1444
Anal. Chem. 1982, 5 4 , 1444-1445
Capillary Flow Injection Analysis for Enzyme Assay with Fluorescence Detection Tim A. Kelly' and Gary D. Christian* Department of Chem;stty, Universlty of Washington, Seattle, Washington 98 195
Flow injection analysis (FIA), as introduced in the 1970s (I-@, has undergone substantial growth in the areas in instrumental development and chemical application. Originally, conditions of turbulent flow were considered necessary to obtain thorough mixing of the injected sample plug with the reagent stream (7). It is now recognized that diffusional mixing takes place and can be controlled to a great extent through the manipulation of the parameters affecting the flow dynamics in a particular system (8-10). Residence time (t) and sample size (s) were initially emphasized in their influence on dispersion (8). Diffusion within the solution stream (&, diffusion coefficient), tube length ( L ) ,flow rate (F),and are recognized to influence the necessary pressure drop (AP) residence time. Contributions from injection, detection, and reaction manifolds have also been studied (9-14). The tube radius ( R ) ,however, was underemphasized with the practical limitations on tubing, connections, and pumps a t the time. Theoretically, the Taylor dispersion equation and the tanks-in-series model have proven effective in describing dispersion in FIA. For straight tubes, as derived from Taylor's equation, the practical variables and sampling rate (8)and reagent consumption per peak (8)have the following relationships to the flow parameters (11):
S = (49/R)(D,/t)1/2
(1)
Q = 6aR3L/ (24D,t)1/2
(2)
A strong dependence on tube radius is observed, supporting an advantageous use of narrower tubes. Recently, the trend within chromatography to miniturized systems has motivated some similar considerations within FIA (9, 11, 15). Tijssen has demonstrated that small diameter tubes are the best approach for high sampling rates and low reagent consumption (11). In comparison to the alternative modifications of coiled tubing, packed-bed reactors and segmented flow (12, 13), capillary systems provide the best combination of dispersion control, pressure drop, reagent consumption, and sampling rate for convenient practical analysis (11). Tightly coiled tubes are effective with larger tubing and faster flow rates but involve higher reagent consumption. Packed-bed reactors can be used effectively for relatively fast reactions at high flow rates. Segmented flow analysis requires rigorous attention to reducing residual system dispersion and synchronization of sampling, mixing, and debubbling operations for acceptable performance. Recently, Reijn and co-workers have illustrated a single bead string reactor that embraces mixing without sacrificing dispersion (14) but have not demonstrated the limits of its performance in comparison to these other modifications. A major difficulty in applying FIA to rate limited reactions such as enzyme reactions is the significant dispersion in coiled or packed reactors (12) when the reactions must proceed for long times to produce sufficient change to measure. Stopped-flow techniques (8, 16) may be employed to minimize dispersion, but this limits the rate of sampling. In some cases, sensitive detection systems can be utilized to allow shorter reaction times (17). Reduced tube radii can serve to counterbalance the adverse effect on dispersion of longer residence times. 'Present address: Pacific Lutheran University, Department of Chemistry, Tacoma, WA 98447. 0003-2700/82/0354-1444$01.25/0
Capillary systems have previously been used in FIA (6,11, 18). Reduced sample and reagent consumption along with enhanced sampling rates were the original benefits exploited (6, 18). However, the full potential of the technique, practically and theoretically, has only been investigated recently (11). Utilization of capillary FIA has not been emphasized as an aid to longer reaction times. In this paper we present the use of capillary FIA and its application to enzyme assays. A fluorescence detection system is employed which was previously developed for substrate determination (17). In that system, serum glucose was determined by using glucose oxidase (GOD) and horseradish peroxidase (HRP) to produce a fluorescent dye [dichlorofluorescein (DCF)]. The reaction sequence was as follows, where LDADCF represents the nonfluorescent dye, leucodiacetyldichlorofluorescein: glucose
+ O2
GOD
gluconic acid HRP
LDADCF + H202
DCF
+ H202
+ 2H20
(3) (4)
The kinetics of this enzyme method are readjusted in this study for the investigation of enzyme activity assay a t low levels using capillary FIA to allow the necessary longer reaction times.
EXPERIMENTAL SECTION Instrumentation. The flow fluorimeter described in the previous study (17)was used. A laser is focused on the flow cell. A microscope equipped with cutoff filter and baffle collects the fluorescent radiation and directs it to a photomultiplier tube to be recorded as peaks of voltage deflections on a strip-chart recorder. The flow system is simply constructed of a compressed air pump, loop injector,Teflon and glass tubing, a bubbled-timed flow meter, and a nanoliter sheath flow cell. Excitation from the argon ion laser was at 488 nm. A glass capillary tubing (0.2 mm i.d., 0.4 mm o.d., 2.75 m length) and a 1O-fiLloop injector were used to reduce reagent consumption and sample dispersion and allow for longer reaction times. The capillary tubing was prepared from Pyrex tubing (4 mm i.d., 6.3 mm 0.d.) drawn on a Shimadzu glass drawing machine, Model GMD-1, at 800 "C, resulting in a coil of 11 cm diameter. Preliminary investigations of the indicating reaction were made on a Perkin-Elmer 650-10s fluorescence spectrophotometer, exciting with 488-nm light and monitoring the fluorescence with time at 525 nm. A translation of optimum concentrations from the static to the flow system was arbitrarily made. Reagents. The agents tested for their effect on dye stability were reagent grade (Baker, Mallinckrodt, MC&C, and Sigma). The other reagents (HzOz,LDADCF, GOD, and glucose) were the same as those used in the previous study (17). The buffer used in the final assay method consisted of 0.02 M phosphate, 0.15 M NaCl, and 1% poly(ethy1ene glycol) (PEG-6000, Sigma P-2139) at pH 7.0 (PBS, phosphate buffered saline). In the GOD assay, pressures of 0.2 and 0.6 atm for the sheath and reagent streams gave respective flow rates of 1.55 and 0.075 mL/min. This allowed 120 s for the reaction to occur before reaching the detector. The reagent stream contained glucose (0.5 M) and activated (17) LDADCF M) in PBS (0.5% PEG6000). Samples were aqueous standards of GOD with HRP (0.4 IU/mL) added, in the PBS solution (1%PEG-6000), made by serial dilution. RESULTS AND DISCUSSION Flow System, The reduced tube radius served to maintain conditions of medium dispersion with the smaller sample plugs over the longer reaction times necessary. In the system here, 0 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 8, JULY 1982
1445
I" 14C
s
\
120
0
I Y) n 0
c
IOC
./
sc
0
50
'
100
0.8 F,
,
0
ul/rnin
Flgure 1. Relationship of F,to ,f (time for response to return to base line) for Injected DCF (4.8 ng) samples using glass capillary tubing (0.2 mm I.d., 2.76 min length).
with limited pressures possible, large reductions in reagent consumption were observed (reagent flow F,,0.075 mL/min, a 30-fold decrease in consumption for comparable dispersion using different sample size, tube length, and tube diameter as in the previous study (17)). Even at these flow rates peaks maintain their integrity over the length of the tubing, allowing a reaction to proceed for 120 s (and longer if necessary) to enhance the sensitivity of the system. The behavior of the flow system, as indicated by the peak lbase width (seconds) is shown in Figure 1 for a range of reagent flow rates. The submicroliter flow cell utilized in the present system is ideal for capillary FIA in that added sample dispersion in the detector is minimized. Tijssen (11) noted that, with a 25-1L detector cell, it is necessary to add extra mobile phase via a T piece at the column output to prevent tailing when using a capillary column. While Brady and Frantz (18)used a capillary system allowing injections of 1p L of sample, they employed a 30-pL mixing chamber and a 20-pL flow cell. LDADCF Stability and Activity. A significant blank exists in the fluorescence detection system previously reported (17). This is primarily the result of catalysis of autooxidation of LDADCF by HRP. Several substances were tested for reducing the blank, including oxidizing and reducing agents, complexing agents, and surfactants. Only poly(ethy1eneglycol) (PEG-6000) resulted iin a large reduction of the blank signal. One percent PEG-6000 was chosen as optimum, and its effect was maximum at pH 7.4 in the presence' of the PBS solution. While the sample plug contained 1% PEG-6000, the reagent stream contained 0.5% in order to lower viscosity and improve flow which resulted in better precision. Glucose Oxidase Assay. The behavior of the enzyme system was presented in the previous study (17). Concentrations were altered to optimize the kinetics for an enzyme assay and to demonstrate measurement of low enzyme activity. Figure 2 shows the response curve for the assay of glucose oxidase. An activity of 2 IU/L was the minimum detectable level. A calibration curve over 4 orders of magnitude was
I
I
I
I
L
I
2
3
4
5
Lag
Activity,
lU/I
Flgure 2. Relationship of fluorescence response to activity of glucose oxidase standards.
demonstrated, with an average precision of *2.5% (1.1% for high activities, 5.5% for low activities). Nonlinearity similar to that of the glucose system was observed and is probably not related to employment of the capillary system. The feature of reduced dispersion was used effectively in an analysis requiring relatively long reaction times and resulted in low sample and reagent consumption. The ability to use simple, inexpensive pumps and standard FIA and chromatography fittings demonstrates that capillary FIA is a practical, effective technique of choice for a range of wet chemical methods of analysis. Appropriate designs of injection devices will allow the use of even smaller samples (e.g., 1FL (18))and capillaries.
LITERATURE CITED (1) Bergmeyer, H. U.; Hagen, A. Z.Anal. Chem. 1972, 267, 333. (2) Whlte, V. R.; Fltzgerald, J. M. Anal. Chem. 1972, 44, 1267. (3) White, V. R.; Fitzgerald, J. M. Anal. Chem. 1975, 47, 903. (4) Stewart, K. K.; Beecher, G. R.; Hare, P. E. Fed. R o c . , Fed. A m . SOC.f x p . Blol. 1974, 33, 1439. (5) Ruzlcka, J.; Hansen, E. H. Anal. Chlm. Acta 1975, 78, 17. (6) Stewart, K. K.; Beecher, G. R.; Hare, P. E. Anal. 8iOChem. 1978, 70, 167. (7) Ruzicka, J.; Hansen, E. H. Anal. Chlm. Acta 1975, 75, 145. (8) Ruzicka, J.; Hansen, E. H. Anal. Chlm. Acta 1978, 99, 37. (9) Reljn, J. M.; Van Der Llnden, W. E.; Poppe, H. Anal. Chim. Acta 1980,
114, 105. 10) Vanderslice, J. T.; Stewart, K. K.; Rosenfeld, A. G.; Higgs, D. J. Talanta 1981, 28, 11. 11) Tijssen, R. Anal. Chlm. Acta 1980, 774, 71. 12) Deelder, R. S.; Kroll, M. G. F.; Beeren, A. J. B.; Van Den Berg, J. H. M. J . Chromatogr. 1978, 749, 669. 13) Van Den Berg, J . H. M.; Deelder, R. S.; Egberink, H. G. M. Anal. Chim. Acfa 1980, 774, 91. (14) Reijn, J. M.; Van Der Llnden, W. E.; Poppe, H. Anal. Chim. Acta 1981, 723, 229. (15) Ruzlcka, J.; Hansen, E. H.; Mosback, H.; Krug, F. J. Anal. Chem. 1977, 49, 1858. (16) Ruzlcka, J.; Hansen, E. H. Anal. Chlm. Acta 1979, 706, 207. (17) Kelly, T. A.; Chrlstlan, G. D. Anal. Chem. 1981, 53, 2110. (18) Brady, J. B.; Frantr, J. D. A m . Minerol. 1980, 65, 1249.
RECEIVED for review November 19, 1981. Accepted April 8, 1982.
