Anal. Chem. 1995, 67,2212-2215
Quartz Crystal Microbalance Bioaffinity Sensor for Biotin Mbr Mbrruwr, Kyusik Yun, Tetsuya Haruyama, E i y Kobatake, and Masuo Aizawa* Department of Bioengineering, Faculty of Bioscience and Biotechnobgy, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226, Japan
A quartz crystal microbalance (QCM) bio-ty sensor for biotin, basedon the bioafbinitycomplexbetween avidin and immobilized desthiobiotin, was established. BSAdesthiobiotin conjugate was adsorbed directly onto the gold layer surface of a quartz crystal. 'Ihe crystal was placed in a QCM flow cell. Avidin formed a metastable complex with the BSA-desthiobiotin conjugate on the crystal surface. When a sample solution containingbiotin was injected, the avidin was released from the surface to form a more stable complex with biotin in solution. 'Ihe biotin concentration corresponded to the weight change on the crystal surface that occurredwhen avidin desorbed. Measurements were done in the stopflow mode with a measurement range between 0.05 and 100 &mL and in the continuous-flow mode with a measurement range between 0.2 and 10 000 p g / d The sensor was regenerated without any loss of activity by readsorbing avidin. Two types of biosensors have been developed which involve a biocatalytic sensor and a bioaf6nity sensor.' A biocatalytic sensor is represented by an enzyme sensor, which consists of an enzyme layer on a signal transducing device. Enzyme sensors have been forwarded into third generation of biosensors due to innovative progress. A constant variety and novel principles and technologies have emerged in the development of bioaffinity sensors which is profoundly stimulated by an enormously increased interest and demand. Bioafhity sensors, which are based on the bioafhity binding reaction between a ligand and a biomolecule, have a potential for a much wider range of applications than enzyme sensors. Such sensors can utilize natural bioafhity molecules as well as a specific antibody, which is raised for a specific analyte, whereas enzyme sensors are limited in number. Most of the research efforts in biosensors have been directed toward pursuing signal-transducing principles based on spectre photometric and electrochemical methods. It is, however, d & d t to measure the affinity binding reaction of biomolecules directly and therefore a labeling molecule has been used, and in the case of electrochemicalbiosensors, the labeling molecule is either an or an electrochemicallyactive g r o ~ p Although . ~ ~ ~ these bioaffinity sensors are characterized by high selectivity and (1) Aizawa, M. Anal. Chim. Acta 1 9 9 1 , 2 5 0 , 249-256. (2) Aizawa, M.; Morioka, A; Matsuoka, H.; Suzuki, S.; Nagamura, Y.; Shinohara, R; Ishiguro, I. J. Solid-Phase Biochem. 1 9 7 6 , 1, 319-328. (3) Aizawa, M.; Morioka, A; Suzuki, S. j.Membr. Sci. 1 9 7 8 , 4, 221-228. (4) Aizawa, M.; Morioka, A; Suzuki,S. Anal. Chim. Acta 1 9 8 0 , 115, 61-67. (5) Gleria, K D.; Hill, H. A 0.;McNeil, C. J.; Green, M. J. Anal. Chem. 1 9 8 6 , 58, 1203-1205. (6) Yao, T.; Rechnitz, G. A Biosensors 1 9 8 8 , 3, 307-312.
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Analytical Chemistty, Vol. 67,No. 13, July 1, 1995
sensitivity, they suffer from repeated use because of stable bioaffinity complexation. In our group, we have proposed bioaffinity sensors based on subtle differences in the bioaffinity between an analyte and an analogue compound to the binding protein. These are easily regenerated for continuous measurement and are specifically attractive for the detection of small molecules like vitamins, hormones, and drug compounds. We have reported an electre chemical sensor for biotin based on the displacement of enzymelabeled avidin7and a sensor for thyroxine based on the displacement of enzyme-labeled antibody.s A similar bioluminescence bioafhity sensor for insulin based on the displacement of enzymelabeled antibody was also r e p ~ r t e d . ~ The binding of biomolecules can also be measured by methods which are sensitive to the physical changes that occur on a surface when a biomolecule binds. Such sensors can be based on the changes in the dielectric properties of the surface, which can be detected by impedance.1° surface plasmon measurements," or weight changes on the surface, which can be measured directly or by a quartz crystal microbalance (QCM). The QCM is an oscillating quartz crystal device whose resonance frequency changes with a change in the mass according to the formula12
4=- f o 2 ( 2 / G ) A m
= -fo22.3 x 10-6Am
where 4fis the observed frequency change (in Hz),pq is the shear modulus, es is the density of the crystal, and Am is the weight change on the surface of the crystal (in g cm-9. Recently,I3 it was shown that the method is suitable for sensitive measurements in liquids as well as in air, and one of the advantages of the method is that there is a clear mathematical relationship between the adsorbed mass and the observed frequency change. Several workers have reported on the biospecific adsorption of proteins where the observed frequency change is in the 10-100 Hz order,'4-'6 but to measure the binding of small molecules is more dficult. The binding of a small (7) Ikariyama, Y.; Furuki, M.; Aizawa, M. Anal. Chem. 1 9 8 5 , 57, 496-500. (8) Ikariyama, Y.; Aizawa, M. Proc. 2nd Sensor Symp. 1982,97-100. (9) Ikariyama, Y.; Aizawa, M. Proc. 3rd Sensor Sump. 1 9 8 3 , 14-20. (10) Bataillard, P.; Gardies, F.; Renault, M. J.; Martelet, C.; Colin, B.; Mandrand, B. Anal. Chem. 1 9 8 8 , 60, 2374-2379. (11) Jonsson, U.; Fagerstam, L.; Ivarsson, B.; Johnsson, B.; Karlsson, R; Lundh, IC; Lofas, S.; Persson, B.; Roos, H.; Romberg, I.; Sjolander, S.; Stenberg, E.; Stahlberg, R.; Urbaniczky, C.; Ostlin, H.; Malmqvist, M. Biotechniques 1 9 9 1 , 11, 620-627. (12) Sauerbrey, G. Z. Phys. 1 9 5 9 , 155, 206. (13) Bruckenstein, S.; Shay, M. Electrochim. Acta 1 9 8 5 , 30, 1295-1300. (14) Ebersole, R C.; Ward, M. D. j.Am. Chem. SOC.1 9 8 8 , 110, 8623-8628. (15) Muratsugu, M.; Ohta, F.; Miya, Y.; Hosokawa, T.; Kurosawa, S.; Kamo, N.; Ikeda. H. Anal. Chem. 1 9 9 3 , 65, 2933-2937. 0003-2700/95/0367-2212$9.00/0 0 1995 American Chemical Society
Gold coated crystal
c3
: Avidin
D
0
:Desthiobiotin
Crystal
: BSA
B
:Biotin
Figure 1- Principle of the QCM bioaffinity sensor based on a metastable bioaffinity complex.
.molecule (MW 5 500) to a closed packed protein (diameter 2 10 nm) with a single binding site will give a weight change of 1ng cm-2 or less. Calculation with the above formula shows that the observed frequency change of a 6 MHz-type crystal will be no more than 0.09 Hz,which is too small for detection. In the present work, a QCM bioafhity sensor for biotin has been developed. Biotin (MW 244,vitamin H)forms a very stable complex with avidin. Avidin also binds analogue compounds of biotin such as 2-[ (4hydroxybenzene)azoJ benzoic acid (HABA), lipoic acid, and desthiobiotin. The sensing principle is shown schematically in Figure 1. Avidin binds BSA-desthiobiotin conjugate immobilized on the gold electrode layer covering the quartz crystal surface. When an analyte containing biotin is injected into the cell, the avidin might be desorbed from the surface as the binding to biotin is much stronger than the binding to desthiobiotin. The positive frequency change could then be proportional to the weight of avidin (MW 68 OOO) desorbed. It is noted that the measuring system could be regenerated under very mild conditions simply by reloading avidin. Fast analysis could be obtained with the continuoudlow conditionsalthough the stop flow conditions offered a more sensitive assay, as the reaction times were longer. EXPERIMENTAL SECTION
Materials. D-Biotin, DLdesthiobiotin, lipoic acid, 24 (4hydroxybenzene)azoJ benzoic acid (HABA) and N-hydroxy succinimide (NHS) were supplied by Nacalai Tesque, Tokyo, Japan. P-Nitrophenyl phosphate (PNPP), DMSO, and avidin were purchased from Wako Chemicals, Osaka, Japan. MgCl2 came from Kanto Chemicals, Tokyo, Japan. l-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) was purchased from Dodjindo Labs, avidin-labeled alkaline phosphatase from EY Laboratories, San Mateo, CA, and BSA from Seikagaku Corp., Tokyo, Japan. BSA-Iigand Conjugates. EDC, NHS,and the ligand (biotin, desthiobiotin, lipoic acid, or HABA) were dissolved in 1 mL of DMSO to give a 40 mM solution for each compound. The solution was stirred for 13 min and then 1mL of 0.1 M carbonate buffer, pH 8.5, containiing 30 mg of BSA was added. Precipitated protein was removed by centrifugation after a 4.7 h reaction at 37 "C. The organic solvent was removed from the remaining solution by ultrafiltration with Centricon (Amicon Inc.), concentrators and (16) Ebato, H.; Gentry, C. A; Heron, J. N.; Muller, W.; Okahata,Y.; Ringsdorf, H.; Suci, P. A Anal. Chem. 1994, 66,1683-1689.
4.8 cm Figure 2. Cross section of the QCM flow cell.
the remaining reagent molecules were removed by dialysis in 0.1 M phosphate buffer, pH 7.5. BSA-biotin and BSA-desthiobiotin conjugateswere also made by overnight reaction in solutions with 50 mM reagent concentrations and 17 mg/mL BSA, containing 10%DMSO. The protein recovery was better with this method but the binding of avidin-alkaline phosphatase (Av-AP) was unchanged in the enzyme binding assay. Assay for Iigand Content The biotin and desthiobiotin content were determined by gradually adding 1p L portions to 750 pL of 0.05 M phosphate solution containing 0.5 mg/mL avidin and 0.25 mM HABA, and the reduction in absorbance at 500 nm was recorded. Desthiobiotin standard solution was also measured, and the desthiobiotin content of the protein was calculated. The HABA content was calculakd from the absorbance at 348 nm, where the HABA absorption peak is. The protein concentration was calculated from the absorbance at 280 nm, using a BSA standard. Enzyme Binding Assay. The 0.16 cm2 gold plates were cleaned by sonication for 1h in a 2:l ratio mixture of H2S04 and H202 followed by rinsing with water. The plates were incubated for 1 h in a BSA-ligand solution, at 70 "C, rinsed with buffer, and placed in ELISA plate wells. The following incubations were done at 37 "C. The plates were incubated for 1 h in a 0.1 M phosphate buffer, pH 7.5 (PBS), containing 0.1 mg/mL avidin, and washed three times with buffer. Then they were treated with biotin solutions with 1h before 1h incubation in 20 pg/mL AvAP in pH 7.4,25 mMTris buffer, 2 mM ZnClz, 4 mM MgCl2 ('IRIS 7.4) solution containing 1%BSA. When assayed for Av-Ap binding, only the last incubation was done. The enzyme reaction was carried out for 1h in 1M DEA, 0.5 mM MgCl2, pH 9.8 (DEA9.8), containing 1 mg/mL PNPP. The reaction was stopped by transferring the solutionsfrom the wells with the gold plates. The absorbance of the solutions was measured at 405 nm with a BioRad Model 450 microplate reader. QCM Measurement The QCM flow cell was made by fitting the QCM cell supplied by Hokuto Denko (Tokyo, Japan) with a rubber stopper and glass tubes as shown in Figure 2. The cell was set up in a flow system in a water bath and connected to an ECQCM Controller HQ-1O1B (Hokuto Denko). The crystal was an AT-cut 6 MHz crystal supplied by Hokuto Denko. Analytical Chemistry, Vol. 67, No. 13, July 1, 1995
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Injection in the stopflow mode was done by pumping the solution into the cell for 3 min, with a 1.5 mL/min flow rate, and then stopping the flow. The frequency was recorded 20 min after the start of the injection, and the injection of next sample was started. M e r the BSA-desthiobiotin-coated crystal had been placed in the cell, the unspecific binding was blocked by two injections of 100 pg/mL BSA One regeneratiodmeasurement cycle constituted injection of a 100 pg/mL solution of avidin, injection of PBS buffer, injection of the biotin sample, and a final injection of buffer; the next cycle was started by injection of the avidin solution. The cell was connected to a 1 mL injection loop (from Rheodyne) when operated in the continuouS-nowmode. The flow rate was 0.5 mWmin. M e r blocking of the crystal with BSA, a 500 pg/mL avidin sample was injected, and 10 min later the frequency was recorded and a biotin sample was injected. Ten minutes later the frequency was recorded again and the next regeneration/measurement cycle started by injection of the avidin solution. The difference between the two frequencies was the frequency shift caused by desorption of avidin by biotin. RESULTS AND DISCUSSION
Enzyme Binding Assay and Immobilization Procedure. In the cell supplied by Hokuto Denko, the crystal is sandwiched between two O-rings. With this cell design, a good stability was eventually achieved with less than 1 Hz drift in 1 h. However, just after a new crystal was fitted in the cell it took up to several hours for the frequency to stabilize until the measurement could be started, and it was therefore difficult to test many different conditions for the immobilization of ligand on the gold surface. A different type of cell was also tested where the O-ring was only on one side. In this case, a different pattern was observed where it generally took less than 1h for the frequency to stabilize initially but the subsequent drift was much higher than with the present design. The cell was therefore not used for further measurements. In order to test different immobilizationprocedures,an enzyme binding assay for the binding of Av-AP to modfied gold plates was therefore investigated. Many different conditions could be tested in one assay, and this allowed the optimization of the conditions for the bioaf6nity sensor before starting the experiment with the QCM. Various procedures were tried, most based on thiol monolayer, and subsequent reactions to link biotin to the surface, but the best results in the enzyme binding assay were obtained when ligand-conjugated BSA was directly adsorbed to the gold surface. The advantage of this procedure was that, after the preparation of the BSA-ligand conjugate, the modfied crystal could be prepared simply in a single adsorption step. High temperature (70 "C) was used for the adsorption reaction in order to maximize the binding, but denaturing of the protein should not affect the avidin binding. Enzyme binding results were not significantly different even if lower temperatures were used. The protein concentration of the conjugate in most of the experiments was 90 pg/mL but changing the concentration in the range 9-500 pg/mL did not affect the results. Saturation binding of Av-Ap in the binding assay was obtained with 10 pg/mL concentrations or higher, but 20 ,ug/mL was generally used in the experiments. Ligand Variation and B i o e t y Assay for Biotin. The ligand to BSA ratio in the BSA-ligand conjugate for desthiobiotin and biotin was measured by a HABA displacement assay. Lipoic 2214 Analytical Chemistv, Vol. 67,No. 13, July 1, 7995
Table I. Llgrndr per Molecule and Relative Binding in the Enzyme Blndlng A88ay
relative binding of Av-AP in ligands per KA for binding of the enzyme molecule ligand to avidinu binding assay molecule BSA-biotin 4.7 1 1015 0.99 k 0.05 BSA-desthiobiotin 3.5 2 x 1012 1.00 f 0.13 BSA-lipoic acid 1.4 x lo6 0.14 f 0.05 BSA-HABA
4.1
1.7
105
0.06 f 0.02
From ref 17. acid is, unlike HABA, only weakly adsorbing in the visible range and ligand content could therefore not be measured with a direct absorption measurement. The reaction conditions was the same as in the case of HABA, so similar BSA to ligand ratios should be expected. There were on average 3.5-4.7 ligands bound to each BSA molecule Vable 1). When the molecule is adsorbed on the surface, there should be at least one ligand available for binding to avidin, and nearly a monolayer binding of avidin should be possible. The same binding of Av-AP was observed with immobilized biotin and desthiobiotin, which is consistent with the high affinity constant that avidin has for binding of these compounds. The avidin-ligand complex, on the gold surface, was therefore stable if biotin was not present in the solution. Lipoic acid and HABA have lower affhity constants for binding with avidin, and lower binding of Av-AP to these immobilized ligands was observed. This was due to either low initial binding or desorption of bound Av-AP during the washing of the gold plates, after the incubation with the avidin conjugate. Desthiobiotin was therefore found to be the most suitable ligand for the present assay method as it has 3 orders of magnitude lower aftinity for avidin than biotin, but the complex on the gold surface was sufficiently stable so that the avidin was not desorbed before incubation with biotin. The measurement range for biotin in the enzyme binding assay with desthiobiotin immobilized on gold was 0.1-1 pg/mL (data not shown) or shifted to 10 times higher concentration than in the previous work8 with an electrochemical sensor based on the HABA-avidin complex. This shift was the result of the higher af6nity of desthiobiotin. The gold surface was flat, and the avidin should therefore be expected to bind to a single immobilized ligand through one binding site out of four available ones, as shown in Figure 1, because of the steric restriction. The three-dimensional structure of the membrane, which is used for the electrochemical sensor, will allow the avidin molecule to bind through multiple binding sites. This would explain why the complex between avidin and HABA in the membrane does not dissociate when washed with buffer, as it did when avidin was bound to HABA immobilized on flat gold surface. QCM Bioafl6inity Sensing. The typical readout for QCM bioaftinity sensing with stop flow and continuous flow is shown in Figure 3. When the sensor was operated in the stopflow mode, it took up to 1 h for the frequency to stabilize after the pumping was (17) Aizawa, M. Electrochemical Sensors in Immunological Analysis; Plenum Press: New York, 1990; pp 268-291. (18) Schmid, R D.: Kunnecke, W. J. Biotechnol. 1990, 14, 3-31.
I
I
I
1
I
I
Biotin
T
500 pg/rln
n
%
I
i, C
I 1
I
I
a
50
i
1
I
a
G
t-3
20
Y
II
-
-
-
0 L d aond n*bd 1 0 - ~ 1 0 - ~ 1 i0o- o~ i o 1 i o 2 10' ..s*d
nlEAd
io4 ios
Biotin concentration (pg/ml)
40
I
30
I
Buffer
Avidin
Figure 4. Result of the bioaffinity assay with the QCM bioaffinity sensor done in the stop-flow mode (0)for concentrations between 0.2 and IOO,ug/mL (average of three measurements). The result of the bioaffinity assay, done in the continuous-flow mode (O), are also shown (average of three measurements).
20
4
Buffer
I 0
I
I
I
I
20
40
60
80
I
Time [ minutes ] Figure 3. Typical readout for the QCM measurements. Above is the continuous-flow method, below the stop-flow method. The zero frequency was arbitrarily set for the measurements. The change in the frequency between 20 ,ug/mL injection of biotin and 500 ,ug/mL injection of avidin in the above graph is not continuous, and this was ascribed to noise.
stopped. The measurement range (Figure 4) was between 0.05 and 100 pg/mL, but the reaction time was shorter than in the enzyme binding assay. The frequency shifts shown in Figure 4 are for the change from the end of the buffer injection until the end of biotin injection, or data with a 20 min interval. Other experiments showed that biotin concentrations in the range of the measurement did not have any effects on the frequency of crystal covered with unmodified BSA The observed frequency changes were therefore caused by changes in the mass on the crystal rather than by a change in the solution. The total time required for one measurementhegeneration cycle was 80 min. When the sensor was regenerated by reloading avidin, the resulting frequency was always a few hertz lower than obtained during previous reloading, as is seen in Figure 3. This phenomenon was consistently observed in all the experiments done. This was not due to insufficient saturation of the binding sites. The enzyme binding assay had shown that the avidin concentration was sufficient for full saturation of the binding sites and also that the increase in frequency was constant, independent of how many times the reloading was done. The negative shift in the frequency after the reloading did not affect the positive change in frequency observed after subsequent injection of biotin. M e r avidin was reloaded, it was seen with the continuousflow operation that the frequency drifted slowly back in the positive direction, indicating that some of the bound avidin slowly being
removed from the crystal. Biotin sample was injected exactly 10 min after the injection of avidin, before the frequency had fully stabilized. The frequency was read from the display of the instrument, with 0.1 Hz accuracy, at the time of the injection. Exactly 10 min later the frequency was read again and avidin injected to regenerate the sensor. The difference between the two values was taken as the frequency change caused by desorption of avidin when binding to biotin in the solution. Some biotin-complexed avidin was still desorbing when avidin was injected, as indicated by the continuous increase in the frequency. Although a chemical equilibrium has not been fully reached, the procedure offers good reproducibility, which is a general feature bf flow injection systems.'* The measurement ranges for the continuous-flow system started from 0.2 pg/mL and continuous change was observed up to 10 OOO pg/mL, but higher concentrations could not be measured because of the limited solubility of biotin. The higher limit of detection corresponds to the shorter reaction time. The frequency changes from 0.2 to 10 0o0 pg/mL was approximately 9 Hz or the same as the frequency change from 0.05 to 100pg/ mL in the stopflow method. No loss of activity was detected in either the stopflow or the continuousflow measurementeven after 3 days of operation or after 27 consecutive measurement and regeneration cycles. CONCLUSION A QCM b i o m t y sensor for biotin has been developed for rapid on-line FIA measurements. This bioaffinity sensor principle can be used for detection of small molecules with QCM, with regeneration of the sensor under mild conditions without any loss of activity. The approach seems promising for further application with other systems such as the antibody-antigen system. Received for review January 11, 1995. Accepted April 7,
1995.B AC9500388 @Abstractpublished in Advance ACS Abstracts, June 1, 1995.
Analytical Chemistry, Vol. 67, No. 13, July 7, 1995
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