Anal. Chem. 1994,66, 3797-3805
Selectivity in Electrophoretically Mediated Microanalysis by Control of Product Detection Time Bryan J. Harmon, Inkeun Leesong, and Fred E. Regnier' Department of Chemistry, Purdue University, West Lafayette, Indiana 4 7907- 1393
Differential electrophoretic mobility between an analyte and its product offers electrophoretically mediated microanalysis (EMMA) a unique capability to selectively control thedetection time of the product of a reaction-based chemical analysis. If an analyte and its product differ in migration velocity under the influence of an applied electric field, the apparent velocity and, consequently, the detection time of the product are dependent upon the relative amounts of time the product effectivelytraverses the capillary with the respective mobilities of the analyte and the product. Consequently, by controlling when the analytical reaction is allowed to occur, the detection time of the reaction product can be selectively maneuvered within a time window defined by the mobilities of the analyte and the product. This paper describes the use of this technique to manipulate the detection time of product profiles independently of nonreacting matrix interferants for the determination of both enzymes and substrates by EMMA. Furthermore, the ability to manipulate product detection times allows for simultaneous EMMA determinations of multiple enzymes or substrates. Recent r e p ~ r t s l -have ~ described the use of capillary electrophoretic (CE) systems for ultramicroreaction-based chemical analysis by a methodology known as electrophoretically mediated microanalysis (EMMA). In EMMA, electrophoretic mixing is utilized to merge zones containing the analyte and analytical reagents; the reaction is then allowed to proceed either in the presence or in the absence of the applied electric field; and, finally, the detectable product is transported under the influence of an applied potential to the detector. Our recent mathematical treatment of EMMA4 emphasized utilizing differential electrophoretic mobility as a method to mix distinct zones of chemical reagents under the influence of an applied electric field. However, differential electrophoretic mobility between an analyte and its product also offers EMMA a unique capability to selectively control the detection time of the product profile. In a typical EMMA determination, the product is observed at a time that is dependent upon the net migration velocities (1) Bao, J.; Regnier, F. E. J . Chromatogr. 1992, 608, 217-224. (2) Wu,D.; Regnier, F. E. Anal. Chem. 1993, 65, 2029-2035. (3) Miller. K. J.; Leesong, I.; Bao, J.; Regnier, F. E.; Lytle
r. E. Anal. Chem.
1993, 65, 3267-3270.
(4) Harmon, B. J.; Patterson, D. H.; Regnier, F. E. Anal. Chem. 1993,65,26552662. ( 5 ) Harmon, B. J.; Patterson, D. H.; Regnier, F. E. J. Chromatogr. 1993, 657, 429-434. (6) Patterson, D. H.; Harmon, B. J.; Regnier, F. E. J . Chromatogr. 1994, 662, 389-395. (7) Liu, S.;Dasgupta, P. K. Anal. Chim. Acta 1992, 268, 1-6. (8) Avila, L. Z.; Whitesides, G. W. J. Org. Chem. 1993, 58, 5508-5512. (9) Xue, Q.;Yeung, E. S.Anal. Chem. 1994, 66, 1175.
0003-2700/94/0366-3797$04.50/0 0 1994 Amerlcan Chemical Soclety
of both the analyte and the product as well as the relative amounts of time effectively spent at each of the two velocities. Upon the application of an electric field, the analyte traverses a portion of the capillary from the injection point to the position at which reaction occurs (&n) &n.
= uAtA = (&p,A + P w )
(1)
where UA and Pep,A are the net migration velocity and electrophoretic mobility, respectively, of the analyte; pe0 is the electroosmotic mobility; E is the electric field strength; and t~ is the time during which the analyte migrates under the influence of the applied electric field prior to undergoing reaction. A chemical reaction, either in the presence or in the absence of an applied potential, then forms the product, which migrates the remaining distance to the detection position under the influence of an applied electric field. Therefore, the product is detected in a total applied potential time period (&Jet), which is equal to the sum of the times during which it was effectively transported with the velocity of the analyte and during which it migrated with the velocity of the product tdet = tA
drxn 1 - drxn + tp = +P' vA
where tp is the applied potential time period required to transport the product from drXn to the detection position; 1 is the separation length of the capillary (i.e., distance from injection inlet to detection position); u p and pep,p are the net migration velocity and electrophoretic mobility, respectively, of the product; and A p e p , p - ~is the difference in electrophoretic mobility between the product and the analyte. As indicated by eq 2, if the analyte and product differ in electrophoretic mobility (Le., A p e p , p - ~# 0), the time at which the product is observed can be selectively altered by regulating the value of tA (i.e., when the analytical reaction occurs). In contrast, a nonreacting interferant must traverse the entire capillary with its single net migration velocity ( V I ) 1
tdet= - = u1
1
bepJ + Pm)E
(3)
where I ~ ~ is~ the , I electrophoretic mobility of the nonreacting interferant. It is this capability to control the detection time of an analyte's product profile independently of interferants Analytical Chemistty, Vol. 66, No. 21, November 1, 1994 3797
by controlling when the analytical reaction occurs which leads to selectivity in EMMA. This methodology is not dependent upon the particular separation mechanism (e.g., free solution, gel, and micellar forms of CE) that imparts the required differential electrophoretic mobility between the analyte and the product. This paper examines this selective control of product detection time as a method to resolve EMMA product profiles from nonreacting matrix interferants and to perform simultaneous multiple EMMA determinations of both enzymes and substrates. While the quantitative aspects of the EMMA methodology have been addressed p r e v i o ~ s l y , ~ -it~ should J-~~~ be noted that the manipulation of product detection time does not inherently affect quantitation. In traditional CE, since temporal peak width is inversely proportional to the velocity of the detected species at the detection position, peak area is directly proportional to migration time if the analyte traverses the capillary with a single velocity. However, in an EMMA determination, the product profile area is determined by the velocity of the observed product at the detection position regardless of its temporal position within the analyte's product detection window (Le., rather than the apparent migration velocity indicated by its migration time). Consequently, the manipulation of the detection time of an EMMA product profile does not alter its area.
EXPERIMENTAL SECTION Instrumentation. Polyimide-coated, fused silica capillaries of 50 pm inner diameter and 360 pm outer diameter were utilized. The total lengths of the capillaries were 50 cm for the leucine aminopeptidase (LAP) determinations and 24 cm for all other analyses. The separation lengths of the capillaries were 25 and 35 em for the LAP determinations depicted in Figure 1 and Figures 2 and 3, respectively, and 19.4 cm for all other analyses. All LAP assays were performed using a CE system built in-house. Electric fields were applied with a Spellman (Plainview, NY) Model FHR 30P 60/EI power supply, and detection was achieved using an 1x0 (Lincoln, NE) CV4CEvariable-wavelength absorbance detector. Data were collected by an i486 personal computer interfaced with a PC-LPM-16 1 / 0 board and NI-DAQ DOS software (National Instruments Corp., Austin, TX). All other EMMA determinations were performed using a BioFocus 3000 capillary electrophoresis system (Bio-Rad Laboratories, Hercules, CA). Reagents. Porcine kidney microsomal LAP, leucine-pnitroanalide, Sigma enzyme control, bovine intestine alkaline phosphatase (ALP),p-nitrophenyl phosphate, Escherichia coli @-galactosidase, o-nitrophenyl @-galactopyranoside, yeast alcohol dehydrogenase (ADH), nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), bovine heart malic dehydrogenase (MDH), malic acid, tris(hydroxymethyl)aminomethane, tris(hydroxymethy1)aminomethane hydrochloride, and glycine buffer solution (500 mM, pH 9.0) were purchased from Sigma Chemical Co. Monobasic and dibasic potassium phosphate were obtained from Fisher Scientific (Fair Lawn, NJ) while absolute ethanol was purchased from Midwest Solvents Co. of Illinois (Pekin, IL). A 10 mM pH 7.2 phosphate buffer and a 100 mM pH 8.0 Trizma buffer were prepared by dissolving monobasic and dibasic potassium phosphate and tris(hy3798
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droxymethy1)aminomethane and tris(hydroxymethy1)aminomethane hydrochloride, respectively, in degassed, doubledistilled, deionized water. A 200 mM pH 9.0 glycine buffer was made by diluting the Sigma glycine buffer solution with degassed, double-distilled, deionized water. The buffer solutions were adjusted to the proper pH with either 1.0 M HCl or 1.O M NaOH. The analytical reagent solutions were prepared by dissolving the appropriate analytical reagents in the prepared buffer solutions and adjusting to the proper pH with 1.O M HCl or 1.O M NaOH: (a) for the determination of LAP, 8.3 mM leucine-p-nitroanalide in 10 mM pH 7.2 phosphate; (b) for the simultaneous analyses of ALP and /3-galactosidase, 10 mM p-nitrophenyl phosphate and 5 mM o-nitrophenyl 0-galactopyranoside in 100mM pH 8.0 Trizma; (c) for the determination of ethanol, 0.5 mg mL-' ADH and 10 mM NAD+ in 200 mM pH 9.0 glycine; and (d) for the simultaneous analyses of ethanol and malate, 0.5 mg mL-I ADH, 0.1 mg mL-' MDH, and 10 mM NAD+ in 200 mM pH 9.0 glycine. Sample solutions were prepared by dissolving the appropriate analyte(s) in degassed, double-distilled, deionized water. EMMA Procedures. The capillaries were treated with 1 M NaOH for 10 min and then rinsed with the appropriate buffer solution for 10 min prior to use. For the determinations of enzymes, the capillary and the buffer reservoirs were initially filled with buffer solution containing the appropriate substrate(s). Hydrodynamic sample injections were then performed either by placing the anodic inlet of the capillary into the sample solution and raising about 10 cm for 10 s for the ALP assays utilizing the in-house system or by the application of pressure for a pressure-time constant of 1 psi s for the simultaneous analyses of ALP and @-galactosidaseusing the BioFocus 3000. The assay was then effected by applying an electric field (100 V cm-I for the determinations of ALP; 300 V cm-l for the simultaneous assays of ALP and @-galactosidase) and monitoring the absorbance at 405 nm. All enzyme determinations were performed either under the influence of a constant electric field or with an intermittent time period at zero potential. All substrate determinations were performed by initially filling the capillary and the buffer reservoirs with buffer solution containing the appropriate enzyme(s) and cofactor. When specified, an ADH-less gap was then created by placing the anodic inlet of the capillary in an analytical reagent solution devoid of ADH (Le., 10 mM NAD+ in 200 mM pH 9.0 glycine for the determination of ethanol; 0.1 mg mL-I MDH and 10 mM NAD+ in 200 mM pH 9.0 glycine for the simultaneous assays of ethanol and malate) and applying an electric field for 1 min. The sample was then hydrodynamically injected by the application of pressure for a pressuretime constant of 1 psi s, and the assay was effected by applying an electric field (250 V cm-I for the determination of ethanol; 300 V cm-I for the simultaneous analyses of ethanol and malate) and monitoring the absorbance at 340 nm. ALP determinations utilizing the in-house apparatus were performed at ambient temperature while the capillary was thermostated by circulating water at 20 OC throughout all assays employing the BioFocus 3000. Between determinations, the capillary was purged with 0.1 M KOH and then refilled with the appropriate analytical reagent solution.
RESULTS AND DISCUSSION EMMA Product Detection Time Window. If the analyte and the product each migrate from the injection point toward the detection position under the influence of an applied electric field, only reaction product that is formed between the point of injection and the detector can be observed in a typical EMMA determination. Product that is formed after the analyte zone passes by the detection position is not detected. As a result, there exists an exclusive “detection time window” during which product may be observed. The temporal limits of this window, Tp and TA,are defined by the mobilities of the product and the analyte, respectively. If the reaction occurs at the point of injection, the product traverses essentially the entire length of the capillary and is observed at a detection time corresponding to its net migration velocity T,=-=1 UP
1 bep,p
+F~JE
(4)
At the opposite extreme, if the reaction occurs just as the analyte reaches the detection position, the analyte itself traverses essentially the entire separation length of the capillary, and the product is detected at a time reflective of the net migration velocity of the analyte
If the product has a greater net migration velocity than the analyte, the first product that can be observed is that which formed first, and eqs 4 and 5 define the lower and upper temporal limits, respectively, of the detection window Tp Itdet I TA However, should the analyte possess the greater net migration velocity, the first product to be observed is that which formed last, and eqs 4 and 5 define the upper and lower temporal limits, respectively, of the detection window
influence of a constant electric field or by performing an intermittent zero potential incubation. In constant potential EMMA determinations of enzymes, the capillary and the buffer reservoirs are typically filled with buffer solution containing substrate (and cofactors, if necessary). Theenzyme sample is then injected, and an electric field is applied. If the enzyme and substrate differ in electrophoretic mobility, the enzyme zone interpenetrates the adjacent substrate region under the influence of the applied potential. Since the enzyme is not consumed, product is formed at a relatively constant rate as the analyte zone traverses the substrate region under the influence of a fixed electric field. Consequently, a relatively constant amount of product can be observed for each value of t A ranging from 0 to TA,and the resulting product profile is a plateau extending throughout the product detection window (ie., between TAand Tp). Therefore, rather than employing a constant electric field to impart selectivity in EMMA determinations of enzymes, the product migration time is most easily maneuvered within its detection time window by utilizing the zero potential mode to selectively produce an accumulation of product with a specific value of t A and corresponding tdct. In this method, an electric field is utilized to electrophoretically merge the enzyme and substrate zones. The electric field is then removed, and product is allowed to accumulate in the absence of an applied potential for a fixed time period. After the zero potential incubation, an electric field is again employed to transport the accumulated product to the detection position. The resulting concentration profile of the product is a peak representing the zero potential incubation superimposed upon the plateau obtained during the applied potential time periods. The area of the zero potential peak serves as a kinetic measure of the activity of the injected enzyme. Since the analyte traverses the capillary with its net migration velocity prior to the zero potential incubation, and the resulting product is transported the remaining distance to the detection position with its net migration velocity, the zero potential accumulation is observed at a total applied potential time determined by the time at which the electric field is removed to begin the incubation (tinc)
(7)
Any product that is formed between the point of injection and the detection position (Le., 0 It A IT A )must be observed at a fdetvalue within this detection time window as determined by t A and eq 2. Therefore, this detection window establishes the temporal limits within which a product profile may be selectively maneuvered in EMMA. The temporal width of this window (Atwindow)is directly proportional to the differential electrophoretic mobility of the analyte and product
As a result, those chemical systems whose analyte and product offer substantial differential electrophoretic mobility present the greatest opportunity for such selectivity. Selectivity in EMMA Enzyme Determinations. EMMA enzyme assay^^^.**^ may be performed either under the
Since tincdefines the time during which the analyte responsible for the zero potential product traverses the capillary, it is equivalent to t A in eq 2. In terms of the detection time window limits, the applied potential detection time of the zero potential product can be estimated as
with the restriction that 0 Itint ITA (Le., the incubation must be performed when the enzyme plug is positioned between the point of injection and the detection position in order for the product to be observed). Equation 10 indicates that a linear relationship exists between the moment at which the potential is removed and the time at which the corresponding zero potential product is observed. Therefore, the detection Analytical Chemistry, Vol. 66, No. 21, November 1, 1994
3799
0.02
D O
0
2
4
6 Time (min)
0
10
12
Figure 1. Control of the zero potential product detection time for the EMMA determinationof leucine aminopeptidase. (A) Constant potential determination; 5 mln zero potential incubation performed (B) at the point of injection prior to the application of an electric field and (C) following the application of an electric field for 5 min. P indicates the zero potential product (pnitroanaline). Experimental conditions stated in text.
/
0
,
2
,
#
4
,
6 Time (min)
8
10
12
Figure 2. Control of the zero potentialproduct detection tlme for the EMMA determination of leucine aminopeptidase in a Sigma enzyme control. (A) Constant potential determination: 10 min zero potential incubationsperformed following the application of an electric field for (B) 1, (C) 2, and (D)3 min. P indicates the zero potential product (pnitroanaline). Experimental conditions stated in text. 0 02-
4 1
time of a zero potential peak can be selectively and predictably manipulated within an analyte's product detection time window by the choice of Zinc. Figure 1 portrays the control of product detection time by use of the zero potential mode. The enzymatic system chosen was the determination of microsomal leucine aminopeptidase (LAP; EC 3.4.1 1.2) by its enzymatic hydrolysis of leucinep-nitroanalide. The product, p-nitroanaline, was monitored by its absorbance at 405 nm as a measure of the extent of reaction. Figure 1A illustrates the plateau obtained for the constant potential determination of LAP. The observed detection time window defined by the plateau extended from approximately 5.8 to 10.2 min thereby establishing the range of available detection times in which to maneuver a zero potential product peak. Figure 1B depicts a 5 min zero potential incubation performed at the time of injection prior to the application of an electric field (Le., tinc = 0). The incubation product (indicated by P), which formed due to diffusional interpenetration of the reagent zones at their interfaces,'~~ traversed essentially the entire separation length of the capillary and was detected at the edge of the detection time window indicative of the migration velocity of the product (i.e., Tp). Since this product accumulation was the first to be detected, the product must have had a greater migration velocity than the analyte. Therefore, the detection time window limits corresponded to net mobilities (Le., pep+ pea) of approximately 7.2 X lo4 and 4.1 X lo" cm2 V-l s-l for p-nitroanaline and LAP, respectively, based upon the experimental separation length of 25 cm and electric field strength of 100 V cm-l. As suggested by eqs 9 and 10, the detection time of the zero potential product could be delayed by allowing the slower migrating analyte to traverse a portion of the capillary prior to performing the zero potential conversion to the faster migrating product. In Figure lC, the potential was initially applied for 5 min during which the analyte migrated approximately 12 cm from the point of injection. Following a 5 min zero potential incubation, the potential was reapplied, and the product was transported the remaining 13 cm to the detection position. As predicted by 3800
Analytical Chemistry, Vol. 66, No. 21, November 1, 1994
-F In
80
li 00161 I
1
0012-
0
2
4
6
E
10
12
Time (min)
Figure 3. Control of the zero potential product detection tlme for the EMMA determination of leucine aminopeptidase in untreated urine. (A) Constant potentialdetermination: (B) 10 mln zero potential incubation performed following the application of an electric fleld for 2 min. P indicates the zero potential product (pnitroanaline) while I slgnifles a matrix Interferant with electrophoreticsimilar mobllHy to the product. Experimental conditions stated in text.
eqs 9 and 10, the incubation product was observed at approximately 7.9 min, thereby indicating that the zero potential accumulation could be selectively positioned within the detection time window by the choice of tinc with the restriction imposed that 0 Itinc I10.2 min in order to observe the zero potential product. Figures 2 and 3 demonstrate the ability to maneuver such zero potential peaks independently of nonreacting matrix interferants in order to aid resolution of the product accumulation. Figure 2 depicts the EMMA analysis of LAP in a Sigma enzyme control, a mixture of more than 10 different enzymes in a nonhuman protein matrix. The constant potential determination of LAP is shown in Figure 2A. Due to the relatively low activity of LAP in the sample, the product plateau was not clearly defined. The continual separation of the product from the analyte zone under the influence of an applied potential prevented sufficient product to accumulate for detection. However, during zero potential incubations the analyte and product were not electrophoretically separated
thereby allowing ample product accumulation to permit observation. In Figure 2B-D, tint was altered to selectively maneuver the detection time of a 10 min zero potential incubation peak (indicated by P) within the detection time window. Linear regression of the selected values of tinc and the resultant observed values of fdet yielded fdet = O . 5 3 t i n c + 7.0 with a correlation coefficient of 0.992, thereby confirming the linear relationship predicted by eq 10. Based upon the coefficients of eq 10, the experimental product detection time window must have extended from 7.0 (i.e., Tp) to 15 min (Le., TA). These results indicated that the zero potential product peak could be selectively positioned within these temporal limits by controlling the value of tinc between 0 and 15 min. However, since the nonreacting matrix components migrated with the same velocity prior to and following the zero potential incubation, their observation times were not affected by the chosen value of tint. Figure 3 depicts the EMMA determination of LAP in an untreated urine sample. Urine samples typically exhibit lower LAP activity than serum samples, and the presence of yellow chromogens makes detection of the product difficult in untreated specimens. However, the ability to manipulate the detection time of an EMMA zero potential product peak allowed LAP activity to be assayed directly from untreated urine samples. As shown in Figure 3A, low LAP activity resulted in an indiscernible product plateau for a constant potential determination. Furthermore, when a zero potential incubation was performed at the point of injection, the resulting product comigrated with a matrix interferant (indicated by I) possessing a similar electrophoretic mobility, and the resulting electropherogram was indistinguishable from Figure 3A. However, in Figure 3B, a 10 min zero potential incubation following a 2 min application of the electric field yielded a resolved product peak (indicated by P). Based upon their differential electrophoretic mobility (i.e., 4.4 X lo4 cm2 V - l s-l), the initial application of the potential gradient allowed the analyte zone and the interferant to separate by approximately 5.3 cm prior to the zero potential incubation. Consequently, although the resulting product and the interferant were then transported at similar velocities, they reached the detection position approximately 1 . 1 min apart. Simultaneous EMMA Enzyme Determinations. The capability to incorporate multiple substrates into the running buffer solution and to manipulate product detection time by use of the zero potential mode also allows for the simultaneous determination of multiple enzymes by EMMA. If two enzymatic analytes (Le., A1 and A2) react with appropriate substrates to yield products (i.e., P1 and P2, respectively), their zero potential accumulations exhibit a difference in detection time ( A f d e t ) of
where tdet,P1 and fdet,R are the applied potential detection times of p1 and p2, respectively; p c p , ~ i ,Pcp,A2, Pep,P1, and PCP,PZare the electrophoretic mobilities of A l , A2, P1, and P2, respectively; and ApCp,p2-p1 is the difference in electrophoretic
mobility between P2 and P1. As defined by eq 1 1 , if the analytes and/or their products differ in electrophoretic mobility, the magnitude of the separation of the product peaks is dependent upon the choice of tinc. When the analytes have indistinguishable electrophoretic mobilities pep,^) while the products differ in migration velocity, eq 1 1 reduces to
Equation 12 indicates that the temporal separation of the zero potential accumulations increases as the value of tinc is decreased since the differential mobilities of the products facilitates a postreaction separation. Maximal separation (Atdct,m,x) is obtained when the zero potential incubation is performed at the point of injection &e., tint = 0) “dct,max
A&p,PZ-PI
=
I
(PCP,Pl+ Pco)(cLep,P2
+ ”)E
(13)
However, if the analytes differ in electrophoretic mobility while the products are electrophoretically indistinguishable (pcp,p), eq 1 1 reduces to A~epA2-Altinc
Atdct
=
+ II,
IIcp,p
where A p e p , ~ 2 - ~isl the difference in electrophoretic mobility between A2 and A l . Equation 14 suggests that as tint is increased, the temporal separation of the zero potential product peaks is also increased since the differential electrophoretic mobilities of the analytes support a prereaction separation. Maximal separation is obtained if the zero potential incubation is performed just prior to the first analyte zone (e.g. A2) reaching the detection position
In systems for which both the analytes and the products differ in electrophoretic mobility, the greater of the respective differential electrophoretic mobilities dominates the optimal choice of tinc. Figure 4 depicts the simultaneous free solution EMMA determination of alkaline phosphatase (ALP; EC 3.1.3.1) and P-galactosidase (EC 3.2.1.23)previously reported by Wu and Regnier2 utilizing capillary gel electrophoresis. ALP was assayed by its hydrolysis ofp-nitrophenyl phosphate to produce p-nitrophenol while &galactosidase was determined by its hydrolysis of o-nitrophenyl /3-D-galactopyranoside to form o-nitrophenol. Both p-nitrophenol and o-nitrophenol were monitored at 405 nm. ALP and p- galactosidase exhibited net mobilities of 3.5 X 10-4 and 2.3 X lo4 cm2 V-’ s-l, respectively, while both products demonstrated net mobilities of approximately 1.6 X 10-4 cmz V - l s-l. Consequently, for the experimental separation distance of 19.4 cm and electric field strength of 300 V cm-I, the product detection time windows for ALP and @galactosidase extended from 3.1 to Analytical Chemistry, Vol. 66, No. 21, November 1, 1994
3001
0 016
I
0
2
4
6
8
Time (min)
Flgure 4. Control of the zero potential product detection tlme for the simultaneous EMMA determinatlons of alkaline phosphatase and &galactosidase. A 10 min zero potential incubation performed (A) at the point of injection prior to the application of an electric field and (B) following the application of an electric field for 2 min. P and 0 Indicate the product accumulationsdue to the reactlons of alkaline phosphatase (pnltrophenol) and &galactosidase (mitrophenol), respectively. I signifies the product formed at the enzyme and substrate zone Interfaces prior to the application of an electric field. Experimental conditions stated in text.
6.8 and 4.6 to 6.8 min, respectively, as defined by eq 7. For Figure 4A, a 5 min incubation was performed prior to the initial application of the potential (Le., tinc = 0). As predicted by eq 14, no separation of the zero potential products was obtained as each was observed at their similar values of T p . The similar electrophoretic mobilities of the products required that a prereaction separation of the analytes be performed prior to initiating the zero potential mode. In Figure 4B, the electric field was initially applied for 2 min, thereby allowing the differential migration velocities of ALP and 8-galactosidase to separate the analyte zones by approximately 4.3 cm prior to undergoing simultaneous 5 min zero potential incubations. Consequently, although the resulting product accumulations migrated at similar velocities, the detection times of the peaks representing ALP (indicated P) and 8-galactosidase (indicated by G) were separated by approximately 1.5 min, as predicted by eq 14. The accumulation ofp-nitrophenol and o-nitrophenol observed at Tp (signified by I) in Figure 4B formed due to diffusional interpenetration at the reagent interfaces during the approximately 40 s required by the BioFocus 3000 between injection and the initial application of the electric field. Selectivity in EMMA Substrate Determinations. EMMA determinations of substrate^^.^ are typically performed at constant potential. As a nonamplifying end point method, EMMA substrate analysis cannot easily exploit the zero potential mode discussed for the kinetic determination of enzymes. Consequently, product detection time in constant potential EMMA determinations of substrates must be regulated by controlling the temporal and spatial engagement of the reagents. In a typical EMMA substrate determination, the capillary and the buffer reservoirs are filled with a solution containing the appropriate enzyme (and cofactors, if necessary), a sample plug of substrate is injected adjacently, and a constant electric field is applied. Therefore, the reaction proceeds immediately (Le., t~ = 0) at the point of injection, and this initial product 3802
Analytical Chemistty, Vol. 66, No. 21, November 1, 1994
is observed at a detection time corresponding to the velocity of the product (i.e., T p ) . As the substrate plug traverses the enzyme zone, product is formed until the analyte is essentially depleted. If a total reaction time of trxn is required to consume the analyte, product is formed for values of ?A ranging from 0 to trxn,and if the substrate plug is fully depleted prior to passing by the detection position, product is observed at corresponding detection times of
when the product has a greater migration velocity than the analyte or
in cases for which the analyte has the greater migration velocity. However, since product formed after the analyte plug passes by the detection position cannot be observed, the maximum reaction time (trx,max) available to fully deplete the analyte is equal to the time required for the analyte to migrate from the injection point to the detection position
If the substrate is not fully reacted prior to passing by the detection position, the product profile is abruptly truncated at TA (i.e., product is observed from Tp to TA),and its area is no longer directly proportional to the quantity of analyte injected . In EMMAend point substratedeterminations, iftheanalyte and its product differ in electrophoretic mobility, the product profile can be maneuvered within its detection time window by delaying the initial engagement of the substrate and enzyme zones, thereby allowing the analyte to traverse a portion of the capillary with its velocity prior to undergoing reaction. The methodology chosen to control the initial engagement of the reagents is depicted in Figure 5 , which assumes the substrate has a greater migration velocity than the enzyme. The capillary is initially filled with the enzyme solution (Figure 5A). However, rather than injecting the substrate sample immediately adjacent to the enzyme zone, the anodic inlet is placed in a solution devoid of the enzyme, and a reagentless “gap” between the substrate and enzyme regions is created (Figure 5B)by the application of an electric field (E8ap)prior to injecting the analyte dgap
= URtgap =
(Pep,R
+ PwlE 8aP 8aP
(19)
where dBapis the spatial width of the created reagentless gap region; tgap is the time for which the potential is applied; and U R and k c p ,are ~ the net migration velocity and electrophoretic mobility, respectively, of the enzyme reagent zone. Consequently, following sample injection (Figure 5C) and the application of an electric field (Eassay), the faster analyte zone
B
F
Therefore, if the substrate is fully consumed prior to passing by the detection position, eqs 21 and 22 define the temporal limits between which product is observed. However, if the analyte is not depleted prior to passing by the detection position, the product profile is truncated at TA(i.e., product is observed between Tdet,i and TA). Consequently, delaying the initial engagement of the substrate and enzyme zones decreases the maximum available time to deplete the analyte to that time required for the substrate to migrate from the point of engagement (dengagement) to the detection position (Figure 5G).
G
'rxn,max
C
D
E
-
- dengagement VA
--
- 'A'engagement VA
A Det
Flgure 5. Method utilized to control the engagement of the substrate and enzyme zones for the EMMA determinationof a substrate with a greater migration velocity than its enzyme. (A) The capillary is initially filled with the enzyme solution (light shading). (B) The inlet end of the capillary (left) is placed in a buffer reservoir devoid of the enzyme, and an electricfield is appliedto create a reagentlessgap region(no shading). (C) The substrate sample (dark shading) is injected.(D) Upon application of an electric field, the faster migrating substrate zone catches up to the slower enzyme region. (E) The substrate plug engages the enzyme zone, and the initial reaction product is formed (not indicated). (F) The reaction proceeds as the substrate plug traverses the enzyme region. (G) The substrate zone passes by the detection position (indicated by Det) thereby endingthe available reaction time as product formed after this point cannot be observed.
must traverse a portion of the capillary prior to engaging the slower enzyme region (Figure 5D). The analyte zone initially interpenetrates the enzyme region at a time (tengagement) dependent upon the spatial width of the gap and the differential velocity of the substrate and enzyme (AuA-R) dgap 'engagement
"A-R
'pep,A-REassay
where Apep,A-R is the difference in electrophoretic mobility between the substrate and enzyme. As a result, the first product is formed at a t A value of ?engagement and is detected at a corresponding time (tdet,i) Of
Following zonal engagement, product formation continues as the substrate traverses the enzyme region (Figure 5F). Assuming a required reaction time of trxn to fully deplete the substrate, the final product is formed at a t A value equal to (tengagement + trxn) and is detected at a corresponding time (tdet,f) of
For systemsin which the analyte has a lesser migration velocity than the enzyme zone, the substrate is initially injected, and a reagentless gap region is then created "behind" the analyte zone prior to the introduction of the enzyme into the inlet buffer reservoir. Alternatively, a gap lacking a required cofactor, rather than the enzyme itself, can be utilized. Figure 6 illustrates the control of product detection time for EMMA substrate determinations. The enzymatic system chosen was the catalytic oxidation of ethanol to acetaldehyde by alcohol dehydrogenase(ADH; EC 1.1.1.1). The concurrent reduction of the coenzyme NAD+ to NADH was monitored by the increase in absorbance at 340 nm as a measure of the extent of reaction and, therefore, the amount of ethanol injected. At experimental conditions, ethanol and NADH exhibited net mobilities of 3.8 X lo4 and 2.0 X l P cm2 V-l s-l, respectively. Therefore, for the experimental separation length of 19.4 cm and electric field strength of 250 V cm-l, the NADH detection time window extended from 3.4 to 6.4 min, as defined by eq 7. For Figure 6A, the capillary was filled with buffer solution containing ADH and NAD+, a sample containing both ethanol and NADH was injected at the anodic inlet, and the electric field was applied. The interpenetration of the adjacent ethanol and ADH zones and the resulting reaction proceeded immediately,and the observed product (indicated by P) extended from Tp to approximately 5.7 min, a detection time that corresponded to a required incubation period of roughly 0.9 min to fully deplete the ethanol sample, as estimated by eq 17. Since the initial NADH formed in the reaction traversed essentially the entire capillary with its velocity, it comigrated with the interferant NADH originally contained in the sample. However, eq 2 1 suggested that, since the analyte had a greater migration velocity than the product, the product profile could be selectively maneuvered to earlier detection times by delaying the engagement of the ethanol and ADH zones by utilization of the reagentless gap technique. In Figure 6B-E,the capillary was initially filled with buffer solution containing ADH and NAD+, the Analytical Chemistry, Vol. 66, No. 21, November 1, 1994
3803
"
--- I
0 006
0
2
6
4
8
Flgure 6. Control of the constant potential product detection time for the EMMA determlnatlon of ethanol. Reagentless gaps of (A) 0, (B) 1.6, (C)3.2,(D) 4.9, and (E) 6.5 cm were utilizedto control the engagement of the ethanol and ADH zones. P Indicates the product NADH while I slgnlfies the interferant NADH originally contained in the ethanol sample. Experimental conditions stated in text. Table 1. Calculated Parameters for the Control of Product NADH Detection Tlme In the EMMA Determlnatlon of Ethanol
6A 6B 6C 6D 6E
Egap
dgrp
ten agcmcnt
den gemrnt (m)
0 100 200 300 400
0 1.6 3.2 4.9 6.5
0 1.0 2.0 2.9 3.9
0 5.6 11 17 22
( ~ c m - 1 ) (cm)
&in)
tdcti
trxnmax
6.4 5.5 4.6
3.4 2.4 1.4 0.5 0
(mil;)
3.7
(min)
anodic end of the capillary was placed in a buffer solution containing only NAD+, and an electric field was then applied for 1 min. As indicated by eq 19, the magnitude of the applied electric field (Le., Egap) determined the spatial width of the ADH-less gap created. The sample of ethanol and NADH was then injected, and an electric field of 250 V cm-' was applied. The effects of thegap width are summarized in Table 1, which includes calculated estimates for the spatial width of the reagentless gap created, the time and position of the initial engagement of the ethanol and ADH zones, and the maximum available reaction time. These values are based upon the experimental net mobilities of ethanol, NADH, and ADH (2.7 X lo4 cm2 V-l s-l) and the stated experimental parameters for Figure 6. As predicted by eq 21, the product profile was shifted to earlier detection times as the initial engagement of the ethanol and ADH zones was delayed by the use of a wider gap. While the detection time of the product NADH was dependent upon the engagement of the ethanol and ADH zones, the interferant NADH was always transported the entire length of the capillary with the velocity of the product and was invariably observed at Tp (indicated by I). The gaps utilized in Figure 6B,C yielded well-resolved NADH peaks. However, in Figure 6D, the ethanol zone did not engage the ADH region until the analyte had traversed nearly 17 cm of the 19.4 cm separation length, thereby leaving only about 0.5 min of available reaction time, as estimated by eq 23. This incubation period proved to be insufficient to fully deplete the substrate, and the product profile was abruptly truncated at TA as the remaining unreacted ethanol passed by the detection position. As indicated in Table 1, the gap created in Figure 6E was too wide for the ethanol zone to 3804
0
E
/?
/I ,
2
4
E
8
I M
10
I
12
Time (min)
Time (min)
figure
I
Analytical Chemistty, Vol. 66, No. 21, November 1, 1994
Figure 7. Control of the constant potentialproduct detection time for the simultaneous EMMA determinations of ethanol and malate. (A) No reagentless gap utilized to delay the engagement of the reagents: (B) ADKless gap employed to delay the engagement of the ethanol and ADH zones while the reaction between the adjacent malate and MDH regions was allowed to proceed Immediately. E and M indicate the product NADHaccumulationsdueto the reactions of ethanoland malate, respectively. Experimental condltlons stated in text.
reach the ADH region prior to passing by the detection position (i.e., trxn,max= 0), and consequently, no product NADH could be observed. Simultaneous EMMA Substrate Determinations. The capacity to incorporate multiple enzymes and cofactors into the running buffer and to selectivelymaneuver product profiles within their respective detection time windows allows for simultaneous substrate determinations by EMMA. While the zero potential methodology typically produces a single valueof tmcforeach simultaneous enzyme analysis, the selective control of the engagement of various reagent zones allows for specific values of t~ to be selected for each individual reaction in a multiple substrate determination. Figure 7 depicts the simultaneous EMMA assays of ethanol and malate. Ethanol was determined via its oxidation by ADH while malate was analyzed through its catalytic oxidation to oxalacetate by malic dehydrogenase (MDH; EC 1.1.1.37). For both reactions, the concurrent reduction of NAD+ to NADH was monitored at 340 nm. In Figure 7A, the capillary was initially filled with buffer solution containing ADH, MDH, and NAD+, a sample containing ethanol and malate was injected adjacently at the anodic inlet, and an electric field of 300 V cm-l was applied. Since both reactions were allowed to occur immediately at the point of injection, the initial NADH formed by both ethanol and malate comigrated at the analytes' similar values of Tp. In Figure 7B, resolution of the NADH product profiles representing ethanol (indicated by E) and malate (indicated by M) was obtained by use of the reagentless gap technique. The capillary was initially filled with buffer solution containing ADH, MDH, and NAD+. A buffer solution containing only the reagents MDH and NAD+ was then placed in the anodic buffer reservoir, and an electric field of 150 V cm-l was applied for 1 min to create an ADHless gap region. Upon injection of the sample containing ethanol and malate and the application of the electric field, the reaction of malate proceeded immediately with theadjacent MDH region while the engagement of the ethanol plug with the ADH zone was selectively delayed in order to move the
ethanol product profile to a sufficiently earlier detection time to allow resolution of the two NADH product accumulations. Malate and NADH exhibited similar electrophoretic mobilities, thereby preventing significant manipulation of the detection time of malate's product profile. This observation confirmed the necessity indicated by eq 2 for differential electrophoretic mobility to selectively maneuver an analyte's product detection time in EMMA.
CONCLUSIONS Analytical determinations are frequently performed in life science laboratories by the direct instrumental measurements of analytes. When an interfering substance precludes direct measurement in traditional reaction-based chemical analysis, a sample pretreatment step or chromatographic dimension is typically added, or the analytical reaction is linked to one or more secondary reactions until a product with uniquedetection characteristics is formed. However, in EMMA determinations, these additional steps can frequently be eliminated due to this capability to selectively maneuver the detection time of reaction product profiles independently of nonreacting matrix components. Furthermore, this selective control of product detection time allows for the simultaneous on-line
determination of multiple analytes without the necessity of unique detection characteristics. Although EMMA determinations typically do not exhibit the extremely large numbers of theoretical plates associated with CE methods, the ability to produce high degrees of selectivity can negate the need for tremendous efficiency. The coupling of this unique selectivity with the ability to analyze micros ample^^*^ due to the small dimensions of the CE systems employed makes EMMA a potentially powerful tool for reaction-based chemical analysis. As an example of the types of analyses of which EMMA is capable, Xue and Yeung9have recently used the zero potential technique to separate and assay isoenzymes of lactate dehydrogenase in single human erythrocytes.
ACKNOWLEDGMENT Financial support was received from the National Institute of Health (Grant 2543 1) and PerSeptive Biosystems. We gratefully acknowledge the generous loan of the CV4 CE detector from Isco. Received for review April 11, 1994. Accepted August 9, 1994." a
Abstract published in Advance ACS Abstracts. September 15, 1994.
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