Anal. Chem. 1994,66, 4295-4301
On-Line Coupling of Liquid Chromatography to Biochemical Assays Based on Fluorescent-Labeled Ligands A. J. Oosterkamp, H. Irth,* U. R. Tjaden, and J. van der Greef Leiden/Amsterdam Center for Drug Research, Division of Analytical Chemistry, University of Leiden, P.O. Box 9502, 2300 RA Leiden, The Netherlands
The on-line coupling of liquid chromatography (E) to a biochemical detection (BCD) technique based fluoresceinlabeled ligands as reporter molecules is described. In a first step, &ity proteins such as antibodies or avidin are added to the LC effluent to react with ligands (analytes) eluting from the LC column. Unbound &ity proteins react, in a second step, with an excess of fluoresceinlabeled ligand to titrate the remaining free binding sites. Prior to detection of the labeled liganaprotein complex, free and bound label are separated on the basis of the considerable difference in molecular weight. A short (10 x 4.0 mm i.d.) column packed with a restricted-access support is used to trap the free labeled ligand at the hydrophobic inner surface of the pores. The high molecular weight labeled liganaprotein complex passes this column unretained and is detected by means of fluorescence detection. The interaction between biotin and avidin was chosen as a model system. A detection limit of 160 fmol was obtained for biotin using reversed-phase LC-BCD. An equilibrium and kinetic model is described which relates the detector response to the concentration of &ity protein, fluorescent label, and reaction time. The combination of liquid chromatography (Lc) with biochemical detection (BCD) methods greatly enhances the performance of both techniques and provides analytical methods which are characterized by high selectivityand sensitivity.' LC has been successfully coupled to catalytic2 or affinity-based biological detection techniques3s4using enzymes and antibodies,respectively, for analyte recognition. The separation power of LC systems, particular in combination with automated sample cleanup techniques, can solve the problem of cross-reactivity which is characteristic for most biological assays. On the other hand, the inherent selectivity and sensitivity of bioassays extends the applicability of LC in, for example, environmental analysis, clinical analysis, or bioanalysis. Catalytic and af&inity-based detection techniques differ fundamentally in the way the binding of analytes to the target protein is monitored. Catalytic systems involving enzyme-substrate (1) De Frutos, M.; Regnier, F. E. Anal. Chem. 1993,65, 17A (2) Marko-Varga, G. Electroanalysis 1992,4, 403. (3) Irth, H.; Oosterkamp, A J.; van der Welle, W.; Tjaden, U. R; van der Greef, J. J. Chromatogr. 1994,633, 65. (4) Oosterkamp, A J.; Irth, H.; Beth, M.; Unger, K K; Tjaden, U. R; Van der Greef, J. J. Chromatogr. 1993,653, 55. 0003-2700/94/0366-4295$04.50/0 0 1994 American Chemical Society
reactions are mainly detected by electrochemical detection5v6 monitoring electron transfers which occur during the breakdown of the substrate. Affinity-based detection techniques generally rely on reporter molecules such as radioactive and luminescent labels or enzymes to detect the binding of analytes to, for example, antibodies or receptors.' Both catalytic and affinity-baseddetection techniques can be carried out in continuous-flow systems and can principally be implemented in flow injection (FI)and LC systems. However, the dynamic nature of these techniques restricts the type of suitable assays to those detection principles which (i) provide short reaction times, i.e., less than 5 min, and (ii) are able to generate a continuous signal without the need of regeneration. If coupling to LC is intended, the bioassay has to be compatible with the LC mobile phase constituents, particularly with organic modifiers such as acetonitrile or methanol. Various detection principles based on both c~mpetitive~,~ and noncompetitive3J0-13 assays which principally meet these requirements were reported. Kusterbeck et al.s,gdescribed an FI assay based on the displacement of labeled antibodies bound to an immobilized antigen support. A noncompetitive FI enzyme immunoassay has been reported by Kronkvist et al.1° that uses a protein G column to separate free and antibody-bound enzyme labels. Recently we described an immunochemical detection (ICD) system coupled to LC in which fluorescein-labeled antibodies were added to the LC effluent to monitor the presence of antigenic a n a l y t e ~ . ~ . ~ Optimum reaction times of 1-2 min were obtained, which are of the same order of magnitude as those characteristic for chemical postcolumn derivatization techniques. Moreover, organic modifiers such as acetonitrile and methanol were tolerated up to 10 and 35%,respectively. The immunoreagentused for this assay consisted of fluoresceinlabeled Fab fragments of anti-digoxigenin antibodies which were immunopurified and commercially available. We realized, however, that the commercial availability of purified, labeled antibodies (5) Turner, A P. F.; Karube, I.; Wilson, G. S., Eds. Biosenson; Proceedings of a Royal Society Discussion Meeting held on 28 and 29 May 1986; The Royal Society: London, 1987. (6) Scheller, F.; Schubert, F. Biosensoren; Akademie-Verlag: Berlin, 1989. (7)Hammila, I. A Applications of Fluorescence in Immunoassays; Chemical Analysis 117; Wiley-Interscience: New York, 1991. (8) Kusterbeck, A W.; Wemhoff, G. A; Charles, P. T.; Yeager, D. A; Bredehorst, R; Vogel, C-W.; Ligler, F. S. J. Immunol. Methods 1990,135, 191. (9) Whelan, J. P.; Kusterbeck, A. W.; Wemhoff, G. A; Bredehorst, R; Ligler, F. S. Anal. Chem. 1993,65, 3561. (10)Kronkvist, K Ph.D. Dissertation, Lund, Sweden, 1993 part V. (11) Gunaratna, P. C.; Wilson, G. C. Anal. Chem. 1993,65, 1152. (12) Freytag, J. W.; Lau, H. P.; Wadsley, J. Clin. Chem. 1984,30, 1494. (13) Nilsson, M.; Hakanson, H.; Mattiason, B. J. Chromatogr, 1992,597, 383.
Analytical Chemistry, Vol. 66, No. 23, December 1, 1994 4295
1 A
+BF
A-BF
41
Figure 1. Scheme of the BCD system. Key: 1, eluate of the HPLC column; 2, reagent pump for avidin solution; 3, reaction coil; 4, reagent pump for fluorescein-biotin solution; 5, column packed with (218-silica restricted-access support; 6, fluorescence detector. A, avidin; B, biotin; BF, fluorescein-biotin.
is the exception rather than the rule. In most cases, antibodies against drugs, for example, are available as unlabeled, only crudely purified antiserum. Although labeling and purification schemes for antibodies and other affinity proteins are well documented, the use of commercially available antisera without any pretreatment should be considered as well. In the present paper we describe an on-line LC-BCD system which uses labeled ligands instead of labeled proteins as reporter molecules. A theoretical model was developed to determine the influence of reaction time, afsnity protein and label concentration, and association rate constant on the detector response. The interaction between biotin and avidin was chosen as a model system. Avidin is a well-characterized protein of 75 kDa which consists of four subunits, each possessing one binding site for biotin. The interaction of biotin and avidin is extremely strong (affinity constant, Ka = 0.6 x 1015L/mol) , with a fast association rate and a slow dissociation rate, Le., k+1 = 2.4 x lo7 Lmol-l s-l and k-1 = 0.4 x s-l, re~pective1y.l~ EXPERIMENTAL SECTION
Materials. Avidin, fluorescein-labeled avidin, biotin, and fluorescein-labeledbiotin were purchased from Sigma (St. Louis, MO) . Sodium chloride and sodium phosphate were from Merck (Darmstadt, Germany). Acetonitrile was obtained from Rathburn Chemicals (Walkerbum, U.K.). All other organic solvents were purchased from Baker (Deventer, The Netherlands) and were of analytical grade. Restricted-accessprecolumns were a gift of Prof. Boos (University of Munich, Germany). Binding buffer consisted of sodium phosphate (10 mmol/L, pH 8.0) containing 0.5 mol/L sodium chloride. Blocking reagent (Boehringer Mannheim, Mannheim, Germany) was prepared according to the manufacturer's specifications. Apparatus. The FI- and LC-BCD system (see Figure 1) consisted of two Kratos-AB1 (Ramsey,NJ) Spectroflow400 pumps and a Pharmacia (Uppsala, Sweden) P3500 pump used to deliver the mobile phase, the labeled biotin, and the avidin solution, respectively, a Gilson 231 autosampler equipped with a Rheodyne six-port injection valve (injection loop, 20 yL), and a Merck (Darmstadt, Germany) 1080 fluorescence detector (excitation wavelength, 486 nm; emission wavelength, 520 nm). The FI carrier solution consisted of binding buffer and was pumped at a flow rate of 0.4 mL/min. LC separations were carried out on a 100- x 3.0-mm i.d. stainless-steel column packed with Nucleosil (14) Green, ru'. M. Methods Enzymol. 1990, 184, 51.
4296 Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
(5 ym particles, Macherey-Nagel, Duren, Germany) using methanol/aqueous triethylammonium acetate (10 mmol/L, pH 7.0) 10:90 v/v, as mobile phase. The avidin (34 nmol/L) and fluorescein-biotin (200 nmol/L) solutions were prepared in binding buffer and added to the LC or FI carrier solution via inverted Y-type mixing unions. The avidin solution was pumped at a flow rate of 0.4 mL/min for both LC- and FI-BCD systems. The fluorescein-biotin solution was delivered at a flow rate of 0.4 mL/min using FI-BCD and 0.8 mL/min using LC-BCD. Knitted 0.5 mm i.d. poly(tetrafluoroethy1ene) reaction coils with an internal volume of 800 and 400 yL for reactions I and 11, respectively, were used. The reaction was performed at 20 "C. Separation of free and avidin-bound fluorescein-biotin was performed using a 10- x 4.0-mm i.d. column packed with c18 alkanediol silica15 donated by Prof. Boos (University of Munich, Germany). The analytical system was controlled by Gilson 605 GSIOC driver software connected to a personal computer. Batch Experiments. The performance of the BCD system under equilibrium conditions was tested in the FI mode by incubating 0.4 mL of avidin (34 nmol/L) in batch for 15 min with 0.4 mL of different biotin concentrations. To this was added 0.4 mL of fluorescein-biotin (200 nmol/L), and the sample was incubated for another 15 min. The amount of avidin/fluoresceinbiotin complex formed was measured by injecting the reaction mixture into an FI system in which the injection valve was connected directly to the restricted-access reversed-phase (RAW) column and the fluorescence detector. Stopped-FlowExperiments. The influence of the reaction time on detection sensitivity was measured in the stopped-flow mode by connecting coil 3 of reaction I (see Figure 1) to a Must (Spark Holland, Emmen, The Netherlands) switchingunit equipped with a six-port Rheodyne injection valve. After injection of biotin, reaction coil 3 was switched off-line to achieve reaction times of 30, 60, 120, and 180 s. Calculationof Theoretical Detection Limits. The computer simulation was performed on a personal computer using TurboPascal (Borland, Scotts Valley, CA). The detector noise (N) was calculated using eq 9 (see Results and Discussion) with nf= 0.005 and n, = mol/L. The signal (S) was calculated according to eq 8 (see Theory). The LOD was determined by an iteration procedure in which the biotin concentration was varied in increments of 10-lO mol/L until a signal-to-noise ratio of 3 was c 1 8
(15) Boos, IC-S.; Walfort, A; Lubda, D.; Eisenbeiss, F. German Patent DE41 30 475 Al, 1991.
obtained for a given avidin concentration. The procedure was repeated for avidin concentration ranging from to lo-' mol/L at increments of lo-" mol/L. THEORY The LC-BCD system using labeled ligands as reporter molecules is based on a two-step afiinity reaction (see Figure 1). The performance of the BCD system has been investigated using avidin as afiinity protein, biotin as ligand, and fluorescein-labeled biotin (fluorescein-biotin) as labeled ligand. In the following, the four binding sites of avidin are considered as individual, independent subunits. Avidin concentrations are calculated as concentration of free binding sites. In the first step of the BCD system, avidin (A) is added to the LC effluent to react with biotin (B) or biotinylated compounds eluting from the LC column to form avidin-biotin complexes (AB) in concentrations proportional to the biotin concentration, provided that [AI I [Bl:
In the second step, an excess of fluorescein-labeled biotin (BF) is added to the reaction mixture to saturate the binding sites of unbound avidin molecules: k+i
A + BF * A-BF
(I9
k-i
d[A-Bl = [AI [BlrE.+,t
The avidin/fluorescein-biotin complex (A-BF) formed is separated from free fluorescein-biotin prior to fluorescence detection on the basis of the difference in molecular size (separation principle; see below). The detection response decreases with increasing biotin concentrations. Similar to the immunochemical detection system reported earlier, which employed fluorescein-labeledantibodies? the present BCD technique is solely based on fast association reactions of biotin or fluorescein-biotin with an avidin binding site. An equilibrium model describing the relationship between detector response, i.e., the concentrationof the avidin/fluorescein-biotin complex, and the concentration of biotin is derived from the two sequentialbiochemical reactions. In this model, band broadening due to dispersion is not considered. The amount of avidin/biotin complex formed in reaction I is calculated
KD =
([AI, - [A-BI) ([Blo - [A-BI) [A- B1
where [BFlo is the fluorescein-biotin concentration added. Substituting eq 2 in eq 3 provides a relationship between the biotin concentration ([BJ) and the avidin/fluorescein-biotin concentration ([A-BF]) as a function of the avidin and fluoresceinbiotin concentration and the binding constant, KD,assuming that the total system is in equilibrium. Preliminary experiments (see below) have shown that the biochemical reaction between biotin and avidin does not reach equilibrium under the reaction conditions chosen (reaction time, avidin concentration) despite the high association rate constant. This resulted in a lower detection response than predicted by the equilibrium model. We therefore developed a kinetic model which relates the detector response to the biotin concentration under nonequilibrium conditions. The model is based on the assumption that dissociation of the avididbiotin and avididfluorescein-biotin complexes is negligible within the time range of the postcolumn reaction (1-2 min). This assumption is true for most bioafiinity interaction systems with dissociation constants on the order of 10-2-10-3 s-1. The model developed does not consider band broadening introduced by the reaction coil, although this may play a role at longer reaction times. The association of biotin with the avidin is described by secondorder kinetics:
(1)
(4)
Assuming that [A-BI,o = 0, [AI+o = [Alo, and [BIt=o= [Blo, integration of eq 4 results i d 6
From eq 5, the concentration of avididbiotin complex after reaction I can be derived:
The concentration of free avidin after reaction I can be calculated according to eq 2. Equation 6 is used to calculate the concentration of avidin/fluorescein-biotin complex formed in reaction I1 by replacing [AI0 with [AIBand [BIowith [BFIo:
with KD being the binding constant, [AI0 the avidin concentration added, and [A-B] the concentration of avidin-biotin complex formed. The concentration of free avidin ([A]B)after this reaction is
[AIB= [AI, - [A-BI The concentration of avidin/fluorescein-biotin formed can be calculated by
(2) ([A-BFJ)
Combining eqs 6 and 7 provides a formula which relates the detector response to the most important parameters of the BCD system, i.e., the reaction time, the concentrations of analyte and avidin, and the dissociation rate constant. The signal obtained (16)Weiland, G. A; Molinoff, P. B. Life Sci. 1981,29,313.
Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
4297
100
+ + +
-
/----
%
B
I r 1 Figure 2. Direct injection of (a) fluorescein-biotin (10 nmoVL), (b) fluorescein-biotin (10 nmol/L) incubated with avidin (10 nmol/L), and (c) fluorescein-biotin (10 nmol/L) incubated with avidin (10 nmol/L) which was preincubated with biotin (1 pmol/L). by indirect fluorescence detection corresponding to a certain ligand concentration ([Blo) is then described as
S = r([A-BFlO - [A-BFId where r is the response factor of A-BF for a given fluorescence detector, [A-BFIo and [A-BFIB are calculated according to the combined formula of eqs 6 and 7 at B = 0 and B = Bo, respectively. RESULTS AND DISCUSSION Separation of Bound and Free Label. Similar to all heterogeneous batch-type biological assays, free and avidin-bound fluorescein-biotin must be separated prior to detection. Separation can be achieved in a continuous-flow system by exploiting the difference in biochemical activity or molecular mass. This separation should introduce only a minimum of extracolumn band broadening and allow the overall system to be operated continuously without the need of regeneration. In the present system, we chose a separation technique which exploits the considerable difference in molecular mass between free fluorescein-biotin (< 1 kDa) and avidin/fluorescein-biotin (>50 m a ) . For this purpose, a short column (10 x 4 mm id., 5 in Figure 1) packed with a restricted-access reversed-phase (RA-RP) support was placed in the carrier stream prior to the detector. The RA-RP support consists of &-bonded porous silica.15 The pores of the particles are shielded by a hydrophilic, polymerous alkane diol layer which efficiently excludes proteins with a molecular mass above 15 kDa. The free fluorescein-biotin is retained at the hydrophobic inner surface of the pores. The avidin/fluorescein-biotin complex is excluded from the pores and passes unretained to the fluorescence detector. The separation of free and avidin-bound fluorescein-biotin is demonstrated in Figure 2. In Figure 2a and b, repeated injections of fluorescein-biotin and fluorescein-biotin incubated with avidin are shown. While free fluorescein-biotin is completely retained by the RA-RF' column, avidin-bound fluorescein-biotin passes the RA-RP column unretained and without appreciable peak broadening. The specificity of the binding between fluorescein-biotin and avidin is demonstrated in Figure 2c, representing the same experiment as in figure 2b but using avidin preincubated with an excess of unlabeled biotin (1pmol/L). The degree of nonspeci6c binding of avidin/fluorescein-biotin complex to the RA-RP s u p port was derived from the measurements shown in Figure 2c. 4298 Analytical Chemisfiy, Vol. 66, No. 23, December 1, 1994
0
10
20
30
40
50
60
70
Biotin (nmolfl)
Figure 3. Detector signal vs. biotin concentration. (-) Predicted by the equilibrium model using 17 nmol/L avidin; (0)values measured with the BCD system using 17 nmol/L avidin; (- -) predicted by the equilibrium model using 17 nmol/L avidin which was preincubated with 34 nmol/L biotin; (0)values measuredwith the BCD system using 17 nmol/L avidin which was preincubated with 34 nmol/L biotin.
At the very low concentrations of protein used, the untreated RA-RP support exhibited a very high degree of nonspecific binding (>95%) in the particular case of avidin and avidin/fluoresceinbiotin. This is in contrast to the earlier experiments: where the RAW was used in sample pretreatment for direct plasma injections. Nonspecific binding of avidin/fluorescein--biotin was drastically reduced by equilibrating the R A W column with 2 mL of blocking reagent consisting of a casein hydrolysate with an average protein concentration of 70 mg/mL. Moreover, 0.5 mol/L sodium chloride was added to the avidin and fluorescein-biotin solutions. In this way, the nonspecific binding was reduced to 0.5%. Nonionic surfactants, e.g., Tween-10 or Brij-35, which are currently used to efficiently reduce nonspecitic binding of immunocomplexes to affinity support^,^,^ appeared to be not compatible with the RA-RP support. Free fluorescein-biotin was retained efficiently on the Clg bonded inner surface or the RA-RP support. The breakthrough volume of fluorescein-biotin dissolved in binding buffer was larger than 300 mL. Due to the high breakthrough volume, the BCD system could be used for at least 24 h. The column was easily regenerated by rinsing with acetonitri1e:water (40:60 v/v) . Influence of Multivalency. Initial experiments to test the performance of the BCD system were carried out under equilibrium conditions and in the FI mode. Avidin was incubated for 15 min with different concentrations of biotin. Next fluoresceinbiotin was added, and the sample was incubated for 15 min prior to injection in the FI system. Figure 3 shows a graph representing the theoretical response, derived from the equilibrium model, and the fluorescence response measured as a function of the biotin concentration. Both curves are similar at biotin concentrations higher than 10 nmol/L. However, at biotin concentrations lower than 10 nmol/L, they differ significantly. Instead of an increasing indirect fluorescence signal, a decrease was observed. A possible explanation for this observation can be derived from the tetravalency of avidin, assuming that the fully occupied 4:l fluoresceinbiotin/avidin complex has a lower fluorescence quantum yield than the 3:l complex due to internal quenching. On the other hand, the 21 and 1:l complexes have a lower quantum yield than the 4:l complex because of the lower number of fluorescein molecules bound per molecule of avidin. We tested this hypothesis by preincubating avidin with a 2-fold molar excess of biotin
0
5
10 Biotin (nmolh)
15
Figure 4. Detector signal vs biotin concentration at different reaction times for reaction I predicted by the kinetic model at (-) 120 s, (- -) 60 s, and (- -) 30 s and values measured with the BCD system at (0) 120 s, (0) 60 s, and (0)30 s. Avidin concentration, 17 nmol/L; fluorescein-biotin concentration,200 nmol/L; reaction time for reaction ( I , 60 s; other conditions, see Experimental Section.
-
and using the 2:l biotin/avidin complex formed as reagent. The dashed line in Figure 3 shows that preincubation with biotin resulted in a shift of the calibration l i e and a linear increase of the indirect fluorescent signal with increasing analyte concentrations at concentrations down to 1 nmol/L. In all subsequent experiments, avidin was therefore preincubated with a 2-fold molar excess of biotin prior to its use as reagent. Optimization of Reaction Conditions. From eqs 6 and 7 it can be derived that for a given bioafhity system, that is, for constant association and dissociation rate constants, the reaction time and the concentration of avidin are the most relevant parameters to be optimized. The influence of the reaction time on the detector response is demonstrated in Figure 4. The curves are derived from eqs 6 and 7, assuming an association rate constant of 2 x 106 L mol-' s-1, an avidin concentration corresponding to 17 nmol/L of free binding sites, and a label concentration of 200 nmol/L. The reaction time for reaction I1 was 60 s. The theoretical curves closely match the values measured (n = 3) under stopped flow conditions. Both theoretical curves and experimental data indicate that the system has not reached equilibrium at the longest reaction time of 120 s. A further increase of the reaction time did not result in larger peak heights since the gain in sensitivity due to a more complete reaction is counteracted by band broadening caused by dispersion in the open-tubular postcolumn reactor. No attempts were made to use segmented reaction detection systems which are recommended if reaction times longer than 5 min are needed.17 When the BCD is operated at a maximum reaction time, the concentration of avidin is the most important parameter to be optimized. Increasing avidin concentrations results in higher concentrations of both ligand-protein and label-protein complexes as predicted by eqs 6 and 7. Higher label-protein concentrations cause an increase of the background signal and baseline noise, which finally will counteract the gain in sensitivity due to increased ligand-protein concentrations. Quantitative Aspects. In Figure 5, theoretical and experimental (n = 3) calibration values for biotin are shown at three (17) Lillig, B.; Engelhardt, H. In Reaction Detection in Liquid Chromatography; Krull, I. S., Ed.; Marcel Defier, Inc.: New York, 1986; p 1. (18) Mho, S.4.; Yeung, E. S. Anal. Chem. 1985, 57, 2253.
5
0
20
15
10 Biotin (nmolfl)
20
Figure 5. Detector signal vs biotin concentrationat avidin concentrations predicted by the kinetic model at (-) 17 nmol/L, (- -) 4.3 nmol/L, and (- -) 1.7nmol/L and values measured with the BCD system at (0)17 nmol/L, (0) 4.3 nmol/L, and (0) 1.7 nmol/L. Fluorescein-biotin concentration, 200 nmoVL; reaction time for reaction I, 60 s;reaction time for reaction II, 60 s;other conditions, see Expedmental Section.
-
5
v
E
8l
lo'
6 - 1
I
'
e
', '.
-I
O ;
10'0
e
_ _ , * '
--_._.-
I
I
I
10.9
10-8
10.7
Avidin (nmolh)
Figure 6. Limit of detection vs avidin concentration predicted by the kinetic model for (- -) k + ~= 2 x lo6 s-l and (-) k + ~= 2 x 10' s-I; (0) values measured with the BCD system. Fluorescein-biotin concentration,200 nmol/L; reaction time for reaction I, 60 s;reaction time for reaction 11, 60 s; other conditions, see ExperimentalSection.
-
different avidin concentrations. In the concentration range between 1and 20 nmol/L biotin, the indirect fluorescence signal increases linearly with the avidin concentration. To predict the limit of detection &OD) as a function of the avidin concentration, an iterative simulation model was developed. In this model, the detector signal, S, at a certain avidin concentration was calculated according to eq 8. The detector noise, N, was calculated (see eq 9) by a function consisting of a constant part, n,, i.e., the instrument noise in absence of A-BF (shot noise) and a variable part, nf,which depends linearly on the concentration of labelprotein complex (flicker noise):'*
In Figure 6, the theoretical LOD calculated as S/N = 3 is depicted as a function of the avidin concentration for two different BCD systems with association rate constants of 2 x lo6 and 2 x lo7 L mol-' s-l, respectively. The simulation assumes that [BF] >> [A]B to assure near equilibrium conditions for reaction I1 (reaction time, 60 s). The LODs measured were divided by a factor of 4 to correct for dilution due to band broadening. The Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
4299
b.
a.
I
0
4
8
1
I
12
0
C.
4
8
i
I
12
0
d
4
8
12
Time (min) Time (min) Time (min) Figure 7. (a) LC-BCD of a blank sample; (b) LC-BCD of biotin (40 nmol/L); (c) LC-UV detection at 210 nm of biotin (400pmoVL). Conditions, see Experimental Section.
avidin concentration providing the lowest LOD depends strongly on the association rate constant. Of particular interest are the different shapes of the curves at different rate constants. At a low association rate constant, a rather narrow optimum is obtained, and changes of the protein concentration in either direction cause a significant increase of the LOD. At a higher association rate constant (curve 2), the optimum range is much broader, and changes in the avidin concentration are less critical. The optimal avidin concentration of the present system was 17 nmol/L. On-line Coupling to Liquid Chromatography. The BCD system was coupled on-line to reversed-phase LC using &bonded silica as stationary phase. Compared to the FI, the flow rate of the label pump (3 in Figure 1) was increased from 0.4 to 0.8 mL/ min, and reaction coil 2 was shortened to provide a reaction time of 15 s. Chromatograms representing LC-BCD of a blank sample, a sample spiked with 40 nmol/L biotin, and LC-UV detection of a sample spiked with 400 ymollL biotin omitting the postcolumn reaction detection system are shown in Figure 7. The extracolumn band broadening of the BCD system amounts to UBCD = 9.9 s at a total peak width, utot,of 15.0 s, which is acceptable if the increase in selectivity provided by the BCD system is considered. Compared to the FI-BCD system, which is operated with a purely aqueous carrier solvent, the LC mobile phase may contain organic modifiers such as methanol or acetonitrile. We showed earlie6s4 that we could operate a postcolumn immunochemical detection system at an acetonitrile content (after mixing) up to 15%. In the present system, the presence of organic modifier did not hamper significantly the interaction between biotin and avidin. However, a methanol content of 2.5% in the LC mobile phase resulted in an increased background signal, which is possibly caused by the breakthough of polar impurities due to the weaker retention on the RA-RP support. After equilibration for 30 min, a stable baseline was obtained, and the BCD system could be operated for 24 h at a total flow rate of 1.6 mL/min before regeneration of the RA-RP was required. 4300 Analytical Chemistry, Vol. 66, No. 23, December 1 , 1994
Tabte 1
FI
concn WmL) 2 5 20
LOD (fmol)
LC
precision
accuracy
precision
accuracy
0.8 0.8 0.8
95.5 99.7 99.5
16.8 11.0 6.5
92.5 98.9 107.2
(%I
(%I
40
(%I
6)
160
Table 1 summarizes the data on accuracy, precision, and limit of detection obtained with both FI- and LC-BCD. An avidin concentration corresponding to 17 nmol/L free binding sites was used throughout these experiments. The detection limit (signalto-noise ratio, 3:l) using LC-BCD was a factor of 1000lower than the LOD obtained for LC-W. The indirect detector signal was linear in the concentration range of 8-200 nmol/L (20 yL injections, $ = 0.998). The low avidin concentration chosen to obtain the highest sensitivity inherently leads to a rather small linear range of the present method. CONCLUSIONS We are currently adapting the present method to be used in combination with labeled antigens and antisera for LC-ICD of pesticides and their metabolites. Compared to the LC-ICD system reported earlier,3s4the present system has the considerable advantage that it principally permits the use of unlabeled, unpurified affinity proteins. The preparation of labeled antigens is based on the same chemistry which has been used for the coupling of the hapten to the carrier protein used for immobilization. The detection limit of the present indirect detection technique is of the same order of magnitude, i.e., in the femtomole range, which was obtained with LC-ICD using labeled antibodies. (19) Tyssen, P. In Practise and n e o t y of Enyme Immunoassay; Burdon, R H.,
Knippenberg, P. H., Eds.; Elsevier: Amsterdam, 1985; p 130.
Separation of free and protein-bound label using a restrictedaccess reversed-phase support requires rather hydrophobic label for efficient retention. Future research is devoted to alternative separation techniques to achieve the same goal for rather polar labels. The use of short columns, however, is advantageous with respect to band broadening, and the availability of restricted-access supports with different internal surface, e.g., ion-exchange functionalities, should extend the range of suitable labels. From a theoretical point of view, the present method is applicable to all those bioaffinity interactions that are characterized by high binding constants and high association and small dissociation rate constants. Particularly the magnitude of the dissociation rate constant plays a crucial role. If the dissociation rate contant is too high, it is likely that the excess of label added in the second step will displace ligand molecules bound to the affinity protein in the first step, rendering detection impossible.
From the kinetic model described above, it can be derived that, for example, at a dissociation rate constant of s-l and a reaction time of 60 s for reaction 2, the signal is reduced by 50% compared to bioaffinity interaction systems with dissociation rates lower than s-l. Most dissociation rate constants for antibodyantigen interactionsare well below s-l,19allowing the design of ICD methods on the basis of the present detection principle. ACKNOWLEDGMENT We thank Prof. Dr. Karl-SiegfriedBoos (University of Munich, Germany) for providing us with restricted-accesssupport precolumns. Received 1994.a
for
review July 6, 1994. Accepted August 25,
@Abstractpublished in Advance ACS Abstracts, October 1, 1994.
Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
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