- Email: [email protected]
Analytical scale ultrasonic standing wave manipulation of cells and microparticles
Ultrasonics 38 (2000) 638–641 www.elsevier.nl/locate/ultras
Analytical scale ultrasonic standing wave manipulation of cells and microparticles W.T. Coakley *, J.J. Hawkes, M.A. Sobanski, C.M. Cousins, J. Spengler School of Biosciences, Cardiff University, Cardiff CF10 3TL, UK
Abstract The ultrasonic standing-wave manipulation of suspended eukaryotic cells, bacteria and submicron latex or silica particles has been examined here. The different systems, involving plane or tubular ultrasonic transducers and a range of acoustic pathlengths, have been designed to treat suspension volumes of analytical scale i.e. 5 ml to 50 ml for both sample batch and ‘on-line’ situations. Frequencies range from 1 to 12 MHz. The influence of secondary cell–cell interaction forces in determining the cell concentration dependence of harvesting efficiency in batch sedimentation systems is considered. Applications of standing wave radiation forces to (1) clarify cell suspensions, (2) enhance particle agglutination immunoassay detection of cells or cellular products and (3) examine and enhance cell–cell interactions in suspension are described. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Acoustic streaming; Antigen detection; Blood separation; Immunoassay; Radiation pressure; Ultrasonic standing wave
1. Introduction The ability of ultrasonic standing waves to concentrate cells and microparticles at sub-millimetre distances in megahertz-frequency non-cavitating ultrasonic standing waves has been widely studied in recent years [1,2]. Areas of particle manipulation that have proved to be of biological interest include (1) primary separation of cells and suspending phase [3–5] (2) cross-linking of microparticles coated with immunological molecules [6 ] and (3) cell–cell interactions [7]. Filtration of cell suspensions in flow systems has been achieved on the intermediate [3] and large-volume scale [4]. Suspensions have been manipulated on an analytical scale (<5 ml ) where the value is in the information obtained from a sample with regard to the properties of larger volume biotechnological processes [2], water-industry suspensions or body fluids [5].
2. Forces on particles in ultrasonic standing waves Ultrasonic frequencies of more than 1 MHz (l<1.5 mm in water) are preferred for particle or cell * Corresponding author. Tel.: +44-1222-874288; fax: +44-1222-874305. E-mail address: [email protected] ( W.T. Coakley)
manipulation, so that high-pressure amplitudes can be employed without inducing ultrasonic cavitation, with its associated vigorous order-disrupting bubble activity [8]. A particle, cell or droplet, in a phase in which there is a standing wave, acquires a position-dependent acoustic potential energy [9]. Suspended bodies therefore experience a force tending to concentrate them at axial half wavelength intervals, i.e. at positions of minimum particle acoustic potential energy. When the particle diameter is small compared to the ultrasound wavelength, the ‘primary’ radiation force, F , acting on a r body of volume, V , located at a distance, z, from a c pressure node is derived from the gradient of the particle acoustic potential energy [8], and is given by: F =−[P2 V b /(2l)]w( b, r) sin(4pz/l), r 0 c w
(1)
where P is the peak acoustic pressure amplitude, and 0 l is the wavelength of sound in the aqueous suspending phase, which has a compressibility b . The function w w(b, r) equals [(5r −2r )/(2r +r )−(b /b )], where c w c w c w b is the compressibility of the particle and r and r c c w are the densities of the particle and the suspending phase, respectively. Weak transverse primary forces and secondary (e.g. Bjerkness) forces drive the concentrated particles to the local particle potential energy minima, within the pressure nodal planes, to give concentration
0041-624X/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S0 0 4 1 -6 2 4 X ( 9 9 ) 0 0 15 1 - 1
W.T. Coakley et al. / Ultrasonics 38 (2000) 638–641
639
regions that appear as columns of clumps striated at half-wavelength separations in the axial direction [1,10]. While the principal features of particle concentration in a standing wave can be explained by a combination of primary-axial and transverse forces [10], further development is needed to explain phenomena such as the high dependence of the concentration efficiency on biomass concentration [11]. A recent review [2] of published data for a range of cell sizes has shown that a high separation efficiency is achieved when the biomass is in the range of 0.01–15% w/w (wet weight of cells per unit weight of suspension) with efficiency generally falling off outside those limits. We note here, however, that primary separation of whole blood (45% w/w) can be achieved in a small-volume batch system [12].
with a density and compressibility most different from that of the suspending aqueous phase experienced the greater forces [Eq. (1)]. Suspended biological cells have densities and compressibilities significantly, but not hugely, different from that of the aqueous suspending phase [2]. Nucleated (eukaryotic) cells have diameters of the order of 10 mm while bacterial cells are of the order of 1.0 mm. It has been estimated that the maximum axial radiation pressure experienced by cells in a 3 MHz standing wave of peak pressure amplitude 1.0 MPa is the equivalent of being in a 160 g field [2]. While this field value is low compared with what is attainable in a centrifuge, particles need to travel only sub-millimetre distances to a concentration point in an ultrasonic standing wave.
3. Applications
3.2. Separation of concentrated cells from suspension
3.1. Primary particle/suspending-phase separations
Cultures of monoclonal antibody-producing hybridoma have been clarified in a recently developed 75 ml sedimentation ultrasonic filter ( USF ) [1,21] at flow rates of several litres per hour. The standing wave is positioned across the outflow at the top of a fermenter, so that, as the soluble cell product is drawn away, the cells clump and sediment back into the culture to produce more antibodies. This system has also been successfully tested with perfusion-cultured insect cells [22]. Effort is now being applied to increase the effective flow rate so that the filtration system can be applied to larger-scale fermentations. A compact (7.5 ml ) sedimentation USF has been shown to recover yeast with an efficiency of over 99% [3]. Its relatively small volume provides, possibly because of reduced acoustic streaming, 82% recovery of micron-sized E. coli [3] at flow rates of up to 3 ml min−1. Analytical applications require filters with a small active volume. Such reduction has been achieved by, inter alia, shortening the acoustic pathlength between the transducer and reflector. The cells emerge as ordered layers in the outflow channel [23,24]. When the pathlength of the filter is reduced to a single half wavelength, cells are concentrated into one ordered layer. An approach has been developed to harvest the clarified off-axis suspending phase, thus providing a general technique for clarification of turbid samples for spectrophotometric or other analysis [25]. The essential difference between the concepts of the short pathlength flow system and the sedimentation or field-modulated batch or filter systems is that, in the former case, the particles emerge with the outflow but in an ordered state. Cells can be separated with a reasonable efficiency in batch systems since the latter allow cells a longer time of residence in the standing wave. The efficiency of whole-blood separation [12] and of E. coli separation at optimal concentrations [13] is in excess of 99.5%.
Currently, centrifugation and filtration are among the techniques most widely employed for suspension clarification. Limitations of these methods include that centrifuges are not readily compatible with in-line systems or small sample volumes, while solid filters can suffer from blockage. However, ultrasonic standing wave forces can be employed in-line without physical blockage. The general approach to primary (solid/bulk) microparticle separation, as against secondary (subpopulation) acoustic separation, in standing waves has been to form clumps, as described above that can, because of their larger volume, be more easily manipulated than single microparticles. Suspensions have thus been clarified in batch [11,14], or flow [3,4,15] systems by clump collection through electronic phase or frequency modulation of the sound field [11,15–17] or pulse-controlled sedimentation [13]. Flowing suspensions have also been clarified by gravitational sedimentation of cell clumps retained in an ultrasonic filter ( USF ) without electronic modulation [3,4]. In another development, particle manipulation by a multi-electrode line-focused transducer has been employed to move single 16 mm aluminium particles laterally in a controlled manner [18]. The suspending phase of single particles is never totally at rest, so that particles in a standing wave are subject to convection by gravitational–thermal [19] and acoustically generated hydrodynamic streaming currents on scales of both the container dimensions and the internodal separations. Container-scale acoustic streaming can be minimized by reducing the container dimensions [20]. The particulate aluminium, glass, silica, carbon and polystyrene latex manipulated in the above systems ranged in diameter from 16 to 0.2 mm. The large particles
640
W.T. Coakley et al. / Ultrasonics 38 (2000) 638–641
3.3. Interactions of biologically coated microparticles in a standing wave The human immune defence system produces antibodies in reaction to invasion by foreign molecules or microbial envelope molecules (antigens). Detection of antigens is one method to confirm the presence of pathogenic bacteria in human body fluids. When latex microparticles, coated with antibody specific to a particular antigen on the microbial surface, are mixed with a sample droplet containing antigens, the antigens bind to the particles and, if the antigen concentration is sufficient, cross-link and agglutinate the particles in local clusters. In this latex agglutination test (LAT ), sedimented clumps of particles become visible to the naked eye within the droplet. Coated-particle-based tests, as conventionally performed, can take times ranging from a few minutes to several hours, depending on the test format. LATs remain popular in bacteriology laboratories because of their simplicity, speed and relatively low cost [26 ], although they are generally less sensitive (based on the lowest concentration of antigen detectable) than enzyme-linked immunosorbent assays ( ELISAs) [2]. A technique has been developed in which the body fluid test sample, mixed with a coated microparticle suspension, is exposed to an ultrasonic standing wave for times of the order of 1 min. The resulting concentration of particles in the standing wave increases the rate of particle agglutination and sensitivity of antigen detection. The rate is increased because the local concentration of beads is accentuated in a standing wave. The sensitivity of antigen detection also increases because the ultrasonic clumping process maintains the beads in close proximity, thus increasing the probability of crosslinking. Where latex is replaced by silica as a carrier microparticle, secondary ultrasonic forces can reduce the equilibrium separation of microparticles and thus further enhance the sensitivity of reaction [27]. Enhancements of the LAT antigen detection sensitivity of 2500-fold for C-reactive protein, 500-fold for fungal antigen and 32-fold for meningococcal antigen have been reported [2]. Standing wave detection of meningococcal antigen has received most attention in the clinical and laboratory context because of the exceptional need for rapid diagnosis of aggressive bacterial meningitis [5,28] so that informed vaccination policies can be implemented rapidly. It has been shown that ultrasound enhanced latex agglutination gives serogrouping information in four times more cases than are detected by conventional agglutination tests on serum [5]. Retrospective serogrouping of infected serum samples by ultrasound is comparable to that achievable by current polymerase chain reaction (PCR) procedures for detection of bacterial DNA [5]. The simplicity of the ultrasonic technique opens up the possibility that it will provide rapid diagno-
sis in the local bacteriology laboratory and that its epidemiological information can complement that obtainable from, sometimes remote, specialized PCR facilities. 3.4. Ultrasonic enhancement of bioseparation rates in biphasic aqueous systems Highly selective separations can be achieved when cells or biomolecules partition rapidly between the two phases formed when a high concentration of aqueous solutions of polymers such as polyethyleneglycol (PEG ) and dextran are mixed together [29]. The collection of the partitioned cells or solute involves coalescence and density-dependent movement of droplets to form two bulk phases. This process, which can take 30 min or longer at room temperature, is routinely accelerated by centrifugation. Biphasic separation of Saccharomyces cerevisiae and E. coli can be achieved three times faster in a low-power ultrasonic standing wave than with centrifugation [2]. The ability to complete a partition separation more rapidly under a low-ultrasonic power than with centrifugation suggests that ultrasound has the potential required to facilitate in-line bioanalytical applications of aqueous biphasic separations [30]. 3.5. Standing-wave enhancement of cell-surface phenomena Adhesive interactions of cells suspended in polymer solutions have been examined in an ultrasonic standing wave by light microscopy. The standing wave levitated the cells away from any solid boundary [31]. Manipulation of cells in a standing wave has also been employed to draw spleen and tissue culture cells together in suspension prior to cell fusion by an electric pulse. The technique exploited the ability of ultrasound, in contrast to dielectrophoresis, to manipulate cells in physiological ionic strength media [7].
4. Conclusions It has been outlined above that the sophistication, characterization and control of ultrasonic particle manipulations have increased markedly in recent years. While pursuit of the operational advantages in bringing cells [7] or bio-particles [5] into close proximity using ultrasound can proceed on an empirical basis, informed development of such techniques requires a further appreciation and analysis of the relative contributions of the different forces expressed in standing-wave phenomena. The need for such an understanding is signalled by observations that very low concentrations of micronsized bacteria can be harvested from suspension if mixed with larger particles such as yeast [3] and that micropar-
W.T. Coakley et al. / Ultrasonics 38 (2000) 638–641
ticles can, in a resonant ultrasonic field, be filtered onto the surface of structures with a pore size two orders of magnitude larger than the suspended particles [32]. Developments in the combination of microscopic observation, particle motion analysis modelling and resonator design [8,17,33] will contribute significantly to this area and to the general exploitation of the ability to control microparticle manipulation in suspension.
Acknowledgements The authors are grateful to BBSRC, Meningitis Research Foundation and the EU TMR for support of some of the above work.
References [1] M. Gro¨schl, Acustica 84 (1998) 432. [2] W.T. Coakley, Trends Biotech. 15 (1997) 506. [3] J.J Hawkes, M.S. Limaye, W.T. Coakley, J. Appl. Microbiol. 82 (1997) 39. [4] T. Gaida, O. Doblhoff-Dier, K. Strutzenberger, H. Katinger, W. Burger, M. Gro¨schl, B. Handl, E. Benes, Biotechnol. Prog. 12 (1996) 73. [5] R. Allman, W.T Coakley, Bioseparation 4 (1994) 29. [6 ] S.J. Gray, M.A. Sobanski, E.B. Kaczmarski, M. Guiver, W.J. Marsh, R. Borrow, R.A. Barnes, W.T. Coakley, J. Clin. Microbiol. 37 (1999) 1797. [7] D.W. Bardsley, J.E. Liddell, W.T. Coakley, D.J. Clarke, J. Immunol. Meth. 129 (1990) 41. [8] R.K. Gould, W.T. Coakley, M.A. Grundy, Ultrasonics 30 (1992) 239. [9] G. Whitworth, W.T. Coakley, J. Acoust. Soc. Am. 91 (1992) 79.
641
[10] S.M. Woodside, J. Piret, M. Gro¨schl, E. Benes, B.D. Bowen, AIChE J. 44 (1998) 1976. [11] C.A Miles, M.J. Morley, W.R Hudson, B.M. Mackay, J. Appl. Bact. 78 (1995) 47. [12] C.M. Cousins, S.E. Higgins, M.S. Limaye, J.J. Hawkes, W.T. Coakley, P. Holownia, C.P. Price, Acustica 85 (1999) S150. [13] M.S. Limaye, W.T. Coakley, J. Appl. Microbiol. 84 (1998) 1035. [14] S. Gupta, D.L. Feke, I. Manas-Zlocczower, Chem. Eng. Sci. 50 (1995) 3275. [15] S. Peterson, G. Perkins, C. Baker, In: Proc. IEEE 8th Ann. Conf. Eng. Med. Biol. Soc. (1986) 154–156. [16 ] C.J. Schram, Adv. Sonochem. 2 (1991) 293. [17] M. Gro¨schl, Acustica 84 (1998) 632. [18] T. Kozuka, H. Mitome, T. Fukuda, Jpn. J. Appl. Phys. 37 (1998) 2974. [19] J.J. Hawkes, J.J. Cefai, D.A. Barrow, W.T. Coakley, L.G. Briarty, J. Phys. D Appl. Phys. 31 (1998) 1673. [20] J. Spengler, M. Jekel, , A. Tiehm, U. Neiss ( Eds.), TU HamburgHarburg Rep. Sanit. Eng. 25 (1999) 189. [21] H. Bierau, A. Perani, M.. Al Rubeai, A.N. Emery, J. Biotechnol. 62 (1998) 195. [22] J. Zhang, A. Collins, M. Chen, I. Knyazev, R. Gentz, Biotech. Bioeng. 59 (1998) 351. [23] J.J. Hawkes, J.J.D. Barrow, W.T. Coakley, Ultrasonics 36 (1998) 925. [24] K. Yasuda, S.S. Haupt, S. Umemura, T. Yagi, M. Nishida, Y. Shibata, J. Acoust. Soc. Am. 102 (1997) 642. [25] J.J. Hawkes, C. Cousins, W.T. Coakley, P.J. Keay, Acustica 85 (1999) S151. [26 ] D.N. Greenberg, D.P. Ascher, B.A. Yoder, D.M. Hensley, H.S. Heiman, J.F. Keith, J. Clin. Microbiol. 33 (1995) 193. [27] N.E. Thomas, M.A. Sobanski, W.T. Coakley, Ultrasound Med. Biol. 25 (1999) 443. [28] R.A. Barnes, P. Jenkins, W.T. Coakley, Arch. Dis. Child. 78 (1998) 58. [29] K.S.M.S. Raghavarao, N.K Rastogi, M.K. Gowthaman, N.G. Karannth, Adv. Appl. Microbiol. 41 (1995) 97. [30] B.Y. Zaslavsky, Anal. Chem. 64 (1992) A765. [31] H. Darmani, W.T. Coakley, Biochim. Biophys. Acta 1021 (1990) 182. [32] S. Gupta, D.L. Feke, AIChE J. 44 (1998) 1005. [33] D.L. Feke, A.E. Penrod, Acustica 85 (1999) S92.
Analytical scale ultrasonic standing wave manipulation of cells and microparticles W.T. Coakley *, J.J. Hawkes, M.A. Sobanski, C.M. Cousins, J. Spengler School of Biosciences, Cardiff University, Cardiff CF10 3TL, UK
Abstract The ultrasonic standing-wave manipulation of suspended eukaryotic cells, bacteria and submicron latex or silica particles has been examined here. The different systems, involving plane or tubular ultrasonic transducers and a range of acoustic pathlengths, have been designed to treat suspension volumes of analytical scale i.e. 5 ml to 50 ml for both sample batch and ‘on-line’ situations. Frequencies range from 1 to 12 MHz. The influence of secondary cell–cell interaction forces in determining the cell concentration dependence of harvesting efficiency in batch sedimentation systems is considered. Applications of standing wave radiation forces to (1) clarify cell suspensions, (2) enhance particle agglutination immunoassay detection of cells or cellular products and (3) examine and enhance cell–cell interactions in suspension are described. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Acoustic streaming; Antigen detection; Blood separation; Immunoassay; Radiation pressure; Ultrasonic standing wave
1. Introduction The ability of ultrasonic standing waves to concentrate cells and microparticles at sub-millimetre distances in megahertz-frequency non-cavitating ultrasonic standing waves has been widely studied in recent years [1,2]. Areas of particle manipulation that have proved to be of biological interest include (1) primary separation of cells and suspending phase [3–5] (2) cross-linking of microparticles coated with immunological molecules [6 ] and (3) cell–cell interactions [7]. Filtration of cell suspensions in flow systems has been achieved on the intermediate [3] and large-volume scale [4]. Suspensions have been manipulated on an analytical scale (<5 ml ) where the value is in the information obtained from a sample with regard to the properties of larger volume biotechnological processes [2], water-industry suspensions or body fluids [5].
2. Forces on particles in ultrasonic standing waves Ultrasonic frequencies of more than 1 MHz (l<1.5 mm in water) are preferred for particle or cell * Corresponding author. Tel.: +44-1222-874288; fax: +44-1222-874305. E-mail address: [email protected] ( W.T. Coakley)
manipulation, so that high-pressure amplitudes can be employed without inducing ultrasonic cavitation, with its associated vigorous order-disrupting bubble activity [8]. A particle, cell or droplet, in a phase in which there is a standing wave, acquires a position-dependent acoustic potential energy [9]. Suspended bodies therefore experience a force tending to concentrate them at axial half wavelength intervals, i.e. at positions of minimum particle acoustic potential energy. When the particle diameter is small compared to the ultrasound wavelength, the ‘primary’ radiation force, F , acting on a r body of volume, V , located at a distance, z, from a c pressure node is derived from the gradient of the particle acoustic potential energy [8], and is given by: F =−[P2 V b /(2l)]w( b, r) sin(4pz/l), r 0 c w
(1)
where P is the peak acoustic pressure amplitude, and 0 l is the wavelength of sound in the aqueous suspending phase, which has a compressibility b . The function w w(b, r) equals [(5r −2r )/(2r +r )−(b /b )], where c w c w c w b is the compressibility of the particle and r and r c c w are the densities of the particle and the suspending phase, respectively. Weak transverse primary forces and secondary (e.g. Bjerkness) forces drive the concentrated particles to the local particle potential energy minima, within the pressure nodal planes, to give concentration
0041-624X/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S0 0 4 1 -6 2 4 X ( 9 9 ) 0 0 15 1 - 1
W.T. Coakley et al. / Ultrasonics 38 (2000) 638–641
639
regions that appear as columns of clumps striated at half-wavelength separations in the axial direction [1,10]. While the principal features of particle concentration in a standing wave can be explained by a combination of primary-axial and transverse forces [10], further development is needed to explain phenomena such as the high dependence of the concentration efficiency on biomass concentration [11]. A recent review [2] of published data for a range of cell sizes has shown that a high separation efficiency is achieved when the biomass is in the range of 0.01–15% w/w (wet weight of cells per unit weight of suspension) with efficiency generally falling off outside those limits. We note here, however, that primary separation of whole blood (45% w/w) can be achieved in a small-volume batch system [12].
with a density and compressibility most different from that of the suspending aqueous phase experienced the greater forces [Eq. (1)]. Suspended biological cells have densities and compressibilities significantly, but not hugely, different from that of the aqueous suspending phase [2]. Nucleated (eukaryotic) cells have diameters of the order of 10 mm while bacterial cells are of the order of 1.0 mm. It has been estimated that the maximum axial radiation pressure experienced by cells in a 3 MHz standing wave of peak pressure amplitude 1.0 MPa is the equivalent of being in a 160 g field [2]. While this field value is low compared with what is attainable in a centrifuge, particles need to travel only sub-millimetre distances to a concentration point in an ultrasonic standing wave.
3. Applications
3.2. Separation of concentrated cells from suspension
3.1. Primary particle/suspending-phase separations
Cultures of monoclonal antibody-producing hybridoma have been clarified in a recently developed 75 ml sedimentation ultrasonic filter ( USF ) [1,21] at flow rates of several litres per hour. The standing wave is positioned across the outflow at the top of a fermenter, so that, as the soluble cell product is drawn away, the cells clump and sediment back into the culture to produce more antibodies. This system has also been successfully tested with perfusion-cultured insect cells [22]. Effort is now being applied to increase the effective flow rate so that the filtration system can be applied to larger-scale fermentations. A compact (7.5 ml ) sedimentation USF has been shown to recover yeast with an efficiency of over 99% [3]. Its relatively small volume provides, possibly because of reduced acoustic streaming, 82% recovery of micron-sized E. coli [3] at flow rates of up to 3 ml min−1. Analytical applications require filters with a small active volume. Such reduction has been achieved by, inter alia, shortening the acoustic pathlength between the transducer and reflector. The cells emerge as ordered layers in the outflow channel [23,24]. When the pathlength of the filter is reduced to a single half wavelength, cells are concentrated into one ordered layer. An approach has been developed to harvest the clarified off-axis suspending phase, thus providing a general technique for clarification of turbid samples for spectrophotometric or other analysis [25]. The essential difference between the concepts of the short pathlength flow system and the sedimentation or field-modulated batch or filter systems is that, in the former case, the particles emerge with the outflow but in an ordered state. Cells can be separated with a reasonable efficiency in batch systems since the latter allow cells a longer time of residence in the standing wave. The efficiency of whole-blood separation [12] and of E. coli separation at optimal concentrations [13] is in excess of 99.5%.
Currently, centrifugation and filtration are among the techniques most widely employed for suspension clarification. Limitations of these methods include that centrifuges are not readily compatible with in-line systems or small sample volumes, while solid filters can suffer from blockage. However, ultrasonic standing wave forces can be employed in-line without physical blockage. The general approach to primary (solid/bulk) microparticle separation, as against secondary (subpopulation) acoustic separation, in standing waves has been to form clumps, as described above that can, because of their larger volume, be more easily manipulated than single microparticles. Suspensions have thus been clarified in batch [11,14], or flow [3,4,15] systems by clump collection through electronic phase or frequency modulation of the sound field [11,15–17] or pulse-controlled sedimentation [13]. Flowing suspensions have also been clarified by gravitational sedimentation of cell clumps retained in an ultrasonic filter ( USF ) without electronic modulation [3,4]. In another development, particle manipulation by a multi-electrode line-focused transducer has been employed to move single 16 mm aluminium particles laterally in a controlled manner [18]. The suspending phase of single particles is never totally at rest, so that particles in a standing wave are subject to convection by gravitational–thermal [19] and acoustically generated hydrodynamic streaming currents on scales of both the container dimensions and the internodal separations. Container-scale acoustic streaming can be minimized by reducing the container dimensions [20]. The particulate aluminium, glass, silica, carbon and polystyrene latex manipulated in the above systems ranged in diameter from 16 to 0.2 mm. The large particles
640
W.T. Coakley et al. / Ultrasonics 38 (2000) 638–641
3.3. Interactions of biologically coated microparticles in a standing wave The human immune defence system produces antibodies in reaction to invasion by foreign molecules or microbial envelope molecules (antigens). Detection of antigens is one method to confirm the presence of pathogenic bacteria in human body fluids. When latex microparticles, coated with antibody specific to a particular antigen on the microbial surface, are mixed with a sample droplet containing antigens, the antigens bind to the particles and, if the antigen concentration is sufficient, cross-link and agglutinate the particles in local clusters. In this latex agglutination test (LAT ), sedimented clumps of particles become visible to the naked eye within the droplet. Coated-particle-based tests, as conventionally performed, can take times ranging from a few minutes to several hours, depending on the test format. LATs remain popular in bacteriology laboratories because of their simplicity, speed and relatively low cost [26 ], although they are generally less sensitive (based on the lowest concentration of antigen detectable) than enzyme-linked immunosorbent assays ( ELISAs) [2]. A technique has been developed in which the body fluid test sample, mixed with a coated microparticle suspension, is exposed to an ultrasonic standing wave for times of the order of 1 min. The resulting concentration of particles in the standing wave increases the rate of particle agglutination and sensitivity of antigen detection. The rate is increased because the local concentration of beads is accentuated in a standing wave. The sensitivity of antigen detection also increases because the ultrasonic clumping process maintains the beads in close proximity, thus increasing the probability of crosslinking. Where latex is replaced by silica as a carrier microparticle, secondary ultrasonic forces can reduce the equilibrium separation of microparticles and thus further enhance the sensitivity of reaction [27]. Enhancements of the LAT antigen detection sensitivity of 2500-fold for C-reactive protein, 500-fold for fungal antigen and 32-fold for meningococcal antigen have been reported [2]. Standing wave detection of meningococcal antigen has received most attention in the clinical and laboratory context because of the exceptional need for rapid diagnosis of aggressive bacterial meningitis [5,28] so that informed vaccination policies can be implemented rapidly. It has been shown that ultrasound enhanced latex agglutination gives serogrouping information in four times more cases than are detected by conventional agglutination tests on serum [5]. Retrospective serogrouping of infected serum samples by ultrasound is comparable to that achievable by current polymerase chain reaction (PCR) procedures for detection of bacterial DNA [5]. The simplicity of the ultrasonic technique opens up the possibility that it will provide rapid diagno-
sis in the local bacteriology laboratory and that its epidemiological information can complement that obtainable from, sometimes remote, specialized PCR facilities. 3.4. Ultrasonic enhancement of bioseparation rates in biphasic aqueous systems Highly selective separations can be achieved when cells or biomolecules partition rapidly between the two phases formed when a high concentration of aqueous solutions of polymers such as polyethyleneglycol (PEG ) and dextran are mixed together [29]. The collection of the partitioned cells or solute involves coalescence and density-dependent movement of droplets to form two bulk phases. This process, which can take 30 min or longer at room temperature, is routinely accelerated by centrifugation. Biphasic separation of Saccharomyces cerevisiae and E. coli can be achieved three times faster in a low-power ultrasonic standing wave than with centrifugation [2]. The ability to complete a partition separation more rapidly under a low-ultrasonic power than with centrifugation suggests that ultrasound has the potential required to facilitate in-line bioanalytical applications of aqueous biphasic separations [30]. 3.5. Standing-wave enhancement of cell-surface phenomena Adhesive interactions of cells suspended in polymer solutions have been examined in an ultrasonic standing wave by light microscopy. The standing wave levitated the cells away from any solid boundary [31]. Manipulation of cells in a standing wave has also been employed to draw spleen and tissue culture cells together in suspension prior to cell fusion by an electric pulse. The technique exploited the ability of ultrasound, in contrast to dielectrophoresis, to manipulate cells in physiological ionic strength media [7].
4. Conclusions It has been outlined above that the sophistication, characterization and control of ultrasonic particle manipulations have increased markedly in recent years. While pursuit of the operational advantages in bringing cells [7] or bio-particles [5] into close proximity using ultrasound can proceed on an empirical basis, informed development of such techniques requires a further appreciation and analysis of the relative contributions of the different forces expressed in standing-wave phenomena. The need for such an understanding is signalled by observations that very low concentrations of micronsized bacteria can be harvested from suspension if mixed with larger particles such as yeast [3] and that micropar-
W.T. Coakley et al. / Ultrasonics 38 (2000) 638–641
ticles can, in a resonant ultrasonic field, be filtered onto the surface of structures with a pore size two orders of magnitude larger than the suspended particles [32]. Developments in the combination of microscopic observation, particle motion analysis modelling and resonator design [8,17,33] will contribute significantly to this area and to the general exploitation of the ability to control microparticle manipulation in suspension.
Acknowledgements The authors are grateful to BBSRC, Meningitis Research Foundation and the EU TMR for support of some of the above work.
References [1] M. Gro¨schl, Acustica 84 (1998) 432. [2] W.T. Coakley, Trends Biotech. 15 (1997) 506. [3] J.J Hawkes, M.S. Limaye, W.T. Coakley, J. Appl. Microbiol. 82 (1997) 39. [4] T. Gaida, O. Doblhoff-Dier, K. Strutzenberger, H. Katinger, W. Burger, M. Gro¨schl, B. Handl, E. Benes, Biotechnol. Prog. 12 (1996) 73. [5] R. Allman, W.T Coakley, Bioseparation 4 (1994) 29. [6 ] S.J. Gray, M.A. Sobanski, E.B. Kaczmarski, M. Guiver, W.J. Marsh, R. Borrow, R.A. Barnes, W.T. Coakley, J. Clin. Microbiol. 37 (1999) 1797. [7] D.W. Bardsley, J.E. Liddell, W.T. Coakley, D.J. Clarke, J. Immunol. Meth. 129 (1990) 41. [8] R.K. Gould, W.T. Coakley, M.A. Grundy, Ultrasonics 30 (1992) 239. [9] G. Whitworth, W.T. Coakley, J. Acoust. Soc. Am. 91 (1992) 79.
641
[10] S.M. Woodside, J. Piret, M. Gro¨schl, E. Benes, B.D. Bowen, AIChE J. 44 (1998) 1976. [11] C.A Miles, M.J. Morley, W.R Hudson, B.M. Mackay, J. Appl. Bact. 78 (1995) 47. [12] C.M. Cousins, S.E. Higgins, M.S. Limaye, J.J. Hawkes, W.T. Coakley, P. Holownia, C.P. Price, Acustica 85 (1999) S150. [13] M.S. Limaye, W.T. Coakley, J. Appl. Microbiol. 84 (1998) 1035. [14] S. Gupta, D.L. Feke, I. Manas-Zlocczower, Chem. Eng. Sci. 50 (1995) 3275. [15] S. Peterson, G. Perkins, C. Baker, In: Proc. IEEE 8th Ann. Conf. Eng. Med. Biol. Soc. (1986) 154–156. [16 ] C.J. Schram, Adv. Sonochem. 2 (1991) 293. [17] M. Gro¨schl, Acustica 84 (1998) 632. [18] T. Kozuka, H. Mitome, T. Fukuda, Jpn. J. Appl. Phys. 37 (1998) 2974. [19] J.J. Hawkes, J.J. Cefai, D.A. Barrow, W.T. Coakley, L.G. Briarty, J. Phys. D Appl. Phys. 31 (1998) 1673. [20] J. Spengler, M. Jekel, , A. Tiehm, U. Neiss ( Eds.), TU HamburgHarburg Rep. Sanit. Eng. 25 (1999) 189. [21] H. Bierau, A. Perani, M.. Al Rubeai, A.N. Emery, J. Biotechnol. 62 (1998) 195. [22] J. Zhang, A. Collins, M. Chen, I. Knyazev, R. Gentz, Biotech. Bioeng. 59 (1998) 351. [23] J.J. Hawkes, J.J.D. Barrow, W.T. Coakley, Ultrasonics 36 (1998) 925. [24] K. Yasuda, S.S. Haupt, S. Umemura, T. Yagi, M. Nishida, Y. Shibata, J. Acoust. Soc. Am. 102 (1997) 642. [25] J.J. Hawkes, C. Cousins, W.T. Coakley, P.J. Keay, Acustica 85 (1999) S151. [26 ] D.N. Greenberg, D.P. Ascher, B.A. Yoder, D.M. Hensley, H.S. Heiman, J.F. Keith, J. Clin. Microbiol. 33 (1995) 193. [27] N.E. Thomas, M.A. Sobanski, W.T. Coakley, Ultrasound Med. Biol. 25 (1999) 443. [28] R.A. Barnes, P. Jenkins, W.T. Coakley, Arch. Dis. Child. 78 (1998) 58. [29] K.S.M.S. Raghavarao, N.K Rastogi, M.K. Gowthaman, N.G. Karannth, Adv. Appl. Microbiol. 41 (1995) 97. [30] B.Y. Zaslavsky, Anal. Chem. 64 (1992) A765. [31] H. Darmani, W.T. Coakley, Biochim. Biophys. Acta 1021 (1990) 182. [32] S. Gupta, D.L. Feke, AIChE J. 44 (1998) 1005. [33] D.L. Feke, A.E. Penrod, Acustica 85 (1999) S92.