Anal. Chem. 1999, 71, 5271-5278
A Receptor-Based Bioassay for Quantitative Detection of Gallium H. Xu, E. Lee, and O. A. Sadik*
Department of Chemistry, State University of New York at Binghamton, Binghamton, New York 13902 R. Bakhtiar
Novartis Pharmaceuticals Corporation, East Hanover, New Jersey 07936 J. Drader and C. Hendrikson
National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahasse, Florida 32310
The detection of gallium in biological samples is required due to its role in the diagnosis of tumor and for possible treatment of malignancies. However, the use of purely instrumental techniques is unsuitable for detection of low levels of gallium in biological matrixes. We have synthesized new protein conjugates based on 4-(2-pyridylazo) ligands. The conjugates were successfully employed for the detection of gallium in biological matrixes using a nonantibody-based sandwich assay format. The recovery level obtained was between 97 and 101.3 with a relative standard deviation of less than 5%. The assay resulted in a detection limit of 5 × 10-8 M and a remarkable selectivity for gallium(III) relative to other metals investigated. The new method provided adequate accuracy for gallium applicable for animal physiology and clinical toxicology.
The use of Ga-containing compounds to diagnose tumors and for possible treatment of malignancies has been well-documented.1-5 The antitumoral therapeutic effects of gallium nitrate have been widely demonstrated in laboratory animals, and the low degree of toxicity would appear to suggest its suitability for the treatment of various tumors. Several workers have shown that when the concentration of Ga is increased, tumor mass is decreased.2,3,6 However, the mechanism of gallium uptake during tumor detection is still largely unknown. It appears that the nature and types of anionic species present are the determining factors responsible for the distribution of gallium in the various organs. In view of the widespread medical application, analytical methods are * Corresponding author: (fax) (607) 777-4478; (e-mail)
[email protected]. (1) Collery, P.; Morel, M.; Millart, H.; Perdu, D.; Lavaud, F.; Anghileri, L.; Pluot, M.; Choisy, H.; Pechery, C. Metal Ions in Biology and Medicine; Collery, P., Poirier, L., Etienne, J., Eds.; Libbey Eurotext: Paris, 1994. (2) Collery, P.; Millart, H.; Chiosy, H. Anticancer Res. 1992, 12, 1920. (3) Edwards, C. L.; Hayes. R. L. J. Nucl. Med. 1965, 10, 103. (4) Wong, H.; Terner, U. K.; English, D.; Noujaim, B. C.; Hill, J. R. Int. J. Nucl. Biol. Med. 1980, 7, 9. (5) Hart, M. M.; Smith, C.; Yancy, S.; Adamson, R. H. J. Natl. Cancer Inst. 1971, 47, 1121. (6) Hart, M. M.; Adamson, R. H. Proc. Natl. Acad. Sci. U.S.A. 1971, 68, 1623. 10.1021/ac990902m CCC: $18.00 Published on Web 10/30/1999
© 1999 American Chemical Society
required that are capable of accurately determining gallium concentrations in different biological samples. In addition, most of the available instrumental methods for determining gallium are expensive, unsuitable for gallium detection in biological samples, and exhibit poor selectivity due to interference from other metals. These include inductively coupled plasma spectroscopy (ICP), atomic absorption spectroscopy (AAS), and neuron activation coupled with high-resolution γ-spectrometry.7-9 A comparative investigation of the suitability of AAS, emission spectroscopy with arc, and hollow-cathode excitation sources for detection of gallium (µg/g) in biological samples indicated that all three methods are significantly influenced by matrix effects.7 Hollow-cathode emission was found to be influenced, to a lesser extent, by matrix effects and to be more precise than the other two.7 Antibodies formed against ethylenediaminetetraacetic acids (EDTA) have been used to detect several metals, including mercury, indium, and cadmium.10-18 These antibodies (Abs) are usually produced from haptens obtained from the derivatization of EDTA with large proteins such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), or synthetic peptides. However, such derivatization often significantly alters the properties (7) Caroli, A.; Alimonti, A.; Femmine, P.; Shukla, S. Anal. Chim. Acta 1982, 136, 225. (8) Caroli, A.; Alimonti A.; Femmine, P. Spectrosc. Lett. 1979, 12, 871. (9) Alimonti, A.; Caroli, S.; Violante, N. Spectrosc. Lett. 1980, 13, 313. (10) Williams, C. A., Chase, M. W., Eds. Methods in Immunology and Immunochemistry, Academic Press: New York, 1967; Vol. 2, p 343. (11) Chakrabarti, P.; Hatcher, F.; Blake, R. C. Anal. Biochem. 1994, 217, 70. (12) Love, R. A.; Villafranca, J. E.; Aust, R. M.; Nakamura, K. K.; Jue, R. A.; Major, J. G.; Radhakrishnan, R.; Butler, W. F. Biochemistry 1993, 32, 10950. (13) Reordan, D.; Meares, C. F.; Goodwin, D. A.; McTique, M.; Davie, G. S.; Stone, M. R.; Leung, J. P.; Bartholomew, R. M.; Frincke, J. M. Nature 1985, 316, 265. (14) Wylie, D. E.; Lu, D.; Carlson, L. D.; Carlson, R.; Babacan, K. F.; Schuster, S. M.; Wagner, F. W. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 4104. (15) Szurdoki, F. Hammock, B. D. In Immunoanalysis of Agrochemicals: Emerging Technologies; Nelson, J. O., Karu, A., Wong, R., Eds.; ACS Symposium Series 586; American Chemical Society: Washington, DC, 1995. (16) Bekheit, H.; Lucas, A.; Szurdoki, F.; Gee, S.; Hammock, B. J. Agric. Food Chem. 1993, 41, 2220. (17) Hayes, F.; Halsall, B.; Heineman, W. Anal. Chem. 1994, 66, 1860. (18) Meares, C. F.; et al. Anal. Biochem. 1984, 142, 68.
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of the antigen. The resultant Abs, particularly polyclonal antibodies, are likely to possess heterogeneous molecular composition comprising a number of “isoforms” which may be different in molecular structure, biological potency, and physiological functions. In addition, the assay technologies largely depend on the availability of Abs, which are expensive to produce and limited by different antibody specificity and cross-reactivity patterns.19,20 Therefore, nonantibody based detection methods will provide an attractive alternative to immunochemical assays. The objectives of this work are (i) to demonstrate the feasibility of preparing nonradioactive metal-protein conjugates using 4-(2pyridylazo)resorcinol (PAR) and (ii) to explore the analytical basis for possible discrimination of the resulting conjugates for detection of gallium. PAR has been used extensively for the analysis of metals and possesses a variety of useful spectroscopic and luminescence properties.21-27 To our knowledge, (2-pyridylazo)resorcinol and its derivatives such as 1-(2-pyridylazo)-2-naphthol (PAN) have not been employed for the preparation of biological conjugates. Compared to PAN, PAR was chosen as a modifier in our work because of its relative stability and higher solubility in water.21 We hereby report a novel, nonantibody-based detection system using protein-modified PAR ligand as biorecognition element. PAR was derivatized with ovalbumin, BSA, and alkaline phosphatase (AP) to generate the respective conjugates. The synthesis was carried out using water-soluble carbodiimide and N-hydroxysuccinimide coupling techniques. The results of characterization experiments using electrospray mass spectrometry, UV/visible, and Fourier transform infrared (FT-IR) confirmed that a new class of protein conjugates has been synthesized. The conjugates were used for the detection of gallium in a sandwich enzyme-linked immunosorbent assay (ELISA) format. The detection format did not include the use of antibody contrary to conventional immunoassay techniques. This method resulted in a remarkable selectivity for gallium(III) relative to other metals investigated, including Fe(II), Zn (II), In(III), Hg(II), Tl(III), and Pb(II). ASSAY PRINCIPLE The detection of Ga using the PAR conjugate was based on a two-site (sandwich) chelate principle as shown in Figure 1. In one site, the gallium metal was captured by an immobilized PARprotein conjugate known as the capture chelator. In the second site, the chelate was detected using an enzyme-labeled PAR conjugate known as the detection chelator. The sandwich assay format was set up first by coating the solid phase with the capture chelator, i.e., PAR-OVA, and the analyte was incubated with the immobilized according to (19) Sadik, O. A.; Van Emon, J. M. CHEMTECH 1997, 27, 38. (20) Xu, H.; Sadik O. A. Abstracts of Papers, 217th National Meeting of the American Chemical Society, Anaheim, CA; American Chemical Society: Washington, DC, 1999; Vol. 39 (No. 1) pp 470-472. (21) Flaschka, H. A.; Barnard, A. J. Chelates in Analytical Chemistry; Marcell Dekker: New York, 1972; Vol. 4, p 127. (22) Sarzanini, C.; Porta, V.; Mentasti, E. New J. Chem. 1989, 13, 463. (23) Gao, J.; Guanglin, H.; Kanf, J.; Bai, G. Talanta 1993, 40, 195. (24) Rao, A.; Malik, A.; Kapoor, J. Talanta 1993, 40, 201. (25) Khoo, S.; Ming, S.; Cai, Q.; Khan, M.; Guo, S. Electroanalysis 1997, 9 (1), 2. (26) Ivanov, V.; Morozko, S. J. Anal. Chem. 1996, 51, 989. (27) Bae, Z.; Park, Y.; Lee, S.; Jeon, W.; Chang, H. Bull. Korean Chem. Soc. 1996, 17 (11), 995.
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Figure 1. Principle of metal detection using a PAR bioconjugate. k1
s1 + M y\ z s1-M k 2
(1)
where s1 represents the binding sites of the capture chelator while k1 and k2 are the association and dissociation rate constants, respectively. All other sample constituents were washed out and the bound metal analyte was quantitated during the second step by adding the excess detection chelator (i.e., PAR-AP). After incubation, the PAR-AP was bound to a different site on the metal-molecule according to k3
s1-M + s2* y\ z s1-M-s2* k 4
(2)
where s2* represents the binding sites of the labeled conjugate and k3 and k4 are the rate constants. Any unbound detection chelator (PAR-AP) was washed out. The signal was detected by reacting the chelator with a substrate, which produced a measurable color change at 405 nm. The signal was directly related to the metal concentration in the sample. Since this assay involved two chelators, it provided an improved specificity over the competitive assay because the cross-reactivity substances that normally interfered with the competitive assay produced no signal in the sandwich assay format. EXPERIMENTAL SECTION Reagents. Analytical reagent (AR) grade chemicals were used throughout unless otherwise stated. All solutions were made from Nanopure water having a resistivity of 18 MΩ/cm or higher. The following chemicals were purchased from sources as indicated: 98% (4-(2-pyridylazo)-2-resorcinol (PAR) and 99.99% 4-(2-pyridylazo2-naphthol from Acros (Pittsburgh, PA); N-hydroxysuccinimide (NHS), ovalbumin (OVA, 98%; Lot 37H7015), BSA (>97%, Lot 102H9308), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), Ga (NO3)3, sodium dihydrogen phosphate (NaH2PO4 (99%) from Sigma. Others include alkaline phosphatase, (Lot 96120639) and p-nitrophenyl phosphate disodium (PNPP, 5 mg/tablet (Lot 96111570) from Pierce (Rockford, IL); CusO4‚5H2O, Co(NO3)3‚ 6H2O, Ni(NO3)3, KNO3, ScCl3, and In(metal) from Fisher; MgCl2‚ 6H2O (99%) La(NO3)3‚6H2O, TlCl (99%), TlCl3‚4H2O (98%), and FeCl3‚6H2O from Aldrich Chemicals; and Pb(NO3)3, BaCl2‚2H2O (99.1%), NH2OH‚HCl, and FeSO4‚(NH4)2SO4‚6H2O from Baker.
To remove all traces of metal ions, all glassware were soaked in 3 M HNO3 for 3 days and in water prior to use for 1 day. Instrumentation. The characterization of all protein conjugates was performed using a Hewlett-Packard diode-array (model 8453) UV/visible spectrophotometer equipped with a 1-cm pathlength cell. Protein concentrations were determined using E1%280 with a molecular weight of 45 000 for ovalbumin and 66 400 for BSA. Optical densities were measured using a 100-fold dilution to give absorbance readings of 0.1-0.3. Fourier transform infrared spectra of lyophilized samples in KBr pellets were obtained using Perkin-Elmer spectrophotometer (model 1600 FT-IR). For the mass spectrometry characterization, an API IIIplus triple-quadrupole mass spectrometer (PE-Sciex, Thornhill, ON, Canada) equipped with a pneumatically assisted electrospray interface (also referred to as an “ion spray” interface) was employed.28,29 The ELISA system used for the analysis of Ga consisted of Maxisorb I 96well microplate, and microplate reader ELX 800 UV plate reader from Bio-Tek Instruments. The plate reader’s “instrument control and data analysis” software was from Biotek KC4 Software. The pipets used were capable of delivering 1 mL and were adjustable in the range 20-200 µL single-channel pipet, 0-25 µL adjustable positive displacement pipet, eight-channel 50-200 µL adjustable pipet; borosilicate glass tubes (12 × 75 or 13 × 100 mm). Labconco, FreeZone 12 L, model 77540 was used for freeze-drying. An analysis to confirm the presence of gallium in biological samples was achieved using inductively coupled plasma atomic emission spectroscopy (ICP-AES) technique. The ICP instrument employed was Varian Analytical Instruments from Liberty Spectrometer System (Mulgrabe, Australia). The gallium detection line of 294.364 nm was employed having lead as the internal standard (line 283.306 nm). The flow rate of the argon gas supply was kept at 15 L/min with auxiliary flow rate of 1.5 L/min. Stock Solutions. Stock solutions of metals (1.0 × 10-2 M) was prepared by dissolving a calculated amount of the salts (99.99%) in an appropriate amount of 1 M nitric acid. These were diluted to the desired volume and then further diluted to 1 × 10-5 M before use. A buffer solution of acetate was used for pH adjustment at 0.1 ( 0.01 M. A stock solution of 0.01 M Ga(NO3)3 was prepared from 0.2557 g of Ga(NO3)3 plus two drops of dilute HNO3 and this was made up to 1 L. The following buffers were used for the preparation of ELISA standards and enzyme tracer dilution: carbonate coating buffer having pH 9.6; 0.32 g of Na2CO3, 0.533 g of NaHCO3, and 0.2 g of NaN3 dissolved and diluted to 1 L; phosphate-buffered saline (PBS) pH 7.2, 8.0 g of NaCl, 0.123 g of NaH2PO4, and 1.67 g of Na2HPO4‚7H2O; 0.1 M acetate buffer, pH 5.5, 13.6 g of sodium acetate and 1.05 mL of glacial acetic acid diluted to 1 L in a volumetric flask. The substrate solution was made from 9 mL of acetate buffer and 1.0 mL of diethanolamine, 0.020 g of MgCl2‚6H2O, and two PNPP tablets. The 100 mM sodium acetate buffer, pH 5.5, was used for both washing and incubation of standards and samples. The metal solutions used for interference tests were prepared using different amounts of Ga(III) and interference ions. (28) Chapman, J. R., Ed. Protein and Peptide Analysis by Mass Spectrometry; Humana Press: Totowa, NJ, 1996. (29) Snyder, A. P., Ed. Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry; ACS Symposium Series 619; American Chemical Society: Washington, DC, 1996.
Preparation of PAR-Protein Conjugates. PAR-protein conjugates were prepared using a modified procedure previously described by Tijssen.15,30 This procedure was carried out in two steps. In step 1, the NHS derivative of hydroxyl-containing PAR was generated. This was either used directly or stored until needed. In step 2, the reaction of activated PAR with protein was achieved. Briefly, the procedure employed is as follows: in step 1, 0.0235 g of (108.5 µmol) PAR was reacted with 0.020 g (173 µmol) of NHS with 0.0249 g (130 µmol) of EDC. The mixture was added to 7 mL of dry DMF at room temperature with constant stirring for 6 h. NHS-activated PAR was formed as the carbodiimide became transformed to urea. This was later isolated after dilution with water and extracted with ethyl acetate to remove carbodiimide, which could deactivate the protein in the subsequent step. During step 2, the NHS-activated PAR and ovalbumin were mixed together at 273 K for 1 h in 0.1 mmol/L PBS (pH 7.2). The mixture was allowed to react at 4 °C or ice-cooled and left overnight. The mixture was extensively dialyzed against phosphate buffer (pH 7.2) to remove the unreacted PAR before being freezedried. Standard reaction procedures were used to generate the enzyme-labeled chelator (PAR-AP).31,32 CHARACTERIZATION OF PAR CONJUGATES The UV/visible, IR, and mass spectra (MS) of the PAR-protein conjugate were measured to determine the qualitative difference before and after the conjugation. MS calibration was performed on both the first quadrupole (Q1) and the third quadrupole (Q3) using a solution of polypropylene glycol (PPG) in 3 mM ammonium acetate. Samples were delivered via a 50-µm-i.d. fusedsilica capillary to the ion spray tip which was held at a potential of +4.6 kV. A syringe pump (model 22, Harvard Apparatus, South Natick, MA) controlled the delivery of the sample at a rate of 5.0 µL/min. The spray tip was positioned 1.0 cm off-axis and 1.2 cm away from the ion sampling orifice (∼65 µm in diameter). Compressed air (with pressure set at 45 psi) was employed to assist liquid nebulization. A curtain gas (nitrogen) flow of 1.1 L/min was used in order to prevent moisture from reaching the orifice and the quadrupole guidance lens. The interface (atmosphere-to-vacuum) heater was set at 55 °C. Mass spectra were obtained with a dwell time of 2 ms for each step (0.5 Da) in the scan. About 5-10 scans were summed to improve the signal-tonoise ratio. The orifice potential was maintained at 55 V in the positive ion detection mode. Determination of Gallium. The gallium assay described in this work utilized a two-site chelate format as described above. A 100-µL metal-free, PAR-OVA ligand (1:100 dilution) in carbonate buffer (pH 9.6) was first adsorbed for 12 h at 4 °C onto a 96-well polystyrene microtiter plate having high protein affinity. The plate was washed with acetate buffer (pH 5.5). Solutions of gallium standards were added to each well, and the plate was incubated for 1 h and then washed in acetate buffer (pH 5.5). Several dilutions of PAR-AP were added to each plate and the incubation was repeated for 1 h. The plates were washed, and an appropriate (30) Tijssen, P. Practice and Theory of Enzyme Immunoassays; Elsevier: New York, 1985. (31) Butler, J. Perspectives, Configurations & Principles. In Immunochemistry of Solid-Phase Immunoassay; Butler, J., Ed.; CRC Press: Boca Raton, FL, 1991; p 41. (32) Harlow, E., Lane, D., Eds. Antibodies: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1988.
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Scheme 1. (a) Equilibrium Forms of PAR at Different pHs and (b) Structures of the 1:1 and 1:2 PAR Chelates
Chart 1: Structures of PAR and PAN
dilution of enzyme-substrate (PNPP) was added. The product of enzyme-catalyzed conversion of the substrate was measured spectrophotometrically at 405 nm. To optimize the overall assay performance, the effects of pH and the stoichiometry of the PAR chelates were investigated. RESULTS AND DISCUSSION The assay principle reported here relies on the high-affinity chelation of the novel PAR bioconjugate and on a simple immunoassay-based technique to develop a highly sensitive and selective analysis of gallium(III). The use of compounds of 2-pyridylazo and their derivatives are suitable for this purpose because their selectivities can be controlled through an appropriate adjustment of the pH and their strong spectroscopic and luminescence properties. Both PAR and PAN in Chart 1 have been widely used as chromogenic agents for determination of metals.21-24 In contrast to PAN, PAR and its metal complexes are more water soluble, thus offering great advantages as chromogenic agents and metal indicators in aqueous media. Compounds of 2-pyridylazo are not selective, either as chromogenic agents or as extractants. However, with appropriate pH adjustment and the use of masking agents, their selectivity can be improved. In this case, hydrogen ion competes with the metal ion to combine with the 2-pyridylazo compound (Scheme 1a). Consequently, the higher the stability of the metal complex, the lower the pH at which it can exist. Also, the lower the pH, the fewer the number of metals that are complexed. The control of pH alone, or in combination with masking agents, has been used both for selective color formation and extraction. As shown in Scheme 1b, PAR forms tridentate 1:1 and 1:2 metal complexes which are coordinated at the pyridine nitrogen, the azo nitrogen further from the heterogeneous cyclic ring and the ortho hydroxyl group. The most stable form of the PAR complex is the tridentate 5274 Analytical Chemistry, Vol. 71, No. 23, December 1, 1999
Figure 2. UV/visible spectra of PAR and protein conjugate. Conjugate was synthesized as described in the Experimental Section. Condition: (a) 2 × 10-5 M PAR, (b) PAR conjugated to ovalbumin (PAR-OVA), and (c) PAR conjugated to alkaline phosphatase (PARAP), phosphate buffer (pH 7.2).
1:2 chelate. As a result of the potential spectrophotometric advantages and chelate formation of 2-pyridylazo and its derivatives, we decided to investigate its use for conjugation with proteins for specific and selective detection of gallium. Synthesis and Characterization of Protein Conjugates. Protein conjugates were synthesized as described in the Experimental Section. The UV/visible spectra recorded for PAR-protein conjugates are shown in Figure 2. Two absorption maximums were obtained at 280 nm for protein and 420 nm for PAR. Results indicated that PAR was successfully conjugated to the proteins. It is expected that PAR should be conjugated to the proteins through its para hydroxyl group, since it is less hindered. As the para hydroxyl group is conjugated, the ortho hydroxyls are free for complexing with the metal. From the absorbance measurements, the relative ratio of OVA to PAR was estimated to be 3:1 and 1.5:1 BSA to 1 PAR (i.e., mixtures of 1:1 and 1:2 complex) was recorded. Electrospray ionization mass spectrometry (ESI-MS) was used to determine the molecular weight and fragment ions of the synthetic protein adduct and the accompanying neutral molecules formed during fragmentation. ESI-MS results indicate that PAR was conjugated to the proteins. The ratio of PAR-protein adduct
Table 1. Vibrational Frequencies of PAR Ligands and Conjugates (cm-1) aromatic ring vibrations νCdO
νNdNs(νNsN)
νCarsOH
νCarsN
1250
760
1447
1654
2304 (1150) 2365
1452
1654
2354
1650
2440
compound
νCsC
νCdC
νCsN
PAR (C11H9N3O2)
1472
1570
1589
PAR-OVA PAR-BSA PAR-AP
was further confirmed using fast protein liquid chromatography (FPLC) coupled with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Results of the FPLC revealed welldefined signals for the protein, which corresponded very well with the molecular weight of the sample. Shortly after the protein eluted, an increase in the conductivity of the solution was observed. This could be attributed to the elution of the salts from the column. The separation of the salts from the protein after solidphase cleanup helped to improve the quality of the mass spectra, eluting the PAR-ovalbumin fraction with a corresponding shift in mass corresponding to the PAR-protein adduct. Using a molecular weight of 66 428 for BSA, a 1:1 PAR-BSA conjugate was estimated from the ESI-MS measurements. This also corresponded with the degree of conjugation estimated from the UV/ visible measurement. The PAR(OVA)3 corresponded to ∼128 300 amu, exceeding the allowable dynamic range (m/z) of the triple quard MS instrument. FT-IR experiments were carried out to determine the presence of major amide functional groups in the conjugates. The spectra range was within 4000-200 cm-1. Table 1 is a list of the absorption bands obtained. From this table, the most important PAR functional groups are those having absorption bands of stretching vibration frequencies within 1200 and 1230 cm-1. These are assigned to the C-OH vibrational modes and NdN- functions (1140-1160 cm-1). The presence of both symmetric and asymmetric N-H stretching bands within the range 3550-3450 and 3450-3350 cm-1 were observed for the PAR-OVA, PAR-BSA, and PAR-AP conjugates. In addition, amide II bands with medium intensities arising from deformation modes at 1650-1580 cm-1 were also present. The presence of CdO at 1654 cm-1 and NH at 1530-1539 cm-1 showed the characteristic bands of amides derived from the conjugated proteins which were absent from the PAR spectra. The IR spectra of the protein-bonded PAR exhibited a conspicuous feature of strong, broad bands of C-H for protein (3100 cm-1), OH (3300 cm-1), and CdO (1100 cm-1) functional groups that are wider and more intense as a result of the presence of the proteins. Influence of pH and Evaluation of Formation Constants. The stability of many metal complexes is dependent on pH.32,33 The effect of pH on the complexation characteristics of PARprotein conjugated with Ga was examined using buffered metal solutions in the range 4.5-8.0. Evidence of the stability of the conjugates was obtained from the formation constants. At pH 4.0, PAR produced a well-defined complexation effect with Ga as (33) Jolly, W. L. Modern Inorganic Chemistry; McGraw-Hill Book Co.: New York, 1984.
1447 1452 1258
νNsH(νOsH)
νNsCdO
3444 1534 (3313) 1539 (3422) 3423
1534
Figure 3. Influence of pH recorded for Ga-PAR. The pH of the chelate is over the respective absorbance peaks.
Figure 4. Quantitative detection of Ga at different pH using the PAR-OVA conjugate.
evidenced by the PAR peak at 410 nm and a wavelength shift at 498 nm (Figure 3). By changing the pH and subsequent titration with Ga, this peak increased significantly up till pH 5.5 and later decreased at above pH 6.0. Similarly, using the microplate assay format, the PAR-OVA produced a well-defined complexation with Ga. Figure 4 shows the results obtained for the quantitative detection of Ga with changes in pH. As the binding slope increased with pH, a peak was formed around pH 5-5.5. At pH >8.0, the quantitative determination of Ga rapidly decreased, which could be attributed to a direct consequence of hydrolysis. An optimum pH of 5.5 obtained was and then used for further analysis. The formation constants under the above conditions were calculated from the absorbance data using the method of continuous variation. The color formation was instantaneous, and the absorbance values remained constant even up to 72 h. No significant change occurred when the order of addition of reagent Analytical Chemistry, Vol. 71, No. 23, December 1, 1999
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Table 2. Formation Constantsa of the Conjugates ligand
pH
log β
Ga-2PAR Ga-2(PAR-OVA) Ga-2(PAR-BSA) Ga-2(PAR-AP)
7.2 7.2 nab 7.2
10.3 ( 0.7 10.2 ( 0.5 na 9.9 ( 0.3
a Temperature 25 °C; measurement was conducted in phosphate buffer. b na, not available.
Figure 5. Suggested structure of the PAR-Ga bioconjugates used in the assay. The metal shows a coordination number of six and the binding is through phenolic oxygen, a nitrogen of the pyridine group, and a nitrogen of the azo group.
was altered. Using the method of continuous variations, Ga-PAR, Ga-PAR/OVA, and Ga-PAR/BSA conjugates were estimated to have the composition 1:2 at pH 5.5, which was confirmed by the mole ratio method. The values of the formation constants of the ligands are summarized in Table 2. The structure of the reagents used in the gallium assay is shown in Figure 5. Ga forms a strong binding with PAR and the protein conjugates with value comparable to literature data.35 Analysis of Gallium Using PAR-Protein Conjugates. PAR-OVA and PAR-BSA conjugates were used for the analysis of gallium to demonstrate the two-site chelate assay principle described above. The gallium metal was captured by the immobilized PAR-protein conjugate (capture chelator) and this was detected using a second enzyme-labeled PAR conjugate (detection chelator) at 405 nm. Figure 6 represents the standard curve obtained for Ga analyzed using a four-parameter logistic curve fitting. The assay was found to exhibit a characteristic sigmoidal shape, and the signal increased with the analyte concentration. At low analyte concentrations, no significant binding occurred and the response was close to that of a zero-gallium concentration. As the concentration was increased, the binding became proportional to the analyte concentration. A plateau was reached when the capture chelator became saturated. Of particular analytical significance was the C value, which represented the value at which the system response was at 50% (or IC50). In immunochemical terms, this is a direct indicator of the general concentration level at which the assay is capable of functioning. This study showed that gallium ions could be conveniently detected with IC50 value of 10-6 M, a linear concentration range of 5 × 10-7-1 × 10-4 M, and a correlation coefficient of 0.9980. The assay detection limit, which is defined as three standard deviations above the zero standard, was recorded to be 5 × 10-8 M. (34) Riley, T., Watson, A. Polarography and Other Voltammetric Methods; J. Wiley & Sons: London, 1987. (35) Dwivedi, C. D.; Munshi, K.; N.; Dey, A. K. J. Inorg. Nucl. Chem. 1966, 28, 245.
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Analytical Chemistry, Vol. 71, No. 23, December 1, 1999
Assay Selectivity. The assay sensitivity can be controlled by changing the nature of the immobilized capture chelator. The sensitivities of the quantitative chelate assays for different concentrations were determined using PAR-OVA, PAR-BSA, and PAR as the immobilized chelator as shown in Figure 7. PAROVA and PAR-BSA conjugates were found to exhibit significant selectivity for gallium while PAR by itself showed no significant absorbance values beyond that of the blank. The increased sensitivity of PAR-OVA conjugate relative to PAR-BSA could be attributed to the higher ratio of coupling during conjugation. The selectivity of the PAR(OVA)3 conjugates originated from a stronger attachment to the plate, thus decreasing the loss of PARprotein conjugates during the assay. This attachment stabilized the ligand in the active site, and the resulting protein conformational change constitutes the basis for the selectivity observed. In contrast, the relatively weaker attachment of PAR-BSA or PAR(BSA)2 to the plate could result in higher loss of PAR-protein molecules. The response of the assay to various metals was characterized with respect to gallium. This response is expressed as the concentration of the gallium required to reduce or inhibit the assay response by 50%. This is defined as 50% inhibition level or IC50. The result of the cross-reactivity is presented in Table 3 for metals such as Al3+, In3+, Zn2+, Cu2+, Co2+, Fe2+, Tl3+, Pb2+, and Ni2+, . The data showed that none of the potential cross-reactants exhibited greater that 2500 nM cross-reactivity relative to gallium(III). Moreover, the remarkable selectivity exhibited by the bioconjugate may be attributed to the “size-to-charge” (r/z) ratios of the metals. Metals exhibiting r/z comparable ratios to that of Ga3+ should display similar characteristics. The presence of the pyridine nitrogen, the azo nitrogen, and the ortho hydroxyl group enables strong binding with the metal, which is greatly influenced by the charge. In addition to the size-to-charge ratio, it appears that three other factors may have affected the selectivity for determination of Ga(III). These factors include the binding constant, the pH, and the stoichiometry. Also, the remarkable assay selectivity observed using the PAR-protein conjugate is substantiated by actual independent data obtained for the complexation of the different metals tested using the native PAR reagent. These data are shown in Table 3, and this also indicates the binding constants (Log K) and optimal pH conditions for the formation of metal-PAR complexes as well as the stoichiometry for different metals investigated. Assuming that the binding between the metal ions and the PAR-protein conjugates is similar to that of the metal ions and the native PAR, then the observed selectivity can be related to those three factors. For example, Al3+, In3+, Sc3+, Zn2+, Cu2+, and Pb2+ ions form 1:1 complexes with PAR and are not expected to produce measurable signals with the PAR-protein conjugates. These metals do not interfere with the receptor assay as summarized in Table 3. Only metals that form 1:2 complexes with PAR are expected to give rise to measurable signals in this type of assay scheme. However, Co2+, Ni2+, and Ni2+, which formed 1:2 ratios with PAR (under optimal pH conditions farther from the pH of 5.5 used in the detection scheme), produced no interference. Finally, metals such as Zn2+, Pb2+, Tl+, K+, and Na+, having ionic radii that are smaller than those of Ga3+ and Fe3+, produced no signals in the assay. Overall, there is a trend between
Figure 6. Four-parameter fit calibration curve obtained for the detection of Ga3+ using the PAR-OVA conjugate in acetate buffer (pH 5.5). Table 3. Cross-Reactivity Data for Ga Determinationa metal-PAR complex metal ion Ga3+ Al3+ In3+ Zn2+ Cu2+ Pb2+ Fe3+ Co2+ Fe2+ Tl3+ Tl+ Na+ K+ Mg2+ La3+ Sc3+ Ni2+ Ba2+
metal concn (nM)
Z
r (pm)
z/r × 100
Log K
stoichiometry
optimal pHb
1 × 104 1 × 105 1 × 106 1 × 107 1 × 106 1 × 107
