Dynamic Multicuvette Fluorometer-Spectrophotometer Based on the GeMSAEC Fast Analyzer Principle T. 0. Tiffany, C. A. Burtis, J. C. M a i l e n , and
L. H. T h a c k e r
Molecular Anatomy ( M A N ) Program, Oak Ridge National Laboratory, Oak Ridge. Tenn. 37830
A dynamic multicuvette fluorometer-spectrophotometer based on the GeMSAEC Fast Analyzer principle has been developed. Inherent in the establishment of a design criterion for such an analyzer is a consideration of the relationship between excitation intensity and point of observation of the emitted fluorescence signal within the cuvette. A discussion of the inner filter effect using as parameters the molar absorptivity of reduced nicotinamide adenine dinucleotide and fluorescein is presented as a practical demonstration of the advantage of using a frontal fluorescence detection optical configuration. A brief discussion of the instrument and a description of its current instrumental configuration are presented. The current detection limit of the fluorescent Fast Analyzer with respect to fluorescein was 0.8 ng/ml, with a useful analytical range above 10 ng/ml. A few examples of the many potential applications of fluorescence analysis to the Fast Analyzer are presented; these include calcium, cortisol, morphine, and an enzymic fixed-time analysis for glucose.
The GeMSAEC Fast Analyzer, developed by Anderson (1-5), has provided an alternative direction in clinical chemistry instrumentation by offering a means of performing large-scale parallel kinetic enzyme activity assays (6-8) and kinetic substrate concentration determinations (9-11) on semimicro sample and reagent volumes. In addition, multiple parallel end-point analyses can be performed and several methods have been adapted for use on the Fast Analyzer (12,13), In simplest terms, the Fast Analyzer is a multicuvette spectrophotometer or photometer which uses millisecond reference updating and digital averaging of transmission or absorbance signals to provide a stable drift-free optical system. Centrifugal force is used to rapidly add and mix each sample and reagent and introduce the resulting combinations simultaneously into the appropriate cuvettes. This establishes a unique initial reaction starting time, with all reactions proceeding essentially in parallel under similar conditions of time, temperature, and reaction composition. Norman G. Anderson,Anal. Biochem., 28, 545 (1969). Norman G . Anderson,Anal. Biochem., 32, 59-69 (1969). Norman G .Anderson, Clin. Chem. Acta, 25, 321 (1969). Norman G . Anderson, Fed. Proc., 23, 533 (1969). ( 5 ) Norman G. Anderson, Science. 166, 317 (1969). (6) T. 0. Tiffany, G . F. Johnson, and M . E . Chilcote, Clin. Chern.. 17, (1) (2) (3) (4)
715 (1971). (7) 8.E. Statland and A. L. Louderback, Clin. Chern.. 18, 845 (1972). (8) D. L. Fabiny-Byrd and G . Ertingshausen, Clin. Chem.. 18, 841 (1972). (9) T. 0. Tiffany, J . C. Jansen. C. A . Burtis, J . 8. Overton. and C. D. Scott, Clin. Chern., 18, 829 (1972). (10) D. L. Fabinyand C. Ertingshausen, Clin. Chem.. 17, 696 (1971). (1 1) T. R. Koch and M . E. Chilcote, Clin Chern.. 18, 687 (1972) (12) J. A. Daly and G . Ertinghausen, Clin. Chem., 18, 263 (1972). (13) G. Ertingshausen, D. L . Fabiny-Byrd. T. 0. Tiffany, and S. T. Casey, Clin. Chem., 18, 688 (1972).
1716
A N A L Y T I C A L C H E M I S T R Y , VOL.
To date, the Fast Analyzer has been designed primarily for operation with either a photometric or a spectrophotometric optical system. Consequently, a multicuvette Fast Fluorometric Analyzer that could be used both as a fluorometer and as a spectrophotometer would enhance the multiple-sample analytical versatility of the Analyzer in the clinical laboratory as well as increase its potential for becoming a unique type of fluorescence instrument because of its capability for rapid, essentially simultaneous monitoring of multiple cuvettes. The first objectives have been to demonstrate that such an instrument is feasible and to define the parameters required for optimization of concentration measurements using a fluorometric detector. A mathematical consideration of the inner filter effect as it is related to right-angle and frontal or surface fluorescence measurements is a first approach of a suitable design criterion. Although a discussion of the inner filter effect has been presented previously in texts and reviews (14,15) and in some detail by Brand and Witholt (16), a quantitative estimation of the inner filter effect through the use of simulated model systems, where parameters of biochemically important solutes have been used for its evaluation, has not been presented. Thus, because of its relevance to the discussion on instrumentation, it is presented here. A few examples of possible applications of the Fast Fluorometric Analyzer have been investigated. These include the simultaneous determination of serum calcium by EDTA titration using calcein as an indicator and by the direct addition of the serum sample and standards to the calcein reagent, all in a single rotor for comparative purposes, the determination of morphine as pseudomorphine, and the determination of glucose using a hexokinase glucose-6-phosphate dehydrogenase enzymic fixed-time method. A discussion of the Analyzer and these analytical applications is presented.
EXPERIMENTAL All the chemical compounds used in these studies were standard reagent grade. Reduced nicotinamide adenine dinucleotide (NADH), Type 111, was purchased from the Sigma Chemical Company, St. Louis, Mo. 63178. Sodium fluorescein was obtained from the MCB Manufacturing Chemists, Norwood, Ohio 45212. The enzymic glucose analysis was performed using a kit produced by Calbiochem, San Diego, Calif. 92112. Standard solutions of sodium fluorescein and NADH were pipetted by hand into a Teflon (Du Pont) transfer disk using a Biopette Automatic Pipette obtained from Schwartz/Mann (Division of Becton, Dickinson, and Company, Orangeburg, N.Y. 10962). A volume of 600 r l was used in each case. For the fluorescent glucose analysis, 56.2-pI volumes of 1:lOO dilutions of glucose standards and serum samples were dispensed automatically into the Teflon transfer disk, followed by a 198-rl washout with glass-distilled HzO. The glucose reagent was prepared according to the manufacturer's specifications, and 300-rl volumes of the reagent were automatically dispensed into the reagent positions of the (14) C. E. White and R. J . Argauer, "Fluorescence Analysis, A Practical Approach," Marcel Dekker. New York, N . Y . , 1970. (15) C. A. Parker and W . T. Rees, Analyst (London). 85, 587 (1960). (16) L. Brand and B. Witholt, "Methods in Enzymology." vol X I . C. H. W. Hirs. Ed., Academic Press, New York, N . Y . , 1967, p 776.
45, NO. 9, AUGUST 1973
Figure 1.
Fast Analyzer transfer disk. cuvette, and rotor assembly showing the analytical module in various stages
( A ) Stationary with reagent and sample separated, (8) initial acceleration and transfer of fluids, (C) continued acceleration and transfer of flulds into the cuvettes, ( D )rotor at speed with all solutions in cuvettes
P,
- P,e-icd)
P,e-ccdjtcdy
Teflon transfer disk. The automatic dilutor used for this work has been described elsewhere ( I T ) . The transfer disk has 15 positions, with an inner well a t each position for reagent and a n outer well for sample. Each position corresponds to a cuvette in the analytical rotor. A cut-away diagram of the transfer disk in position in the analytical module of the Fast Analyzer is shown in Figure 1. This demonstrates the sample and reagent wells of the transfer disk and indicates also how solutions are transferred upon acceleration into the cuvette of the rotor.
will be absorbed. The approximation in Equation 2 is always valid when the quantity tc dy is infinitely small. Integrating between y1 and yz, the number of photons absorbed a t y2 in a region of length Ay = yz - y1 is found to be: - - K 1 > (1 - e-"A,) N , ( N = P,e (3)
INSTRUMENTATION The Inner Filter Effect. If one considers the relationship between excitation light intensity and the position in the cuvette from which fluorescence emission is detected by the photomultiplier tube, it is possible to establish a certain criterion for the design of a fluorescence analyzer which is to be used for concentration measurements. In a discussion of inner filter effects, Brand and Witholt (16) have presented the requirements for optimization of fluorescence measurements to extend the measurable concentration range. If Po is the intensity, in quanta available a t the cuvette sample interface a t a given wavelength A, then the intensity a t any given distance y in the cuvette is:
The physical interpretation of Equation 3, in terms of instrumental parameters, is for a right-angle detection system in which l y is equivalent to the emission slit width while y1 indicates the position of the slit relative to the surface of the cuvette being impinged upon by the excitation light. The measured fluorescence a t X is proportional to the total emission and to the number of photons absorbed (Na). From Equation 3, it should be observed that the emitted fluorescence signal can never be exactly linear with respect to concentration. However, by controlling the instrumental parameters, the measurable concentration range can be extended. For example, when y1 in Equation 3 is set equal to zero, the resulting relationship,
P,
= P,,e-'('
N,(x)
(1)
=
P,(I - e-"A')
(2)
(4)
can be expanded, using Taylor series, to give
Within a volume element dVof length dy, an amount (17) C. A. Burtis, W. F. Johnson, J . C. Mailen. and J. E. Attrill. Clin. Chem.. 18, 433 (1972).
N,(h)
=
Po[tcAy - 1 (tcAy)'
+ 61 (tcAyI3 -
ANALYTICAL CHEMISTRY, VOL. 45, NO. 9, A U G U S T 1973
(5) 1717
Table I. Calculated Deviation from Linearity Due to Inner Filter Effecta Right angle y , = 0 5 c m Ay = 0 1 cm NADH concn ( M ) v1
m
a
x
1 1
-
.
x
x 5 x 1 x 5 x 1 x 1 x 1
\
L
MOLAR CONCENTRATION l M / l i t e r l
Figure 2. Plot of photons absorbed vs. concentration as a function of position in the cuvette using chemical parameters ( a ) fluorescein; e
= 16,OO X 2.303, ( b ) NADH; e = 6220
X 2.202
and Equation 5 reduces to the idealized case when tcily is very small:
N,(X)
=
P,fcAy
(6)
When tcAy is very small, N,(X) is proportional to some fraction of the total emission and is linearly related to solute concentration. Equation 6 can be approached by observing the fluorescence emission a t the surface of the cuvette ( i e . , by setting y1 = 0); and by holding ccAy very small, which is satisfied when the concentration is very low, or by reducing l y when working a t higher solute concentrations. In this manner, the measurable linear concentration range can be extended for fluorescence measurements. The extent of the deviation from linearity when fluorescent measurements are made a t different concentrations can be assessed by evaluating expression 3 for Na(X) as a function of concentration through the use of reasonable experimental parameters. Both right-angle and surface (frontal) fluorescence measuring detectors can be evaluated mathematically. To provide greater utility from such an evaluation, the molar absorptivities of biochemical compounds of interest can be used. For example, Figure 2 shows the results from such an evaluation using the molar absorptivity of fluorescein and NADH as model systems. The deviation from linearity for both the frontal and right-angle detectors is tabulated in Table I as a percentage decrease in the slope (d[N,(X)]/dc) using the molar absorptivity of NADH as a model system. The most important thing to note in this table is the deviation from linearity of the right-angle system even a t 1 0 - 6 mole/liter for optical settings of y1 = 0.2 cm and ily = 0.05 cm. From Equation 3, it can be determined that the linear concentration range can be enhanced an order of magnitude by reducing the slit width by a factor of 10. From the foregoing instrumental considerations, it is apparent that the design criterion of an analytical fluorescence analyzer which is to be used primarily for concentration measurements over an extended range ( e . g . , the dynamic range of concentrations encountered in a clinical chemistry laboratory when enzyme activity assays or enzymic substrate analyses of serum samples are performed) should be directed toward the use of a frontal or surface 1718
10-8 10-7 10-6 10-6 10-5 10-5 10-4 10-3
% of slope
100.00 99.97 99.39 96.50 92.58 67.57 19.92 0.41
aEvaluated from N,(X) X 2.303.
= 6220
% Dev
0 0.03 0.51 3.50 7.42 32.43 80.48 99.59
Right angle y , = 0 2 cm, Ay = 0 05 cm % of slope
100.00 100.00 99.82 98.38 96.64 83.75 70.08 30.00
% Dev
0
0 0.18 1.62 3.36 16.25 29.47 70.00
Frontal, y , = 0, Ay = 0 1 crn % of slope
100.00 100.00 100.00 99.77 99.46 96.67 93.33 53.24
O/O
Dev
0 0 0 0.23 0.54 3.33 6.66 46.76
= Poe-LCY1 ( 1 - e-ecaY) where Po = 1000,
fluorescence detection system. To complete the discussion, it should be noted that the primary limitation of a frontal detection system is the increase in intensity of scattered light. The intensity of scattered light from nonpolarized excitation light is a t a minimum when observed at an angle of 90" to the incident excitation light. The intensity of scattered light from a nonpolarized source is never equal to zero, and it is only approximately twofold less than the intensity of scattered light observed a t an angle 30" to the angle of incident light ( 1 8 ) . Larger particulate matter, which could cause scattering, is obviously removed by the centrifugal force of the spinning analytical cuvette module. Instrumental Design. A multicuvette Fluorometric Fast Analyzer has been developed from an existing G-IIC Fast Analyzer system (19,20). A diagram of the modified analytical module is shown in Figure 3. The Analyzer, a 15-cuvette system, operates with a surface fluorescence detection system. The angle of incidence of excitation is 60" to the cuvette surface, and the emitted signal is detected a t an angle normal to the rotating cuvette surface. An example of the relative intensity of emitted light, with respect to increasing concentrations of concentrated sodium fluorescein solutions, from the surface Fluorescence Analyzer as compared to that obtained with a right-angle system is shown in Figure 4. This demonstrates experimentally the difference between a frontal and a rightangle detection system when the same solutions are observed under as closely controlled similar conditions as possible. The effect of path length reduction on linearity is demonstrated in Figure 5 . Reduction of path length in the Fast Analyzer can be achieved in several ways: by reducing the physical dimensions of the cuvette window siphon spacer, by providing a fluorescence shelf of decreased path length in the cuvette configuration, and by providing insertable cuvette path length delimiters to define a reduced path length. Figure 5 represents the last approach, in which small metal shims have been inserted in the cuvette wall (see Figure 3) to reduce the path length to 2 mm. This modification allows one to use the system as a spectrofluorometer with the flexibility of being able to decrease the path length as needed, yet permits it also to be used as a spectrophotometer with a 1-cm path length. Figure 3 shows that the bifunctional characteristics of the analytical module can be obtained by (18) C. Tanford, "Physical Chemistry of Macromolecules," John Wiley and Sons, New York, N.Y., 1967. p 280. (19) D. N. Mashburn, R . H. Stevens, D. D. Willis. L. H. Elrod, and N. G. Anderson. Ana/. Biochem.. 35, 76-1 12 (1970). (20) C. A. Burtis, W. F. Johnson, J. E. Attrill, C. D. Scott, N. Cho. and N. G. Anderson, Clin. Chem.. 17, 686 (1971).
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 9, AUGUST 1973
PHOTOMETER OR SPECTROPHOTOMETER LIGHT PIPE AND MIRROR ASSEMBLY
(1
(0
-
9-
8 -
I 1 I I A SPECTRO FLUOROMETER (AMINCO BOWMAN1 bATH LENGTH: 0 5 cm EXCITATION: 4 7 0 nm (XENON) EMISSION: 5 3 0 nm S L I T ' 0.5 m m
Ii
,
I
1
A GeMSAEC SURFACE FLUORESCENCE PATH LENGTH 0 2 crn 7
GeMSAEC SURFACE FLUORESCENCE EXCITATION 470 nm I X E N C N I EMISSION 5 0 0 - 600 r m FILTER SLIT 0 5 - 1 G r n m
GaMSAEC SURFACE FLUORESCENCE PATH LENGTH: 0 . 2 c m EXCITATION: 4 7 0 nm (XENON) EMISSION: 5 0 0 - 6 0 0 nm FILTER SLIT: 0 . 5 - i . O m m
.~
,
I
7
f
2.5
z
g 6
= c W 2
W
2.0
; 5
a a
Y J
4
: c s 0
1.5 0
e
3 t.0
E
P u a Y
2
0.5 1
a 0
0 0
20
40 60 80 FLUORESCEIN ( p g / m l l
(00
420 0
:
I
I
Experimental comparison of the fluorescence emission from a frontal and right-angle fluorometer Figure 4.
switching from one quartz optical fiber to the other. The analog transmission and emission signals from the same spinning rotor containing increasing concentrations of fluorescein solutions demonstrate the dual nature of the analyzer, as seen in Figure 6. The linearity of absorbance of
fluorescein solutions when observed a t 490 nm in the spectrophotometric mode is demonstrated in Figure 7 .
ANALYTICAL CHEMISTRY, VOL. 45, NO. 9, A U G U S T 1973
1719
7
6
0
- -HV 2 4 i F 111 ,.r' r" FLJOHESCEIN -JObJ?EEITiLTIO"i :pglmli
~-
w
2
2 J W
n
Figure 6. Fluorescence emission and per cent transmittance of fluorescein solutions in the same rotor
"-
0 40 80 120 FLUORESCEIN CONCENTRATION ( n q ' m i I
Figure 8. Relative fluorescence intensity of low-concentration fluoresceinsolutions ( a ) FIuorescence Fast Analyzer. ( b ) Aminco-Bowman Spectroliuorarnefer [Relative Intensity = (Meter Reading) (Scale Factor) (lOOO)]
0
2 4 6 B IO FLUORESCEIN CONCENTRATION Ip g / m l l
I2
Figure 7. Spectrophotometric performance of the Fluorometric Fast Analyzer at 490 n m Two excitation sources have been used in the development of this instrument: a 22-W quartz-iodine lamp driven by a stable regulated dc power supply, and a 150-W xenon lamp (Bausch and Lomb, No. 33-86-20-01, Rochester, N.Y.) which has been modified to reduce the 60-cycle ac ripple component to less than 15 m V rms when the lamp is operated a t 20 V and 7.5 A. A Bausch and Lomh high-intensity gradient monochromator (Bausch and Lomh 33-86-07, 200-700 nm) with adjustable entrance and exit slits has been used for excitation. Sharp-cut or specially designed emission filters have been used for the isolation of the emission signal. Quartz light pipes, 0.4 cm in diameter, purchased from Schott Glass Company (Du1720
ryea, Pa. 18642) were employed for directing the excitation light. Detection Limits and Instrumental Noise. The major developmental efforts have heen focused on defining the optical system and reducing the noise of the excitation source. The fluorescence analyzer is interfaced to a Digital Electronics Corporation (Maynard, Mass. 01754) PDP-8 computer, and digital averaging of the intensity emission signal is performed routinely to reduce instrumental noise. The initial evaluation of the detection limit of the analyzer was carried out at a n excitation wavelength of 470 nm using sodium fluorescein. Solutions of this compound ranging in concentration from 20 to 100 ng/ml were prepared in 0.01N NaOH by diluting a stock solution (100 mg/ml), and the relative fluorescence emissions of these solutions were determined. A filter specifically designed for fluorescein (Baird Atomic Barrier Aniography filter) was used to isolate the emission signal, and the photomultiplier voltage was set at 1.1kV. (Regulation of the photomultiplier high voltage is accomplished with a HewlettPackard 6515A high-voltage dc power supp1y:The photomultiplier tube is an RCA IP-l28A P.M. tube.) The emission data were obtained from ten successive reading intervals a t 10 sec per interval, with digital averaging of 30 readings per cuvette per reading interval. A plot of the relative emission intensity us. fluorescein concentration is shown in Figure 8a. The same samples were analyzed
ANALYTICAL CHEMISTRY, VOL. 45, NO. 9. AUGUST 1973
I
, 1
2.0
EDTA T I T R A T I O N USING CALCEIN A S AN INDICATOR
z
[EDTAj=2.20x10-3 M [ C a + ' ] = [ Z . Z O X I I J ' ~ M ] [ 4 . 0 mg d l - l / l ~ J - ~M; [ C a + ' ] = 6 8 mg/dl
0
A
I
1
0
E
a z 0
$
//
I
I
DIRECT ADDITION OF CALCIUM TO CALCEIN [Co+'] = 6.9 mg/dl
PM VOLTAGE = i . 4 kV N=30READINGS PER CUVETTE
'or----rij I
C
0.4
5 0 E W
a
0 0
2
4
6
8
100
RELATIVE INTENSITY ( v o l t s )
Electro-optical noise v s . relative intensity from relative intensity data for low-concentration fluorescein samples (Figure
Figure 9. 8a)
0
ANALYTICAL APPLICATIONS Serum Calcium. The fluorometric analysis of serum calcium is of interest because of its sensitivity (analyses can be performed on serum sample-reagent volume dilutions of l:50 and 1:100),and competitive divalent cation interference. particularly magnesium, can be minimized or eliminated by control of the p H of the reagent (21). (21) H. Diehl. "Calcein. Calmagite, and 0.0'-Dehydroxyazobenzene Titrimetric, Colorimetric and Fluorometric Reagents for Calcium and Magnesium." The G. Fiederick Smith Chemicai Cb., Columbus, Ohio, 1964, p 73.
4
6
6
I0
I2
E D T n C D N C E N T R A T I O ~(10-3 moies/lirer) C A L C I e M CONCENTRATION ( p g / d l l
Figure 10.
using an Aminco-Bowman spectrofluorometer a t an excitation wavelength of 470 nm and an emission wavelength of 530 nm, using the two-pass mirror system and a slit width set at ,5 mm. Readings were made with the sensitivity range set at 0.01 or 0.03; the resulting plot of relative intensity us. concentration is shown in Figure 8 b A plot of the standard deviation of emission signal, in millivolts, as a function of emission signal potential for the centrifugal Fluorescence Analyzer was made as a first approximation in determining the detection limit of the Analyzer under the conditions described above. This plot, along with a plot of per cent relative standard deviation us. signal potential, is shown in Figure 9. The noise level a t extreme dilutions is of the order of 15 to 20 mV, and the slope of the plot in Figure Sa is 64 mV per concentration unit (ngiml). If' one defines the detection limit as twice the background noise level, then the detection limit is of the order of 0.6 ngiml, with a useful analytical range greater than 10 ng/ml. The detection limit, expressed in terms of moles per milliliter, is 1.60 X mole/ml, which is equivalent to 9.6 x 1010 molecules per milliliter of solution. The detection limit for the Aminco-Bowman spectrofluorometer under the conditions described was of the order of 0.3 ng/ml, with a useful analytical concentration range above 5 ng/ml. This is based on an uncertainty in the reading of the intensity scale of four divisions a t a full sensitivity setting of 0.001. The comparison of the two instruments was provided strictly for a relative evaluation of the sensitivity of the fluorescence centrifugal analyzer, based on the reliable performance of an accepted spectrofluorometer operating under instrumental parameters established to achieve maximum sensitivity. An effort is currently being made to increase the sensitivity of the centrifugal fluorescence analyzer.
2
Determination of serum calcium using fluorometric
procedures There are two analytical approaches that can be taken: titration of the serum with standarized EDTA solution and detection of the end point using calcein as an indicator, or the direct addition of serum and standards to calcein reagent. Both procedures were carried out in the same rotor on the same serum sample for comparative purposes; the results are shown in Figure 10. Determination of Morphine as Pseudomorphine. The oxidation of morphine to pseudomorphine, using dilute solutions of potassium ferricyanide, greatly enhances the detectable fluorescence emission (22), and the application of simplified extraction techniques by Goldbaum e t al. (23) has extended the application of this analytical technique to the analysis of morphine in urine. A review of this subject and of the analytical methods for drug detection has recently been published (24). We have examined the feasibility of using the multicuvette Fast Fluorometric Analyzer to detect morphine levels a t physiological concentrations that are expected to be found in urine after drug administration. The advantage of this system would be that alt,ernate cuvettes could be used for simultaneous detection of pseudomorphine and of the extracted blank fluorescence. The results of analyzing aqueous solutions of morphine ranging in concentration from 0.2 to 10 pg/ml are shown in Figure 11. In this experiment, duplicate 400-p1 aliquots of morphine solution were dispensed into adjacent inner sample wells and alternate 20-pl aliquots of dilute K3Fe(CX)G and HzO were dispensed into the outer reagent wells. Reagent, sample, and HzO were mixed simultaneously as the rotor was accelerated, and the pseudomorphine and the morphine blank were determined in parallel. Since the morphine blank and the pseudomorphine were in adjacent cuvettes, the rapid comparison of the two could be achieved. The excitation wavelength was set a t 287 nm (absorbance maximum of pseudomorphine), and the emission signal was isolated through the use of a 400-nm sharp-cut filter. Although aqueous solutions of morphine were used, the system is applicable to the analysis of urine extracts, and (22) H. Kupferberg. A. Burkhalter. and E. L. Way. J Pharmacoi. Exp. Ther., 1 4 5 , 247 (1964) (23) L. Goldbaum. P. H. Santinga. and A. Bomingues, 23rd Annual Meeting, American Academy of Forensic Sciences. Phoenix, Ariz., February 25, 1971 (24) P. Santinga, "The Application of Fiuorescence and Gas Chrornatography to Mass Drug Screening," fluorescence N e w s . vol. 6 (1971).
A N A L Y T I C A L C H E M I S T R Y , VOL. 45.
NO. 9 ,
AUGUST 1973
1721
BACKGRCLND SIGhAL
k
'A
z r
5 Y
?
c
4 2 L
a
.
I
,
,
.
0.5 1.0 2.0 4 0 6.0 8 0 io.0 p g / m INCREASING M O R P H I N E CONCENTRATION --L.
Figure 11. Morphine fluorescence (as pseudomorphine) on the Fast Fluorometric Analyze1
Figure 13. Fixed-time absorbance change 8s a function 01 glucose concentration and fixed-time interval (At) EXCITATlON-34Onm(IkOW XENON
450tEM;S610N-400nm
3
SOURCE1
(SHARPCUT FILTER1
TiME (set)Figure 12. Enzymic fluorometric determination of glucose with a
hexokinase glucose-&phosphate dehydrogenase coupled assay provides the necessary simultaneous blank and pseudomorphine determination to prevent the false identification of morphine in urine. Enzymic Substrate Analysis. The subject of enzymic substrate analysis on the spectrophotometric Fast Analyzer has heen previously discussed (9) in terms of application of both enzymic integral and kinetic rate analyses to the determination of substrate concentration. The determination of glucose by a hexokinase glucose 6-phosphate dehydrogenase method (25) has heen selected as an interesting application of the fluorometric Fast Analyzer because of the increased sensitivity it affords and the effect on the kinetics of the reaction resulting from reduction of the initial substrate concentration. It can be demonstrated that, in the case of a n enzymic reaction in which the initial substrate concentration is small with respect to the Michaelis-Menten constant ( K m ) of the enzyme, the relationship between the initial velocity of the enzymic reaction to the initial substrate concentration is linear and the reaction appears to obey pseudo-first-order kinetics (26). It can also he demonstrated that, for a pseudo-first-order reaction, the change in substrate concentration (AS) between two fixed time readings is directly proportional to the substrate concentration. Furthermore, it can he shown that such a rela( 2 5 ) M. W. Slein. "Melhods of Enzymatic Analysis," H.-U. Bergmeyer. Ed., Academic P r e s , New York. N.Y.. 1963. p 117. (26). 4. D. IngieandS. R. Crouch, Anal. Chem.. 43. 697 (1971).
0
1 i 0
I 1 I 1 I 100 150 2M) 250 300 350 GLUCOSE CONCENTRATION Imq /IO0 ml 1. 10'1 I I I I I I I 5 10 15 20 25 30 35 GLUCOSE CONCENTRATION IM/lilcr 810'1 50
i 400
T 40
Figure 14. Fixed-time relative intensity change a s a function of glucose concentration and fixed-time interval tionsbip holds for series first-order or coupled enzymic reactions obeying pseudo-first-order kinetics (27).A problem arises with spectrophotometric enzymic linear Axedtime substrate analysis since the Michaelis-Menten Constant ( K m ) of the particular enzyme being used is often of the same order a s the initial substrate concentration required for the reaction and the reduction of the substrate concentration to fit the pseudo-fmt-order criterion results in a loss of sensitivity and analytical utility of the fixedtime approach. The availability of a fluorescence detection system for use in enzymic substrate analysis, where the concentration of a fluorescence product or substrate such, a s nicotinamide adenine dinucleotide oxidation (NAD) or reduction (NADH) can be monitored, does allow a decrease of the initial substrate concentration by one or two orders of magnitude and, hence, facilitates the use of linear kinetic enzymic fixed-time analysis. (27) A. Frost and R. Pearson, "Kinetics and MeChmiSmS," John Wiley andsons. New York. N.Y.. 1963, pp165-77.
1722 * ANALYTiCAL CHEMISTRY. VOL. 45, NO. 9. AUGUST 1973
Figure 12 is a curve showing the progress of the reaction involved in the hexokinase glucose-6-phosphate dehydrogenase determination of glucose in a normal serum sample. In this reaction, the production of reduced nicotinamide adenine dinucleotide phosphate (NADPH) is monitored fluorometrically. The Michaelis-Menten constant for hexokinase is 1.5 x 10-4M. The range of glucose concentrations when samples with concentrations up to 400 mg/100 ml are used and the reaction sample volume is 2.8 pl in a total reaction volume of 553 pl (reaction conditions for the spectrophotometric Fast Analyzer) is from 0.1 X 10-4 to 1.11 X 10-4 mole/liter. This results in substrate Krn ratios from 0.075 to 0.75 and should result in a nonlinear relationship between fixed-time absorbance change and initial substrate concentration. Results of the spectrophotometric fixed-time analysis of glucose under the conditions described above, shown in Figure 13, demonstrate the nonlinearity of the reaction due to the unfavorable initial substrate/Krn ratio. Figure 14 illustrates the fluorometric enzymic fixed-time analysis of glucose using a sample volume of 0.55 p1 and 553-pl total reaction volume. The highest sample concentration represents an initial substrate concentration/Km ratio of 0.15. The linearity of the change in relative intensity over all fixed-time intervals chosen, demonstrates the effect of reducing initial substrate concentration to a more favorable level. To ensure accuracy and precision in pipetting of these small sample volumes, 56-pl aliquots of serum glucose samples diluted 1:20 and 1: 100, respectively, for the spectrophotometric and fluorometric analyses were used. The replicate analysis of a serum glucose control sample having a stated glucose concentration of 157 mg/100 ml was 163 f 5.8 mg/100 ml with a relative standard deviation of 3.6%. The excitation wavelength was set a t 340 nm. The photomultiplier voltage was set a t 1.0 kV, and the emission signal was isolated using a 400-nm sharp-cut filter.
CONCLUSIONS The discussion in the preceding sections points out one limitation of the use of fluorescence measurements for concentration determinations. This limitation, the loss of linearity in signal due to the inner filter effect, can be a significant contribution even a t a substrate concentration of 10-6M. One method for improving the emission signal linearity with respect to concentration would be to use frontal or surface fluorescence monitoring. Therefore, a Fast Analyzer incorporating such a fluorometric detection system has been developed.’ An initial evaluation of this analyzer, as compared with a right-angle spectrofluorometric analyzer, has shown increased linearity of emission
signal for the surface Fluorescence Analyzer. In addition, the use of reduced cuvette path length was shown to enhance the linearity even further. The feasibility of developing a Fluorescence Fast Analyzer has been established, and some simple analytical examples have been presented to demonstrate not only the operation of the Analyzer but also a few of the many analytical applications that can be made with such a system. I t was demonstrated that sodium fluorescein solutions a t a concentration of 0.6 ng/ml, or 1.60 X 10-9 mole/l., could be detected. This concentration approaches that desired for fluorescein-labeled antigen antibody studies, and work is in progress to decrease this detection limit by a t least another order of magnitude. The advantage of being able to reduce the initial substrate concentration for kinetic enzymic substrate analysis and still retain the desired sensitivity and analytical application through the use of a fluorescence detection system was demonstrated using the hexokinase glucose-6-phosphate dehydrogenase enzymic fixed-time analysis of glucose. Work is under way to improve the signal/noise ratio of the detection system by increasing the amplification of the emission signal while decreasing the required P M voltage. The objective of this effort is to reduce the analytical variation from 2-390t o below 1%. In conclusion, there is a certain advantage in using a multicuvette Fluorometric Analyzer with essentially simultaneous monitoring of all cuvettes for fluorescence measurements. This is the potential use of an on-board fluorescent standard reference compound that could be included in each analytical run and could be used to approach an absolute fluorescence emission intensity measurement. The use of a small computer in conjunction with the Analyzer does provide digital averaging to reduce instrumental noise, and could be used to correct for intensity variation from cuvette to cuvette due to slightly different optical configurations (the relative standard deviation of cuvette intensity variation for the same solution is about 1% before correcting and less than 0.5% after correction), and to correct the emission intensity relative to the on-board reference standard. Received for review December 5, 1972. Accepted March 8, 1973. The Molecular Anatomy (MAN) Program is supported in part by the National Institutes of General Medical Sciences and the U.S. Atomic Energy Commission. Oak Ridge National Laboratory is operated for the U.S. Atomic Energy Commission by Union Carbide Corporation, Nuclear Division.
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 9, A U G U S T 1973
1723
