Anel. Chem. 1999, 65, 2933-2937
2935
Quartz Crystal Microbalance for the Detection of Microgram Quantities of Human Serum Albumin: Relationship between the Frequency Change and the Mass of Protein Adsorbed Makoto Muratsugu,'*tFumihiko OhtaJ Yoshihiro MiyaJ Toshiaki Hosokawaj Shigeru Kurosawa$*ll Naoki Kamo,i and Hisami Ikedat Department of Laboratory Medicine, School of Medicine, Asahikawa Medical College, Asahikawa 078, Japan, Ibaraki Research Laboratory, Hitachi Chemical Company, Ltd., Hitachi 31 7, Japan, and Department of Biophysics and Physical Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan
We have developed a piezoelectric immunosensor for the detection of microalbumin. Human serum albumin (HSA) in the range 0.1-100 pg mL-l could be detected using a flow cell; the immunosensoris sensitive enough to monitor levels of albuminuria. The immunosensor did not respond to bovine serum albumin, only to HSA, implying that the specificity for HSA was high. We investigated the relationship between the frequency change (AF) and adsorption per unit area of piezoelectrically active quartz crystal (AM). AM was estimated with radioisotope-labeledanti-HSA or HSA. When anti-HSA was adsorbed onto the surface of the crystal or HSA was bound to anti-HSA supported by the crystal, values of l A F / A q were larger than the value predicted from theory (Sauerbrey's equation). Furthermore, lAF/AM for HSA was larger than that for anti-HSA. INTRODUCTION Serum albumin is a major plasma protein and is one of the main proteins excreted in urine. The microdetermination of albumin is important; the increase of urinary excretion of albumin has been seen in diabetic patients early in the course of the disease and is considered to be an incipient sign of diabetic nephropathy.1J The increase, termed "microalbuminuria", is not generally detectable in ordinary testa (for example, a dye-binding methods), but only with methods sensitive to low concentrations of albumin in urine, such as radioimmunoassay (RIA),4-' enzyme-linked immunosorbent assay (ELISA)Pleand so on. These assay methods require a radioisotope- or enzyme-labeled antibodylantigen. However ~~~
~~
~
+ Aaahikawa Medical College.
Hitachi Chemical Co. University. IPresent address: National Institute of Materials and Chemical Research, Tsukuba, Ibaraki 305,Japan. (1)Miles, D. W.; Mogeneen, C. E.; Gundereen, H. J. G. Scand. J. Clin. Lab. Invest. 1970,26,5-11. (2)Mogensen, C. E.N.Eng. J. Med. 1984,310,356-360. (3)Doumse, B. T.; Watson, W. A.; Homer, G. B. Clin. Chim. Acta 1971,31,87-96. (4)Keen, H.; Chlouverakis, C. Lancet 1963,2,913-914. (5) Woo, J.; Floyd, M.; Cannon, D. C.; Kahan, B.Clin. Chem. 1978,24, 1464-1467. (6)Viberti, G. C.; Hill,R. D.; Jarrett,R. J.; Argyropouloe,A.;Mahmud, U.;Keen, H.Lancet 1982,1430-1432. (7)Gaizutis, M.; Peace, A. J.; Lewy, J. E. Microchem. J. 1972,17,327337. (8)Fielding, B. A.; Price, D. A.; Houlton, C. A. Clin. Chem. 1983,29, 365-357. (9)FeldtRasmuasen, B.; Dinesen, B.; Deckert, M. Scand. J. Clin.Lab. Invest. 1985,45,639-544. t
1 Hokkaido
00082700/93/0365-2933$04.00/0
a piezoelectric immunosensor does not require the labeled antibodylantigen, because it can sense a mass change due to antigen-antibody reaction on a surface of quartz crystal. After Sauerbrey's pioneering work,lO piezoelectric quartz crystalshave been used for the measurement of mass. Various hydrocarbons, gas-phase analytes, pollutants, water vapor, and other compounds have been detected (reviewed in ref (11);this method was also applied to electrochemicalsystems (reviewed in refs 11 and 12). This mass-sensing device is referred to as the quartz crystal microbalance (QCM). Sauerbrey gave the following equation relating the change of oscillatingfrequency,AFto the mass change, Am, at the crystal surface.
AF = - (PAmlNAp) (1) where N is the frequency constant, p is the density of quartz, A is the piezoelectrically active area, and F is the basic oscillation frequency of the crystal. For the AT-cut quartz crystal (F = 9 MHz, N = 167 kHz cm, p = 2.65 g cma), numerical substitution in eq 1yields AF = -0.183AM (2) where AM = Am/A and the units of AF and AM are hertz and nanogram per square centimeter, respectively. A was 0.196 cm2 for the quartz crystal used in this study. Thus, if a substance with the weight of 1ng is homogeneously attached to the active area of crystal, a frequency change of 0.933 Hz occurs; the adsorption per unit area (AM = 5.1 ng cm-2) leads to a frequency change of about 1 Hz. The QCM has also been used as an immunosensor to detect antibodiea,Uhaptens,143protein antigen,'5g and microbee.Most of these analytes have been monitored in the liquid phase; antibodylantigen binding quartz crystals bound antigen/ antibody in liquid phase to change the oscillating (10)Sauerbrey, G. 2.Phys. 1959,155,206-222. (11) Ward, M. D.;Buttry, D. Science 1990,249,1000-1007. (12)Buttry, D. A. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1991; Vol. 17, pp 1-86. (13)Shone, A.;Dorman, F.; Najarian, J. J. Biomed. Mater. Res. 1972, 6,565-570. (14)Ftajakovic, L.; Ghaemmaghami, V.;Thompson, M. Anal. Chim. Acta 1989,217,111-121. (16)Ngeh-Ngwainbi, J.; Foley, P. H.; Kuan, S. S.; Guilbault, G. G. J. Am. Chem. SOC.1986,108,5444-5447. (16)Roederer, J. E.;Bastiaana, G. J. Anal. Chem. 198.9,55,2333-2336. (17)Davis, K. A.; Leary, T. R.Anal. Chem. 1989,61,1227-1230. (18)Thompson, M.; Arthur, C. L.; Dhaliwal, G. K. Anal. Chem. 1986, 58,1206-1209. (19)Prueak-Sochaczewski, E.;Luong, J. H. T. Anal. Lett. 1990,23, 401-409. (20) Prusak-Sochaaewski, E.;Luong, J. H. T.; Guilbault, G. G. Enzyme Microb. Technol. 1990,12,173-177. (21)Muramatau, H.; Kajiwara, K.; Tamiya, E.; Karube, I. Anal. Chim. Acta 1986,188,267-261. (22)Muramatsu, H.;Watanabe, Y.;Hikuma, M.; Ataka, T.; Kubo, I.; Tamiya, E.;Karube, I. Anal. Lett. 1989,22,2166-2166. 0 1993 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 20, OCTOBER 15, 1993
Reservoir
Flgure 1. Schematic diagram of experlmental system. Various solutionswere appliedto the oneside-sealedpieroelectrlcquartz crystal
by a peristaltlc pump. The electric clrcuit for measuring oscillation ~ ~ a universal counter frequency was as described p r e v i ~ u s l y ,using controlled by a microcomputer. TTL, translstor-transistor logic clrcuit.
frequency. It is unclear at present whether lAF/Ah4/=0.183 Hz n g ' cm2,derived from the above theory, is true or not in a piezoelectric immunosensor inserted in a liquid phase. Human serum albumin (HSA) was detected with quartz crystals which bound antihuman serum albumin (anti-HSA) with protein A or polyethylenimine coated on their surfaces. However, this method required a drying stage in air, after HSA bound to the antibody.l'J In the present paper, HSA is detected with a quartz crystal physically precoated with antiHSA and fixed in a flow cell. This system does not required the drying process. The sensitivity of this immunosensor is enough to monitor microalbuminuria. Moreover, we estimate the value of IAF/M.I( in this immunosensor using radioisotopelabeled anti-HSA or HSA. EXPERIMENTAL SECTION Materials. The following materials were obtained as indicated: anti-HSA [developed in rabbit, immunoglobulin G (IgG) fraction] from Sigma ChemicalCo.; human serum albumin from Gland-Pharm; bovine serum albumin (BSA) from GIBCO Laboratories Life Technology,Inc.; AT-cut piezoelectric quartz crystals (9 MHz resonance frequency, 8 8 mm) from Yakumo Tsushin Co.; NalsI from Amersham; protein assay kit from BioRad Labs. Other chemicals used were certified reagents purchased from Wako Pure ChemicalCo. A 50 mmol L-l phosphatebuffered saline, pH 7.5 (formedby mixing NazHPOl and KHzPOd solutions), containing 0.9% NaCl and 0.1% NaN3 (referred to as PBS) was used as assay medium for the piezoelectric immunoassay. Piezoelectric Immunoassay. One side of each piezoelectric crystalwaa sealed with a quartz plate and siliconadhesive (Konishi Co.); thistreatment stabilizesthe oscillation frequencyin solution, as described previously.% The one-side-sealed piezoelectric quartz crystal was set inside a glass flow cell (7-mL volume) thermostated at 25 & 0.1 OC with circulating water and was inserted in a laboratory-made TTL circuit (Figure 1). PBS was passed over the crystal at a flow rate of 7 mL min-l with a peristaltic pump (Model P-1, Pharmacia) and the flow cell thoroughly filled with PBS. The flow was stopped in order to stabilize the oscillation frequency. After stabilization, solutions of antibody or of antigen diluted with PBS were introduced at the same flow rate. After the solution in the cell was completely substituted with the protein solution, the flow was stopped for stabilization. During this process, the oscillation signal was automatically fed to a universal counter (ModelSC-7201,Iwatau Electric Co.) and the frequencychangestored in a microcomputer (Model PC-286U, Epson Co.). Determination of the Amount of Protein Adsorbed onto the Surface of the Piezoelectric Quartz. The amount of antiHSA or HSA adsorbed onto the surface of the piezoelectricquartz was estimated using a radioisotope method. (23) Kurosawa, S.; Tawara, E.; Kamo, N.; Kobatake, Y.Anal. Chim. Acta 1990,230,4149.
Anti-HSA and HSA were iodinated with NalBI by a modification of the chloramine-T method of Bolton and Hunter.%pB The labeled proteins were separated from the free iodine by dialysis, and the dialysis was performed until the radioactivity of the outer solution approached a background count. After the addition of trichloroacetic acid solution to the small amount of dialyzate, the supernatant was separated from the precipitate by centrifugation and the radioactivity of supernatant and of precipitate was measured. The percentage incorporation of radioactivity into anti-HSA and HSA was 97.2 and 97.6%, respectively. The specific radioactivities of [l=I]anti-HSA and [lZI]HSA were 2.1 X 108 and 1.2 X 108 cpm pg-l, respectively. Protein concentration was determined with the Bio-Rad protein assay kit, which contains a bovine y-globulin standard and a BSA standard. The adsorption of labeled anti-HSAwas performed as follows. A silicon seat (thickness, 0.5 mm), with a hole of almost the same size as the electrode on the surface of the crystal, was stuck on thepiezoelectricquartzcrystalwithanadhesive,formingashallow well over the electrode. This ensured the electrode would only make contact with the protein solution. Labeled anti-HSA (40 pL) waa added to the well. Each solution was prepared by the dilution of stocked labeled anti-HSA (209.7 pg mL-1) with PBS. After the well was allowed to stand for 10 min at room temperature, the solution was removed. This stage was carried out three times, in order to compensate for the decrease of concentration of protein due to the adsorption onto the electrode. After the hot solution was removed, the well was washed three times with PBS in accordance with the general immunoassay washing method. After washing, the cryetal was transferred to a test tube, and the radioactivity of the crystal was counted in a y counter (Model5320,Packard). The presence of crystal had no influence on counting efficiency. The amount of labeled antiHSA adsorbed was calculated using the specific radioactivity. This procedure was repeated withvarying concentrations of antiHSA. For the determination of the amount of labeled HSA adsorbed onto the electrode on the crystal, the same procedure was carried out, except that anti-HSA-precoated electrodes were used and the adsorption site not covered with anti-HSA was blocked with BSA. Nonspecificadsorption of labeled HSA was estimated using the electrode precoatedwith anti-C3a instead of anti-HSA. AntiC3a refers to antihuman C3a fragment antibody (IgG fraction), which was developed in rabbit, containing no anti-HSA. The amount of labeled HSA bound was calculated using the specific radioactivity. The experiment was performed in duplicate.
RESULTS AND DISCUSSION Adsorption of Anti-HSA onto Piezoelectric Quartz Crystal. Figure 2 shows the typical change of oscillation frequency when anti-HSA solution flowed over the one-sidesealed piezoelectric quartz crystal. Substituting anti-HSA solution for PBS led to a reduction in frequency. After the flow of the protein solution wm stopped,the frequency change stabilized within about 20 min. T o remove unbound antiHSA, PBS was added until complete substitution occurred. After stabilization of frequency change, the difference (AF) between this stabilized level and the original level was read. This difference is due to the adsorption of anti-HSA onto the crystal. Proteins are predominantly adsorbed onto the surface of substrata that have a relatively small surface energy (7). y is smaller as the contact angle (e) is larger. Absolom et al.26 showed IgG was well adsorbed onto Teflon (y = 20.0 erg cm-2, (24) Bolton, A. E.; Hunter, W. M. In Handbook of Experimental Immunology, Vol. 1. Immunochemistry; Weir, D. M., Herzenberg,L. A,, Blackwell, C., Herzenbrg, L. A., E&.; Blackwell: Oxford, UK, 1986; pp 26.1-26.56. (25) Muratsugu, M.; Kuroeawa, S.; Kamo, N. J . Colloid Interface Sci. 1991,147, 378-386. (26) Absolom, D. R.; Zingg, W.; Neumann, A. W. J. Biomed. Mater. Res. 1987,21, 161-171.
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Flgure 2. Typical frequency change on the additlon of anti-HSA to the crystal. AntCHSA solutlon (200 pg mL-l) was applied (a), and after 4 mln the flow was stopped (b). Then, after 30 mln PBS was applied (c), andatter 4 mln the flow was stopped (d). After 30 mlnthe frequency value was read and the difference of this level and the orlglnal level (In PBS solution)was definedas the osclllatlngfrequency change (AF).
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8 = 108') or siliconized glass (18.7 erg cm-2 and looo,
respectively). The surface of the silver electrode on the crystal, which is generally regarded as an active area, has e = 98'. This value was determined using a drop of water resting on the silver electrode. Hence, the silver surface was able to adsorb anti-HSA (IgG fraction). It was confirmed that the adsorption of anti-HSA onto the crystal was responsible for AF, as follows. The frequency changes (AF)were measured under varying concentrations of anti-HSA. These results are presented in Figure 3. AF depended on the concentration of anti-HSA ranging from 1to lo00pg mL-' but reached a plateau level at more than 100pg mL-1, which indicated the existence of adsorption equilibrium. Binding of HSA to Anti-HSA-Coated Piezoelectric Quartzcrystal. Figure4showsthe typicalfrequencychange on application of BSA or HSA to the piezoelectric quartz crystal precoated with anti-HSA. A one-side-sealed quartz crystal was immersed in 1.6 mg mL-l anti-HSA for 12 h at 4 "C. The quartz crystal was then washed with PBS and set
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CONCENTRATION OF HSAOR BSA/rg m l - ' Flguro 5. Relationship between the concentrationof HSA 01 BSA and frequency change (AF): e,HSA; . BSA. , The vertical bars designate the standard devlatlon from the mean of the experiments denoted In the graph.
in the flow cell. PBS flowed into the cell, and an oscillating frequency was stabilized as described above. i n order to inhibit the nonspecific adsorption of protein, 100 pg mL-1 BSA was subsequently added into the cell, lowering the frequency. After stabilization of the frequency the protein solution was replaced with PBS, causing no change in frequency. This is the so-called 'blocking" procedure in radioimmunoassay, enzyme immunoassay, etc. Thereafter, substituting 10pg mL-1 BSA for PBS solution did not change the oscillating frequency. In contrast, replacing PBS with 10 pg mL-' HSA decreased the frequency. The subsequent replacement of HSA with PBS did not cause a frequency change. These resulta indicated that HSA specificallybound on the quartz crystal through anti-HSA. When HSA or BSA of varying concentrations was applied to the anti-HSA-precoated crystal. AF was plotted against the protein concentration (Figure 5). A F occurred at more than 0.1 pg mL-l HSA, increased with ita concentration, and reached a plateau when the concentration was over 100 pg mL-1. However, the application of BSA caused little frequency change between 10 and 100 pg mL-1. These results indicate that the anti-HSA-coatedpiezoelectricquartz crystal works as an immunosensor which can specificallydetect HSA in the presence of BSA. The crystals precoated with a thin layer of (glycidoxypropyl)trimethoxysilane,(yaminopropy1)triethoxysilane,27 p~lyethyleneimine,'~*~ and other filmshave often been used for immobilization of antibody, but it was found that, without these thin layers, the silver electrode itself was effective for immobilization. HSA in the range 0.1-100 pg mL-l could be detected with the present piezoelectric immunosensor. The normal concentration of HSA in the urine of humans is 8.84 f 9.15 pg mL-1-28 In order to detect microalbuminuria,highly sensitive methods which can measure microgram per milliliter levels are required. Such microgram quantities of albumin have generally been detected with RIA or ELISA. Labeling antibodytantigen with radioisotopes or enzymes is important in these assays, but such a labeling procedure is not necessary for this piezoelectric immunosensor. Moreover, this immunosensor does not require a second labeled antibody, which immunoradiometric assay (IRMA) and ELISA do; only the first antibody is required for this immunosensor. The piezoelectric immunosensor is sensitive enough to monitor microalbuminuria, and we believe this to be a useful method for the clinical laboratory. Relationship between AFand Adsorption of Anti-HSA or HSA on the Surface of the Crystal. We also thought (27) Muramatsu, H.;Dicks, J. M.;Tamiya,E.; Karube, I. Anal. Chem. 1987,59, 2760-2763. (28) Yamagata, K.;Oh,Y.; Nakagawa, S.;Miyazaki, M.; Koyama, T. Clin. Rep. 1991,25,4465-4471.
ANALYTICAL CHEMISTRY, VOL. 85, NO. 20, OCTOBER 15, 1993
2096 N
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CONCENTRATION OF lXI- ANTI-HSA/ pg ml Flgure 6. Adsorption of [ 1*61]anti-HSA onto the crystal. The vertical bars designate the standard deviation from the mean of the duplicate experiments. I
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CONCENTRATION OF 1251-HSA/pg ml-' Flgure 7. Adsorption of [1251]HSA to the crystal precoated with antiHSA. The crystal was precoated with 1.4 mg mL-l anti-HSA. Adsorption of [ 1251]HSAwas corrected by subtracting the nonspecific binding to the crystal precoated with anti-C3a (2.0 mg mL-l). The vertical bars designate the standard deviation from the mean of the duplicate experiments. it desirable to use radioactive-labeling methods to evaluate the validity of lAF/w= 0.183 Hz n g l cm2,as Muramatsu et al. have shown.22 We obtained this value when assuming that only the region under the electrode is piezoelectrically active; at the bare quartz the mass sensitivity is negligible.lOiB In this study, since the one-side-sealedpiezoelectric crystal was inserted into the flow cell, both sides of the crystal were in contact with the surrounding solution. If isotope-labeled protein were applied to such a system, labeled protein could be attached to the area beside the active area of crystal. This procedure accordingly may lead us to underestimate the value of JAF/hMJ. To avoid this problem, we employed a system in which labeled protein made contact with the active area only, as described in the Experimental Section. The relationship between the amount of labeled anti-HSA adsorbed and its concentration is presented in Figure 6. The adsorption of labeled anti-HSA increased with its concentration. AF increased as the concentration of anti-HSA increased (Figure 3). These results supported that AF increased following the increase of adsorption of anti-HSA onto the crystal. The relationship between the amount of labeled HSA adsorbed and its concentration is illustrated in Figure 7. Because nonspecific binding of labeled protein generally occurs in immunoassay, we considered that the binding of labeled HSA to the anti-C3a-precoated crystal was nonspecific. The data in Figure 7 are corrected for this nonspecific amount. The relationship between labeled HSA bound and its concentration remarkably resembled that between @and (29)Ullevig, D.M.;Evans, J. F.; Albrecht, M. G. Anal. Chem. 1982, 54,2341-2343.
300
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'%I- ANTI-HSA OR ,ng cm-2 lXI-HSA ADSORBED Flgure 8. Relationship between antCHSA or HSA adsorbed and , HSA. frequency change (AF): 0, anti-HSA;. the concentration of HSA in Figure 5, implying that AF depends on antigen-antibody reaction. The amount of anti-HSA adsorbed and AF were read at the anti-HSA concentrations of 1-56 pg mL-l(l5 points) in Figures 6 and 3, respectively. For HSA, the values of the adsorbed amount and A F were read at 0.1-21 pg mL-1 HSA (10 points) in Figures 7 and 5, respectively. Within these ranges of concentration, the adsorption and AF increased with concentration. These values were plotted in Figure 8. In this study, in order to estimate lAF/w, the linear regression lines were drawn as shown in Figure 8. The slope, "regression coefficient", was referred as to IAF/AiUl. (AF/ for anti-HSA and HSA were 0.375 f 0.0124 (n = 15) and 0.716 f 0.0660 (n = 10) Hz n g l cm2, respectively, and were significantly larger than the theoretical value, 0.183 Hz ng-1 cm2 (P < 0.01). Davis and Leary developed a piezoelectric biosensor that detected human IgG and antibodies to IgG.I7 The addition of 10 ng of immunoglobulincaused a frequency change of ca. 1 Hz. calculated from the data is smaller than the theoretical value, but since AM is not directly determined, lAF/wmay be underestimated. Muramatsu et al.27 also determined immunoglobulin concentrations using a piezoelectric biosensor and observed that frequencychanges owing to the addition of IgG were greater than the value predicted from eq 2; i.e., I A F / wis larger than the theoretical value, which contradicts the observationsof Davis and Leary. Since AMis not directly determined here, the possibility of its value being underestimated, as above, cannot be excluded. In our study, we observed a significantly larger value of than the theoretical value (P < 0.011, and it is noteworthythat AM was estimated directly by the radioactivelabeling method. This result indicates the presence of factors which cause a frequency shift as well as a mass change. Bruckenstein and S h a P and Kanazawa and Gordon3'v!j2 derived a simplerelationship between the changein frequency of piezoelectric crystal and the viscosity and density of liquid on the operation of a crystal in a liquid phase. Some recent studies have revealed that the oscillation of a quartz crystal in the liquid phase is governed by some interfacial factors: surface r0ughness,3~,~ surface stress,% interfacial slip,%
w's
lM/w
(30)Bruckenstein, S.;Shay, M. Electrochim. Acta 1985, 30, 12951300. (31)Kanazawa, K. K.; Gordon, J. G.,I1 Anal. Chem. 1985,57,17701771. (32)Kanazawa, K. K.;Gordon, J. G.,IIAnal. Chem. 1985,175,99-105. (33)Schumacher, R.Angew. Chem.,Znt. Ed. Engl. 1990,29,329-343. (34)Schumacher,R.;Borgea, G.; Kanazawa, K. K. Surf. Sci. 1985,163, L621-L626. (35)Heusler, K. E.;Grzegorzewski, A; Jaeckel, L.; Pietrucha, J. Ber. Bunsengee. Phys. Chem. 1988,92,1218-1225. (36)Duncan-Hewitt, W.C.;Thompson,M. Anal. Chem. 1992,64,94105.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 20, OCTOBER 15, 1993
hydrophilicity or hydrophobicity,ls etc. In the present work, it appears that the adsorption of protein onto the surface of a quartz crystal can substantially alter the properties of the surface, leading to a change in oscillating frequency besides that induced by the increase in mass owing to the adsorption of protein, although it is unclear which of the surface properties is a main factor at present. In addition, IAF/AMl for HSA bound to the crystal surface through anti-HSA was significantly larger than that for antiHSA on the crystal surface (P < 0.01). The protein assay was performed with a dye-binding method. Because the dye color development is greater with albumin than with IgG, bovine y-globulin and BSA were used as a standard for the determination of the concentrations of anti-HSA and HSA, respectively. The species dependence in color development is negligible (a personal communicationfrom Bio-Rad Labs.). This result also supports the finding that interfacial factors relate to the oscillating frequency change and may be associated with the following facts. Thompson et al.l* observed the differencebetween hydrophilic and hydrophobic surfaces in the time-dependent behavior of oscillating frequency and pointed out that the frequency change caused by antigen-antibody reactions might relate to the increased hydrophilicity during the reaction. The class of anti-HSA used in our work was IgG. Digestion of IgG with pepsin produces F(ab’)z and Fc fragments. The latter fragment is less soluble in water than the former. Thus, IgG has two domains on a molecule; one is hydrophilic and the other is hydrophobic, and each domain is localized on the molecule.37 In contrast, HSA, a hydrophilic protein, does not have such domains. In this study, we estimated the value of lAF/W on the assumption that the mass sensitivity is negligible near the electrode boundary. However, it has been reported that the mass sensitivity was observed beyond the electrode boundary in liquid phase.If the active area is beyond the electrode boundary, the active area consists of different regions; the surface of the electrode (A&,which is silver, and of the bare crystal (A,). Thus, the adsorption rate of protein to these substrata should be considered. Equation 2 can be rewritten as follows:
A F = -0.183
+ MA 4 + Ac
(3)
where M. and AMc are the adsorption of protein per unit (37) O K o , T.;Niahiyama, S.; Shinohara, I.; Akaike, T.; Sakurai, Y. Kobunshr Ronbunshu 1979,36,209-216. (38) Martin, B. A.; Hager, H. E. J. Appl. Phys. 1989,65,263&2635. (39) Ward, M. D.; Delaweki, E. J. Anal. Chem. 1991,63,886-890. (40) Hillier, A. C.; Ward, M. D. Anal. Chem. 1992,64,2539-2554. (41) Kuroaawa, S.; Muratsugu, M.; Kamo, N. Polym. Adv. Technol. l991,2,253-259.
2997
area of electrode and of bare crystal, respectively. Introduction of AiU, = AiUe dM into eq 3 leads to
*
(4)
If we assume that the adsorption of protein per unit area is the same between the bare crystal and the electrode, dM = 0, eq 4 is identical to eq 2, demonstrating that lAF/AMl, experimentally obtained here, does not alter. The adsorption rate of antibody (IgG) may be different between a quartz crystal and a silver electrode, because the adsorption of the F(ab’)2 fragment of IgG to silver is about 3 times as large as that to g l a s ~ . ~Thus, , ~ ~ if the adsorption rate is different between the substrata, dit4 # 0, JAF/M.IJwould differ from that obtained when one assumes that the active area is limited under the electrode. The maas sensitivity exhibits a Gaussiantype distribution and is greatest at the center of the electrode, decreasing monotonically toward the electrode edge. Thus, the mass sensitivity beyond the electrode boundary for the adsorption of antibody may not be so large. In addition, A, is much smaller than A,. Therefore, the contribution of the second term of eq 4 to a frequency change may be shall. If the exact active area and the adsorption rate of antibody for the bare crystal and the electrode can be estimated, we will be able to determine lAF/AMl more accurately. This will be investigated further. However, it should be noted that even if the active area is accurately determined, JAF/AMlfor HSA will still be larger than that for anti-HSA, as is evident from the above discussion. In conclusion, this piezoelectric immunosensorsufficiently sensitive to detect microgram quantitiea of albumin and has a high specificity for HSA. This immunosensor should be considered for clinical use in future. Values of IAF/AMl for some proteins are larger than the value predicted with Sauerbrey’s equation. In addition, IAFIAMl for the immunoadsorption of HSA is larger than that for the physical adsorption of anti-HSA. This result appears to relate to the hydrophilicity or hydrophobicity of proteins.
ACKNOWLEDGMENT We are grateful to Dr. K. Takahashi, Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, for a gift of anti-C3a. This work was supported partly by a grant from the Ministry of Education, Science and Culture (Japan) and from the SuzukenMemorial Foundation.
RECEIVED for review March
24, 1993. Accepted July 22,
1993.’
* Abstract published in Advance ACS Abstracts, September 1,1993.
