corresponds to injection of the fluorescent product generated by 0.02 molecules of alkaline phosphatase. In terms of practical mass limit, the LOD is nine molecules of enzyme in a 1-µL sample volume. The injection volume of 2.4 nL is larger than is typically used for the previous conditions due to the use of a larger diameter capillary. Using a CE-based assay with UV detection of product, Regnier’s group reported concentration and mass LODs of 7.6 × 10-12 M and 5.2 × 10-20 mol, respectively, for alkaline phosphatase.11 A non-CE-based assay, involving the use of alkaline phosphatase bound to an oligonucleotide probe utilizing AttoPhos as a substrate, has yielded a concentration LOD of 5 × 10-16 M and a mass LOD of 5 × 10-20 mol.12 Chemiluminescent assays have been the most sensitive, reporting the detection of 1.6 × 10-17 M alkaline phosphatase in a sample volume of 100 µL.13 CONCLUSIONS We demonstrate detection limits of 15 aM (1.5 × 10-17 M) concentration of alkaline phosphatase in an overnight incubation, which corresponds to nine analyte molecules contained within a 1-µL analysis volume. As demonstrated in the preceding paper in this issue, molecular shot noise is an ultimate limit to chemical analysis (8). The precision of an analytical measurement is ultimately dominated by fluctuations in the number of molecules taken for analysis. (11) Wu, D.; Regnier, F. E. Anal. Chem. 1993, 65, 2029-2035. (12) Cano, R. J.; Torres, M. J.; Klem, R. E.; Casadesus J. J. Appl. Bacteriol. 1992, 72, 393-399. (13) Schaap, A. P.; Akhavan, H.; Romano, L. J. Clin. Chem. 1989, 35, 18631864.
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When dealing with very dilute solutions, large sample volumes are required to produce precise results. For example, a 15 aM sample contains 107 molecules/L; to obtain 1% precision in the amount of alkaline phosphatase by our high-sensitivity assay, it is necessary to assay at least 1 mL of solution. A part-per-thousand precision requires assay of at least 100 mL of solution.
ACKNOWLEDGMENT This work was supported by an operating grant from the Natural Sciences and Engineering Research Council. J.C.Y.W. acknowledges a summer studentship from the Alberta Heritage Foundation for Medical Research. N.J.D. acknowledges a McCalla professorship from the University of Alberta.
Xue and Yeung have detected the fluorescent product generated by isolated, individual enzyme molecules in a modified version of Regnier’s assay.14 That assay represents the ultimate limit of detectionscounting enzyme moleculessand the precision will be strongly dominated by molecular shot noise. (14) Xue, Q.; Yeung E. Nature 1995, 373, 681-682.
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Analytical Chemistry, Vol. 68, No. 4, February 15, 1996
Received for review July 5, 1995. Accepted November 22, 1995.X AC950650Z
X
Abstract published in Advance ACS Abstracts, January 1, 1996.
