ISSN 00063509, Biophysics, 2012, Vol. 57, No. 3, pp. 336–342. © Pleiades Publishing, Inc., 2012. Original Russian Text © O.I. Guliy, B.D. Zaitsev, I.E. Kuznetsova, A.M. Shikhabudinov, O.A. Karavaeva, L.A. Dykman, S.A. Staroverov, O.V. Ignatov, 2012, published in Biofizika, 2012, Vol. 57, No. 3, pp. 460–467.
CELL BIOPHYSICS
Obtaining Phage MiniAntibodies and Using Them for Detection of Microbial Cells with an Electroacoustic Sensor O. I. Guliya, B. D. Zaitsevb, I. E. Kuznetsovab, A. M. Shikhabudinovb, O. A. Karavaevaa, L. A. Dykmana, S. A. Staroverova, and O. V. Ignatova aInstitute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, Saratov, 410049 Russia b
Saratov Branch, Institute of Radio Engineering and Electronics, Russian Academy of Sciences, Saratov, 410019 Russia Email:
[email protected],
[email protected] Received July 22, 2011; in final form, March 12, 2012
Abstract—Phage miniantibodies to bacterial cells of strain Azospirillum brasilense Sp245 were obtained, and the possibility of using them for detection of microbial cells with a lateral field excited piezoelectric resonator was studied. It has been found that the frequency dependences of the real and imaginary parts of electrical impedance of such a resonator loaded with a suspension of A. brasilense Sp245 cells with the miniantibodies differ significantly from the dependences of the resonator with a control cell suspension without minianti bodies. The limit of possible determination of the concentration of microbial cells is found to be 103 cells/mL upon interaction with miniantibodies. It has been ascertained that detection of A. brasilense Sp245 cells with the aid of miniantibodies is possible even in the presence of other cultures, for example, E. coli BLRil and A. brasilense Sp7. Therefore, it has been shown for the first time that detection of microbial cells with an elec troacoustic sensor is feasible. Keywords: Azospirillum brasilense Sp245, phage miniantibody, lateral field excited piezoelectric resonator, detection DOI: 10.1134/S0006350912030086
INTRODUCTION Antigen–antibody interaction is widely applied in sensor systems for detecting various species of micro organisms [1–4]. Traditionally the biological compo nents used for cell determination are represented by polyclonal and monoclonal antibodies, which are widely applied in production of diagnostic test sys tems. In the recent time for solving similar tasks in molecular biology use has been made of geneengi neering technologies of cloning recognizing frag ments—hypervariable regions of immunoglobulins, which appear cheaper and may compete in selectivity with hybridoma technologies. Related thereto, for example, is the phage display technology created by George Smith [5]. For the first time the possibility of obtaining singlechain sequences composed of the variable domains of antibodies in the composition of a hybrid phage coat protein was demonstrated by McCafferty et al. [6]. The authors on an example of antibodies against lysozyme have demonstrated the possibility of highly effective affine selection of clones carrying antigenbinding singlechain antibodies (miniantibodies). At a later time libraries were con structed in which combinations of light and heavy Editor’s Note: I certify that this is a most close equivalent of the original publication with all its factual statements and terminol ogy, phrasing and style. A.G.
chains of antibodies were produced in the composition of a phage protein in the form of singlechain antibod ies, heterodimers or separate soluble molecules. Anti bodies obtained with the aid of the phage display tech nology are successfully used for identification of bac terium [7–9]. For analysis of biological interactions, wide use is made of acoustical methods. Some methods are based on using in the quality of a protein receptor of active layers or membranes applied onto the surface of a piezoelectric acoustic line or resonator [10]. Thus with the aid of immobilization of corresponding antiviral antibodies on the resonator surface an immunosensor was developed for selective detection of herpes viruses in human blood [11]. There exist also acoustical meth ods of analysis of biological interactions immediately in a liquid suspension contacting with the surface of a piezoelectric. This approach is characterized by a sig nificantly smaller detection time as compared with methods using active films. For example, for a resona tor with a longitudinal electric field a possibility was shown of detecting endotoxin [12] and fibrinogen [13] upon addition of corresponding reagents changing the viscosity of suspension and leading to a shift of the res onance frequency. It is known also that some biologi cal interactions lead to a change of the electrical con ductivity of cell suspension. On this property a sensor
336
ELECTROACOUSTIC DETECTION OF MICROBES WITH MINIANTIBODIES
was developed with a transversehorizontal acoustic wave for detection of pH changes connected with hydrolysis of catalyzed enzymes in urea [14]. In recent years great interest of investigators is evoked by piezo electric resonators with a lateral electric field, which, as distinct from traditional resonators with a longitudi nal electric field, are more sensitive to the contacting liquid, inasmuch as they react both to a change of its viscosity and of conductivity. Shown is a possibility of detecting microbial cells Escherichia coli O157:H7 in suspension upon application on the resonator surface of a film immobilizing corresponding antibodies [15]. The aim of the given work presented as obtaining miniantibodies to cells of model strain A. brasilense Sp245 and investigating the possibility of their detec tion with the aid of a lateral field excited resonator. EXPERIMENTAL Microorganisms. In the work use was made of bac terial cells Azospirillum brasilense Sp245, A. brasilense Sp7, Escherichia coli XL1, E. coli BLRil obtained from the collection of microorganisms of IBPPM RAS. Cultivation of microorganisms. E. coli XL1 and E. coli BLRil were grown on a liquid nutrient medium LB of the following composition (g/L): NaCl⎯10; yeast extract⎯5; pepton⎯5. Cultivation was conducted in aerobic conditions on a rotary shaker (160 rpm) at constant temperature 30°C in the course of a day. Cultures were stored on a Petri dish with solid agarized medium. Daily cultures of A. brasilense Sp245 and A. brasilense Sp7 were obtained by means of passaging from a Petri dish into a flask with liquid LB medium. Incubation of cells was actualized on a rotary shaker with stirring intensity of 160 rpm at 30 ± 1°C for 18– 20 h. Cultures were stored on a Petri dish with solid agarized medium. Affine selection of miniantibodies from a phage library. For selection of phages carrying antibodies to whole cells, in the quality of a solid phase for fastening antigens use was made of a plate for immunoenzyme analysis. Into the well we introduced 200 μL of antigen and incubated overnight at 4°C, then blocked the anti genunoccupied space on the plate walls with a 2% solution of dry skimmed milk for 1 h, after which added 200 μL of the phage library of sheep antibodies (Griffin.1, Great Britain) [16] at a concentration of 1012 phage particles/mL, then incubated overnight at 4°C. After the expiry of the incubation time the plate was washed thrice with 0.1 M TrisHCl buffer contain ing 0.05% Tween 20. Elution of phage particles was conducted with triethylamine. Eluted phage particles were used for infecting the cells of E. coli strain XL1. The E. coli cells infected with outselected phages were grown overnight in medium 2TY of the following com position (g/L): trypton⎯10, yeast extract⎯5, BIOPHYSICS
Vol. 57
No. 3
2012
337
NaCl⎯5, containing 100 μg/mL ampicillin and 1% glucose. A 1/100 part (100 μL) of obtained culture inoculated 10 mL medium 2TY and grown on a ther mostated shaker at 37°C to optical density A600 = 0.3 (~1012 mL–1) in the course of 6 h. Then we added helper phage M13K07 and incubated for 1 h at 37°C. After incubation the cells were sedimented by centrif ugation at 2000 g for 10 min. The cell pellet was resus pended in 50 mL of medium 2TY containing 100 μg/mL ampicillin, 50 μg/mL kanamycin and 100 μg/mL isopropyl thiogalactazide, and grown on a thermostated shaker overnight at 37°C. The overnight culture of cells was repeatedly centrifuged 40 min at 3000 g. To the supernantant containing phage particles we added 1/5 volume of solution containing 20% PEG6000, 2.5 M NaCl and incubated on ice for 1.5 h. Phage particles were sedimented by centrifuga tion 10 min at 8000 g, the pellet resuspended in 1/10 of initial volume of culture of TE buffer, pH 7.5 (5 mL). The obtained preparation was clarified by centrifuga tion in the same conditions, after which the phage par ticles were again sedimented by addition of 1/5 volume of PEG6000/NaCl solution (1 mL) with subsequent centrifugation. The pellet was dissolved in 1 mL of buffer solution TE. The concentration of phage parti cles was determined spectrophotometrically, using for calculation the following relationship [17]: number of phage particles/mL = A269 – A320 × 6 ⋅ 1016 / number of nucleotides in the phage genome. Inasmuch as in the genome of phage M13 the number of nucleotides ~6400, then true is the formula: A269 – A320 = 30 ~ 2.8 ⋅ 1014 phage particles/mL. The obtained preparation of phage particles was used for conducting the subsequent two rounds of selection actualized in analogous conditions but with reduction of the incubation time and use of a smaller amount of phage particles at the stage of their interac tion with immobilized antigen (1.5 and 1 h incubation at room temperature and 1011 and 1010 phage particles for second and third rounds respectively). The speci ficity and selectivity of the obtained phages were determined by the method of dot analysis [18]. The titer of serum was determined with the aid of solid phase immunoenzyme analysis by the commonly accepted method [19]. Measurement was conducted on a microplate spectrophotometer Power Wave (Bio Tek Instruments, USA) at a wavelength of 490 nm. Selected were two clones having shown the best sensi tivity to whole cells of azospirilla. Preparation of conjugates of antiphage antibodies with colloidal gold. Antiphage antibodies were obtained by immunization of rabbit with bacterioph age M13 as described in [20]. Colloidal gold with mean particle diameter 15 nm was obtained by method [21], using the reaction of reduction of hydrochloro auric acid (Aldrich, USA) by sodium citrate (Fluka, Switzerland). Reduction was conducted under heating 242.5 mL of 0.01% water solution of hydrochloroauric
338
GULIY et al.
acid in an Erlenmeyer flask on a magnetic stirrer with a backflow water condenser. Then we added 7.5 mL of 1% water solution of sodium citrate. Further we determined the “gold number” for antiphage antibodies (minimal amount of probe pro tecting the sol from salt aggregation). For this in a 96 well microtitration plate we made double dilutions of antibody solution (initial concentration 1 mg/mL, volume 20 μL) in 0.002 M borate buffer. Into each well we added 200 μL of colloidal gold and then 20 μL of 1.7 M NaCl. The minimal stabilizing concentration for the probe constituted 12.5 μg/mL (which corre sponded to the last well (fourth) in which aggregation did not occur). Conjugation was conducted by simple mixing of reagents without using linking agents [22]. Preparation of cells to electroacoustic analysis. Before conducting analysis the cells were washed in distilled water by triple centrifugation at 2800 g over 5 min, then resuspended in a small volume of water (electroconductivity 1.8 μS/cm). The optical density of the prepared bacterial suspension was brought to values D660 = 0.4–0.42. Conduction of electroacoustic analysis. All experi ments on studying the changes in mechanical and electrical properties of cell suspension upon biospe cific interaction of microorganisms with minianti bodies were conducted with the aid of a specially man ufactured sensor on the basis of a lateral field excited resonator in a frequency range 6–7 MHz. The mate rial of the resonator, its orientation, type of acoustic wave, geometrical dimensions of elements and the means of suppressing parasitic oscillations have been determined in the course of preliminary experiments [23]. This resonator was manufactured from a plate of lithium niobate of xcut 0.5 mm thick. On the under side of the plate two rectangular electrodes were applied sized 5 ⋅ 10 mm2 and a gap between them of 3 mm. The area around the electrodes and part of the electrodes were covered with special lacquer that damped Lamb’s parasitic waves and ensured a high enough Q factor ~630. On the top side of the plate, glued was a liquid cell of volume ~1 mL. For conducting the analysis the prepared microbial cells without addition of miniantibodies were intro duced into the abovementioned liquid cell and mea surements were conducted of the real and imaginary parts of electrical impedance of the sensor with the aid of a precision meter of LCRparameters Agilent 4285A. Electron microscopy. For electron microscopic identification of the interaction of A. brasilense Sp245 cells with miniantibodies we prepared preparations a cell concentration of 106 c/mL, then incubated 0.5 mL of a suspension of microorganisms in buffered physiological solution with 10 μL of a solution of homologous antibodies with a concentration of 100 μg/mL for 1 h on a shaker. After which we centri fuged the suspension at 12000 g 5 min and resus pended the cell pellet in 0.5 mL of buffered physiolog
ical solution. Then the suspension was incubated with 100 μL of antiphage antibodies + colloidal gold conju gate (D520 = 0.5) for 1 h, after which we repeated the operations of sedimentation and resuspension. Approximately 20 μL of suspension was applied onto a film (Parafilm, USA) and placed on the drop a nickel grid (200 mesh) with a carbonreinforced nitrocellu lose support for 20 min. We conducted thermal attach ment by holding the grid near an incandescent bulb for 2 min. Excess liquid was removed by touching the grid upon a strip of filter paper. The grid was washed on a drop of deionized water, dried and placed into a con tainer. Analysis was conducted with the aid of an elec tron microscope Libra 120 (Carl Zeiss, Germany). RESULTS AND DISCUSSION In the recent time a new technology has been developed of obtaining highly affine fragments of anti bodies, which consists in constructing libraries of miniantibodies exposed on filamentous phages. In 1999 to IBPPM RAS in the course of executing a joint scientific project with the University of Aberdeen (Great Britain) a phage display of sheep antibodies was passed with protocols and methodical recommenda tions on application of the given technology [16]. Rounds of affine selection (biopanning) are repeated, as a rule, from three to six times. We have conducted three rounds of selection of miniantibodies onto whole cells of A. brasilense Sp245 with the use of clones possessing higher sensitivity and productive activity. The sensitivity of obtained phage particles was determined by the method of dot analysis. At that it was shown that the sensitivity of phage antibodies increased after the third round of selection. The titer of the obtained phage miniantibodies determined by the method of immunoenzyme analysis constituted 1 : 8000. Simultaneously for checking the specificity of interaction of miniantibodies we conducted electron microscopic identification of the interaction of A. brasilense Sp245 cells with the used miniantibodies labeled with colloidal gold. On the electron micropho tograph it is seen that antibodies with colloidal gold interact with the cells of azospirillum, at that gathering of the marker occurs over the whole surface of the cell (Fig. 1). Further with the aid of the electroacoustic sensor we have investigated suspension of bacterial cells A. brasilense Sp245 in interaction with the obtained miniantibodies. First into its liquid cell we introduced a suspension of A. brasilense Sp245 cells (108 cells/mL) and conducted measurement of the real and imaginary parts of electrical impedance of sensor with the aid of a meter of LCRparameters. Then we added specific miniantibodies on the basis of 20 phages per bacterium, after which the measure BIOPHYSICS
Vol. 57
No. 3
2012
ELECTROACOUSTIC DETECTION OF MICROBES WITH MINIANTIBODIES R, Ohm 2000
339
(a)
1500 3 1000
2
500
1
0 Fig. 1. Electron microscopic image of bacteria A. brasilense Sp245 labeled with a colloid gold conjugate of antiphage antibodies upon interaction of cells with mini antibodies (magnification ×10000).
ments were at once repeated. The measured depen dences are presented in Fig. 2a,b. The indicated con centration of miniantibodies was chosen on the basis of preliminary experiments, in which it was estab lished that the maximal change in resonance fre quency is observed upon introduction into a suspen sion of A. brasilense Sp245 cells of specific minianti bodies on the basis of 20 phages per bacterium. In Fig. 2 it is shown that the values of resonance fre quency, and also the values of real and imaginary parts of impedance in the aboveindicated frequency range strongly differ for suspension of cells with minianti bodies (curve 3) and without them (curve 2). It should be noted that in the course of measurements we did not notice changes in resonator impedance with time, i.e. the speed of impedance change was substantially smaller than the time of measurements, which consti tuted ~ 10 min. In this way, it can be said that the indi cated measurements fixed the final state of suspen sion, inasmuch as repeated experiments led to the same results. At the next step it appeared of interest to conduct analysis at lower concentrations of cells in suspension. For this the measurements were conducted at an amount of cells in the cell of 106, 104 and 103 cells/mL. The procedure of preparing the sample, bringing in miniantibodies and conducting measurements was analogous to the abovedescribed one. In Figs. 3, 4, 5 (a and b) we present the measured frequency depen dences of the real and imaginary parts of electrical impedance of the sensor. It is seen that for suspension of cells in the cell 106, 104, 103 c/mL, bringing in the corresponding miniantibodies also substantially changes the values of resonance frequency and the val ues of real and imaginary parts of impedance (curves 2 and 3). In this way, it is shown that the sensor allows distinctly demarcating situations when there occurs interaction of bacterial cells with specific minianti bodies from control experiments when such interac tion is absent. As evident from the obtained data, the BIOPHYSICS
Vol. 57
No. 3
2012
6.14
6.34
6.54
6.74
6.14
6.34
6.54
6.74
6.94 f, MHz f, MHz 6.94
–3500 –4000
(b) 3
–4500 –5000
2 1
–5500 –6000 X, Ohm Fig. 2. Dependences of the real (a) and imaginary (b) parts of electrical impedance on frequency (quantity of cells in the cell 108 c/mL) upon interaction of A. brasilense Sp245 cells with miniantibodies: (1) distilled water; (2 ) suspen sion of A. brasilense Sp245 cells without addition of mini antibodies; 3 – suspension of A. brasilense Sp245 cells with addition of miniantibodies.
minimal changes of the aboveindicated registered parameters upon bringing into the suspension specific miniantibodies correspond to a cell concentration of 103 c/mL. In this way, analysis has shown the possibil ity of detecting cells upon using specific miniantibod ies, at that the detection limit constitutes roughly 103 cells/mL. At that the degree of change in the char acteristics of the resonator depends on cell concentra tion, which open a possibility of conducting not only qualitative but also quantitative analysis of bacteria. Analysis of the obtained results permits making a conclusion about that as a result of specific biological interaction there is a change in the physical properties of the suspension. As it is known [15], the characteris tics of a piezoelectric lateral field excited resonator are extremely sensitive to a change of both mechanical and electrical parameters of the analyzed liquid. Among them are density, coefficients of elasticity and viscosity, and also dielectric permittivity and electrical conductivity. Regretfully, having at our disposal a small amount of analyzed suspensions (~1 mL), we
340
GULIY et al.
R, Ohm 2000
R, Ohm 2000
(a)
1500
(a)
1500 3
1000 500
3
1000
2
2
500
1
1 0
0 6.14
6.34
6.54
6.74
6.14
6.34
6.54
6.74
6.94 f, MHz f, MHz 6.94
–3500 –4000 –4500
6.14
6.34
6.54
6.74
6.14
6.34
6.54
6.74
f, MHz 6.94
–3500 (b)
–4000 3
(b)
–4500
3
2 –5000
6.94 f, MHz
1
2 1
–5000
–5500
–5500
–6000 X, Ohm
–6000 X, Ohm
Fig. 3. Dependences of the real (a) and imaginary (b) parts of electrical impedance on frequency (quantity of cells in the cell 106 c/mL) upon interaction of A. brasilense Sp245 cells with miniantibodies: (1) distilled water; (2) suspen sion of A. brasilense Sp245 cells without addition of mini antibodies; (3) suspension of A. brasilense Sp245 cells with addition of miniantibodies.
Fig. 4. Dependences of the real (a) and imaginary (b) parts of electrical impedance on frequency (quantity of cells in the cell 104 c/mL) upon interaction of A. brasilense Sp245 cells with miniantibodies: (1) distilled water; (2) suspen sion of A. brasilense Sp245 cells without addition of mini antibodies; (3) suspension of A. brasilense Sp245 cells with addition of miniantibodies.
had no possibility of measuring the indicated charac teristics by direct standard methods. Measurement of these characteristics is planned at the following steps of the work. It can only be said that all biological pro cesses proceeded in the volume of suspension without participation of the contacted surface of the resonator. This is explained by that the resonator is executed of a crystal of lithium niobate, which appears chemical resistant practically to all chemical compounds. Besides, the resonator surface, worked to the 14 grade of finish, does not admit any adsorption. Therefore after washing of the resonator on its surface there remain no traces of suspension, and a confirmation of this presents as complete restoration of all its charac teristics after all experiments. One of the important moments in development of acoustoelectric analysis appears as obtaining an ana lytical signal provided interfering factors and, first of all, in the presence of extraneous microflora. There fore at the next step of the work we conducted mea surements of the frequency dependences of the real and imaginary parts of impedance for a cell suspension
of A. brasilense Sp245 upon interaction with mini antibodies in the presence of E. coli BLRil and A. brasilense Sp7cells. These dependences are pre sented in Fig. 6. The choice of the given cultures is stipulated by the semblance of their shape with azospirillum cells (rods). For this to a mixed suspen sion of A. brasilense Sp245, E. coli BLRil and A. brasilense Sp7 (the amount of cells in the measuring cell 104 c/mL) taken in equal relationship (1 : 1 : 1), we entered miniantibodies on the basis of 20 phages per bacterium. In the quality of control we used the mixed suspension of A. brasilense Sp245, E. coli BLRil and A. brasilense Sp7 without entering miniantibodies. Curves 2 and 3 presented in Fig. 6 show that upon interaction of A. brasilense Sp245 cells with minianti bodies in the presence of extraneous microflora there also occurs a change in the abovementioned regis tered parameters. Simultaneously we conducted con trol experiment on the study of nonspecific interaction of miniantibodies with E. coli BLRil and A. brasilense Sp7 cells. It was established that changes of parameters occur only in A. brasilense Sp245 cells BIOPHYSICS
Vol. 57
No. 3
2012
ELECTROACOUSTIC DETECTION OF MICROBES WITH MINIANTIBODIES R, Ohm 2000
R, Ohm 2000
(a)
1500
1500
1000
1000 3 2
500
(a)
3
2
500 1
1 0
0 6.14
6.34
6.54
6.74
6.14
6.34
6.54
6.74
6.94 f, MHz f, MHz 6.94
–3500 –4000
341
6.14
6.34
6.54
6.74
6.14
6.34
6.54
6.74
6.94 f, MHz f, MHz 6.94
–1500 (b)
–2500
–4500
3 2
–5000
–3500
1
–5500
–4500
2
–5500
1
X, Ohm
–6000 X, Ohm Fig. 5. Dependences of the real (a) and imaginary (b) parts of electrical impedance on frequency (quantity of cells in the cell 103 c/mL) upon interaction of A. brasilense Sp245 cells with miniantibodies: (1) distilled water; (2) suspen sion of A. brasilense Sp245 cells without addition of mini antibodies; (3) suspension of A. brasilense Sp245 cells with addition of miniantibodies.
upon interaction with specific miniantibodies, in sus pension of E. coli BLRil and A. brasilense Sp7 cells the changes of resonance frequency under the action of miniantibodies on A. brasilense Sp245 do not occur. It should be noted that curves 3 presented in Figs. 4 and 6 are strongly distinct, and this apparently is connected with distinctions of physical properties of the analyzed suspension in the presence of extraneous microflora and without it as a result of specific interac tion. Nonetheless, and this is the most important thing, bringing specific miniantibodies into the mixed suspension also strongly changes the character istics of loaded resonator. In this way, as a results of conducted investigations it is shown that the frequency dependences of the real and imaginary parts of impedance of a resonator loaded with a suspension of cells with miniantibodies significantly differ from a resonator with a control sus pension of cells without miniantibodies. The graphs presented in Figs. 2–6 vividly demonstrate that in the quality of an informative parameters one may take BIOPHYSICS
3
(b)
Vol. 57
No. 3
2012
Fig. 6. Dependences of the real (a) and imaginary (b) parts of electrical impedance on frequency (mixed suspension of A. brasilense Sp245, E. coli BLRil and A. brasilense Sp7 cells) upon interaction with miniantibodies: (1) distilled water; (2) mixed suspension of A. brasilense Sp245, E. coli BLRil and A. brasilense Sp7 cells without addition of miniantibodies; (3) mixed suspension of A. brasilense Sp245, E. coli BLRil and A. brasilense Sp7 cells with addi tion of miniantibodies.
both the change in resonance frequency and the change in the maximal value of the real part of imped ance or the middle of the linear dip of the imaginary part etc. We determined the limit of possible determi nation of microbial cells, which constitutes ~103 c/mL upon their interaction with miniantibodies. It is established that in the presence of extraneous cultures of E. coli BLRil and A. brasilense Sp7 cells, it is also possible to detect A. brasilense Sp245 cells with the use of specific miniantibodies. The presented results show the possibility of creat ing a biological acoustical sensor for quantitative anal ysis of microbial cells with a detection limit of ~103 cells/mL. ACKNOWLEDGMENTS The work was supported by the Russian Founda tion for Basic Research (100201313a and 1202 01057a).
342
GULIY et al.
REFERENCES 1. R. D. Vaughan, C. K. O’Sullivan, and G. G. Cuilbault, Enzyme Microb. Techn. 29, 635 (2001). 2. K. A. Uithoven, J. C. Schmidt, and M. E. Ballman, Biosensors and Bioelectronics 14, 761 (2000). 3. P. Gascoyne, R. Pethig, J. Satayavivad, F. F. Becker, and M. Ruchirawat, Biochim. Biophys. Acta 1323, 240 (1997). 4. A. H. Peruski and L. F. Peruski, Jr., Clin. Diagn. Lab. Immunol. 10, 506 (2003). 5. G. P. Smith, Science 228, 1313 (1985). 6. J. McCafferty, A. D. Griffiths, G. Winter, and D. J. Chiswell, Nature 348, 552 (1990). 7. V. Nanduri, I. B. Sorokulova, A. Samoylov, et al., Biosens. Bioelectron. 22, 986 (2007). 8. G. Paoli and J. D. Brewster, J. Rapid Meth. Automat. Microbiol. 4, 74 (2007). 9. D. D. Williams, O. Benedek, and C. L. Turnbough, Jr., Appl. Environ. Microbiol. 69, 6288 (2003). 10. D. S. Ballantine, R. M. White, S. J. Martin, et al., Acoustic Wave Sensors: Theory, Design, and Physico chemical Applications (Academic Press, 1997). 11. B. Koenig and M. Graetzel, Anal. Chem. 66, 341, (1994).
12. H. Muramatsu, E.Tamiya, M. Suzuki, and I. Karube, Anal. Chim. Acta 217, 321 (1989). 13. H. Muramatsu, E.Tamiya, M. Suzuki, and I. Karube, Anal. Chim. Acta 215, 91 (1988). 14. J. Kondoh, Y. Matsui, and S. Shiokawa, Jpn. J. Appl. Phys. 32, 2376 (1993). 15. J. F. Vetelino, Proceed of IEEE Ultrason. Symp (San Diego, 2010). 16. K. A. Charlton, S. Moyle, A. J. R. Porter, and W. J. Harris, J. Immunol. 164, 6221 (2000). 17. G. P. Smith and J. K. Scott, Methods Enzymol. 217, 228 (1993). 18. L. A. Dykman and V. A. Bogatyrev, Biokhimiya 62, 411 (1997). 19. J. D. Beatty, B. G. Beatty, and W. G. Vlahos, J. Immunol. Meth. 100, 173 (1987). 20. D. V. Pristenskii, S. A. Staroverov, D. N. Ermilov, et al., Biomed. Khimiya 53, 57 (2007). 21. G. Frens, Nature Phys. Sci. 241, 20 (1973). 22. L. A. Dykman, V. A. Bogatyrev, S. Yu. Shchegolev, and N. G. Khlebtsov, Gold Nanoparticles: Synthesis, Proper ties, Biomedical Application (Nauka, Moscow, 2008) [in Russian]. 23. B. D. Zaitsev, I. E. Kuznetsova, A. M. Shikhabudinov, Tech. Phys. Lett., 37, 473 (2011).
BIOPHYSICS
Vol. 57
No. 3
2012
