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Selective detection of specific bacteria using dielectrophoretic impedance measurement method combined with an antigen–antibody reaction
Journal of Electrostatics 58 (2003) 229–246
Selective detection of specific bacteria using dielectrophoretic impedance measurement method combined with an antigen–antibody reaction Junya Suehiro*, Daisuke Noutomi, Masanori Shutou, Masanori Hara Department of Electrical and Electronic Systems Engineering, Graduate School of Information Science and Electrical Engineering, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan Received 21 October 2002; received in revised form 7 March 2003; accepted 11 March 2003
Abstract This paper describes a new selective detection method for specific bacteria by using a dielectrophoretic impedance measurement method combined with an antigen–antibody reaction. The authors have previously proposed a bacteria detection technique called dielectrophoretic impedance measurement (DEPIM) using positive dielectrophoretic force to capture bacteria in suspension onto an interdigitated microelectrode array. In this paper, the authors propose a selective bacteria detection method using DEPIM combined with an antigen–antibody reaction. A suspension containing Escherichia coli (E. coli) and Serratia marcescens (Serratia) was tested as a sample specimen. Those two bacteria were captured onto a microelectrode by positive dielectrophoresis in almost equal amounts. Agglutination of target bacteria caused by an antigen–antibody reaction was combined with conventional DEPIM in two different ways. As a result of agglutination, target bacteria became larger than nontarget ones and could experience higher dielectrophoretic force. In one method, E. coli and Serratia were trapped together under positive dielectrophoresis and then agglutinated E. coli was selectively left in the electrode gap by washing process. In the other method, agglutination products, which had been produced in advance, were selectively trapped and detected by DEPIM. It was experimentally confirmed that proposed two
*Corresponding author. Tel.: +81-92-642-3912; fax: +81-92-642-3964. E-mail address: [email protected] (J. Suehiro). 0304-3886/03/$ - see front matter r 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0304-3886(03)00062-7
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methods could selectively detect E. coli from the mix suspension as long as E. coli population was more than that of Serratia. r 2003 Elsevier Science B.V. All rights reserved. Keywords: Dielectrophoresis; Impedance; ‘‘DEPIM’’; Bacteria inspection; Antigen–antibody reaction; Agglutination
1. Introduction Recently, bacteria inspection is becoming more important in diverse fields such as bioscience research, medical diagnosis and hazard analysis in food industry. Conventional bacteria inspection methods, such as a colony counting technique, are well established and reliable. However, they require rather long time (typically a few days) and therefore cannot provide a fast diagnosis in case of emergency. The authors have previously proposed a new detection technique for biological cells by using dielectrophoresis (DEP) [1]. The technique called dielectrophoretic impedance measurement (DEPIM) utilizes the positive dielectrophoretic force to trap suspended biological cells onto an interdigitated microelectrode array in the form of pearlchains. Higher cell concentrations result in faster development of the pearl-chains, which are electrically connected in parallel to the electrode gap and hence increase the conductance and the capacitance between the electrodes. By monitoring the temporal variation of the electrode impedance or admittance, the cell population can be quantitatively evaluated. A similar impedance technique for measuring dielectrophoretic collection of biological cells has been proposed by Milner et al. [2]. DEPIM can realize fast and simple bacteria inspection by using only electrical phenomena and instruments. Thus DEPIM is suitable for an automated bacteria inspection system, which is necessary in a food safety system based on Hazard Analysis and Critical Control Point (HACCP) regulation [3]. In bacteria inspection, there are many cases in which a specific bacteria is to be detected according to the species or its physiological state. The authors have previously demonstrated a selective DEPIM technique according to cell viability [4]. It was found that dielectrophoresis of heat-treated bacteria showed strong dependency on viability when applied field frequency was as high as 1 MHz. As a result, viable bacteria could be exclusively collected by positive dielectrophoresis and selectively detected by the DEPIM technique from a suspension also containing heat-treated nonviable cells. However, the selective DEPIM based on this principle is limited to the case where dielectric properties of target bacteria are distinctively different from those of other bacteria in the mixture. The authors have already proposed the other type of selective DEPIM technique, which utilized an antigen–antibody reaction to realize selective detection of bacteria of a certain cell strain [5]. The antigen–antibody reaction was utilized in DEPIM measurements in two different ways. In the first method, antibody was added to the cell suspension after dielectrophoretic trapping of bacteria in order to cause agglutination of the antibody-specific bacteria. The DEP force exerted on agglutinated bacteria, whose apparent size increased, became more dominant than
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hydrodynamic drag force exerted by a liquid flow. In the second method, antibody molecules were immobilized onto the microelectrode surfaces before the DEP cell capture so that only antibody-specific bacteria would be bound to the immobilized antibody. These two methods provided the means whereby specific bacteria could be selectively left between the electrode gap when the electric field was reduced after the preliminary DEP cell collection procedure. However, these methods had been tested only with suspension of one bacteria species (E. coli) and not yet applied to a truly selective bacteria detection from mix suspension of various types of cells. In this work, selective DEPIM inspection of E. coli from a mix suspension of E. coli and Serratia marcescens (Serratia) was demonstrated using agglutination phenomena caused by E. coli-specific antibodies. Two different protocols were proposed and tested. In one method, antibody was directly added to a microelectrode chamber of DEPIM. The other method employed an additional electrode system that served as an agglutination reactor assisted by dielectrophoresis. It was experimentally confirmed that agglutinated E. coli was selectively detected by both methods.
2. Principles Fig. 1 depicts a basic diagram of DEPIM apparatus, which consists of three major parts: a microelectrode, a voltage source and impedance measurement equipment. By energizing the microelectrode with ac signals of appropriate amplitude and frequency, positive dielectrophoresis is achieved near the electrode arrays. As a result, suspended cells are trapped in the electrode gap. If trapped cells possess
Bacteria Dielectrophoresis
Microelectrode
Ac voltage source Impedance analyzer (Lock-in amplifier) Fig. 1. A schematic diagram of DEPIM for bacteria inspection.
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higher electrical admittance than the suspension medium, total admittance of the microelectrode increases as the number of dielectrophoretically trapped cells increases with elapsed time. By analyzing increment rate of admittance, cell concentration in suspension liquid can be quantitatively estimated [1]. Typical DEPIM results are shown in Fig. 2, in which four different bacteria (E. coli, Klebsiella pneumoniae, Serratia marcescens and Pseudomonas aeruginosa), which are similar in their size and shape, were individually measured at the same order of concentration (108 CFU/ml, CFU means colony forming units). In the figure, only a conductance component of the measured admittance increment is shown because a susceptance component increases in the same manner. As explained in the authors’ previous work [1], the conductance increase is due to the presence of bacteria that are trapped and enriched between the electrode gap under positive DEP. It was found that the conductance increase rate at t ¼ 0 was directly proportional to the cell population that was to be quantitatively evaluated by the DEPIM method. Temporal increase of conductance showed similar characteristics and hardly depended on bacteria species. Similar results were obtained with various electric field frequencies ranging from 1 kHz to 1 MHz although the relative order of conductance increase was altered between these bacteria. This result implies that dielectric properties of those bacteria are so similar that their experiencing dielectrophoretic force and electrical impedance do not show distinctive features, which are necessary to realize a selective DEPIM inspection [4].
200 E.coli Klebsiella Pseudomonas Serratia
Conductance, GT (µS)
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Time, t (s) Fig. 2. An example of DEPIM detection results of four different bacteria (E. coli, Klebsiella pneumoniae, Serratia marcescens and Pseudomonas aeruginosa) which are similar in their size and shape. Electric field frequency was 1 MHz.
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One possible way to overcome this limitation and to add selectivity to the conventional DEPIM technique is additional introduction of a biochemical reaction. Especially, an antigen–antibody reaction with high selectivity is possibly suitable for improving DEPIM selectivity. Biological cell membranes contain protein molecules (antigen), which are specific to the cell species or strain. An antibody is a molecule that can be bound with a particular type of antigen. The binding reaction between antigen and antibody is highly selective on a molecular basis, and has been widely employed as a means of identifying unknown microorganism in clinical diagnosis [6]. In this study, agglutination of bacteria caused by an antigen–antibody reaction was utilized to realize a selective DEPIM technique. Agglutination is a binding reaction between many particles or biological cells covered with antigen. Antibody serves as a kind of ‘‘glue’’ that clumps these particles together by a biochemical reaction. Usually, the agglutination products can become large enough to be recognized with the naked eyes. This is a reason why the agglutination has become one of the most widely used immunoassay techniques. Protocols of selective DEPIM utilizing agglutination are schematically illustrated in Figs. 3 and 4. In this study, the following two different methods were proposed and tested.
Nontarget cell
Target cell
Antibody
DEP force Electrode Glass substrate
(a)
(b) Liquid flow
Agglutinated target cells
(c) Fig. 3. A schematic diagram outlining the principle of a selective DEPIM method utilizing agglutination phenomena caused by antigen–antibody reaction (Method A). (a) Preliminary DEP cell trapping to a microelectrode without selectivity. (b) Adding antibody specific to target bacteria in order to cause their agglutination. (c) Washing process to eliminate nontarget bacteria that do not cause agglutination and kept the original size.
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Target cell Antibody Liquid flow DEP force Agglutinated target cells To reservoir or DEPIM chamber Needle electrode
Nontarget cell
(a)
(b) Agglutinated target cells Liquid flow
DEP force
Electrode Glass substrate (c) Fig. 4. A schematic diagram outlining the principle of a selective DEPIM method utilizing agglutination phenomena caused by antigen–antibody reaction (Method B). (a) Preliminary DEP cell trapping to needle electrode tips without selectivity. Antibody specific to target bacteria has been added to the cell suspension in advance so that target bacteria cause agglutination enhanced by positive dielectrophoresis. (b) Washing process. The agglutinated target bacteria and nontarget ones are washed away together by liquid flow and sent to a reservoir for the following DEPIM inspection. (c) Selective DEPIM inspection of agglutinated target bacteria. DEP force and drag force are adjusted so that only agglutination products of target bacteria can be selectively trapped to a microelectrode.
2.1. Method A (Fig. 3) Firstly, suspended bacteria are trapped by positive dielectrophoresis (Fig. 3a). When several different species of bacteria with similar dielectric properties are mixed in the suspension, they will be equally trapped as expected from Fig. 2. After the preliminary DEP trapping, antibody that is specific to the target bacteria is added to
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cause agglutination (Fig. 3b). The apparent size of target bacteria can be increased as a result of agglutination. By adding antibody after DEP trapping of bacteria, agglutination can be secured because trapped cells have previously contacted with each other in the electrode gap. Finally, the electric field strength is decreased and liquid flow is introduced into the chamber so that nontarget cells, which keep their intact shape and size, are removed from the electrode gap by drag force exceeding the DEP force (Fig. 3c, washing process). On the other hand, agglutinated target cells can be left on the electrode even after decreasing electric field strength in liquid flow. For a spherical particle, drag force is proportional to the particle radius a; while DEP force increases with the particle volume or a3 [7]. As a result, relative DEP force to drag force is proportional to a2 : Thus the agglutinated target bacteria can be selectively retained under a dominant DEP force. Markx et al. have proposed a similar separation method of biological cells based on imbalance between DEP and drag forces although the force imbalance was realized by DEP force dependence on their dielectric properties not on their size [8].
2.2. Method B (Fig. 4) In Method A, antibody is added to a microelectrode in which bacteria have been already trapped and enriched by positive dielectrophoresis. One advantage of this method is that one microelectrode can be used for the DEP enhanced agglutination as well as the following DEPIM inspection. However, the DEP trap region achieved in a microelectrode is limited to a rather small area near the thin electrode surface. This implies that the number of agglutinated bacteria may be limited and that resultant impedance change may not be large enough to realize highly sensitive bacteria detection. To overcome this drawback, Method B employs two individual electrode systems as shown in Fig. 4. The first electrode system serves only as an agglutination reactor assisted by dielectrophoresis (Fig. 4a). Contrary to the Method A, antibody is added to suspension before DEP cell trapping. It is expected that agglutination is enhanced by DEP because cells can collide with each other more frequently [9]. In order to increase the number of agglutinated bacteria, the electrode system preferably has a larger configuration such as a needle-to-needle electrode system so that the DEP trap region is enlarged. After the DEP capturing, the electrode potential is switched off so that the agglutinated target bacteria and nontarget ones are washed away together by streaming liquid and sent to a reservoir for the following DEPIM inspection (Fig. 4b). By repeating these procedures, more agglutination products can be obtained and stored. Finally, suspension of these bacteria mixture is introduced to the second microelectrode system, which is used only for DEPIM inspection (Fig. 4c). Agglutinated target cells can be selectively captured by positive dielectrophoresis under a condition that DEP force exceeding the drag force effectively acts only on larger agglutinated target bacteria according to the same principle as Method A.
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3. Materials and methods 3.1. Cells and antibody E. coli strain K12 and Serratia marcescens (Serratia) bacteria were incubated on agar plates. The size of both bacteria was almost 1 mm. Right before the measurements, cells were harvested from agar and suspended in 0.1 M mannitol solution. After several washing by centrifugation, they were individually resuspended in 0.1 M mannitol solution at desired concentrations as determined by a Neubauer haemacytometer or colony counting method. The electrical conductivity of suspension was adjusted to 0.2 mS/m. These cell suspensions were finally mixed in various ratios. E. coli-specific antibody (specific to all ‘‘K’’ and ‘‘O’’ antigenic serotypes, 5 mg/ml concentration) was purchased from Cosmo Bio Co. (Japan) and dialyzed against distilled water for 12 h before use. 3.2. Electrodes Two types of electrode system were used. An interdigitated microelectrode of chrome thin film, which was employed both in Methods A and B, was patterned on a glass substrate by photolithography technique. The electrode had a castle-wall pattern in order to form high and low electric field regions periodically [4]. Each electrode finger had 5 mm length and 5 mm the minimum clearance. The castellations were squares with sides of 50 mm. The 20 electrode fingers formed 19 castellated gaps. The castle-wall electrode was surrounded by a silicon rubber spacer to form a chamber in which cell suspension liquid of 15 ml was stored. In Method B, a multiple needle electrode system was additionally employed for the first DEP-enhanced agglutination process (Fig. 4a). As shown in Fig. 5, four pairs of stainless steel needles (tip radius 10 mm, diameter 1 mm) were placed facing each other so that facing two needle tips were placed with a clearance of 300 mm. The needle electrodes were inserted into a silicon rubber chamber (200 ml volume). The lateral spacing of the needles was 5 mm. 3.3. DEPIM equipment Details of the DEPIM principle and apparatus have been described elsewhere [1,4,5]. Sinusoidal ac voltage was generated by a function generator (FG110, Yokogawa, Japan) and applied to the electrode system. If necessary, the output signal was amplified by a high-speed bipolar amplifier (Model 4055, NF Corporation, Japan). Electrode impedance measurements were carried out using a DSP lock-in amplifier (Model 7280, Perkin-Elmer Instruments, USA). The impedance measurement apparatus was controlled by a personal computer, which also served as a data recorder and analyzer. Visual observation of dielectrophoresis and antigen–antibody reaction was conducted by using an inverted microscope
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Fig. 5. Construction of a needle electrode system used for preliminary DEP trapping in Method B.
(Diaphoto, Nikon, Japan) and a CCD digital camera (COOLPIX 990, Nikon, Japan). 3.4. Experimental procedures 3.4.1. Method A Cell suspension liquid was continuously fed into the microelectrode chamber from a reservoir by a peristaltic pump at a flow rate of 0.9 ml/min. DEP cell trapping to the microelectrode was performed with ac voltage of 100 kHz frequency and 7 V amplitude (peak-to-peak value) for 20 min. Then 200 ml of antibody solution was added to cell suspension in the reservoir (1.8 ml volume) and introduced into the microelectrode chamber to cause agglutination reaction. The final concentration of antibody was 0.5 mg/ml. Twenty minutes later, ac voltage amplitude was decreased to 0.5 V and liquid flow rate was increased by 6 times. During these processes, DEPIM inspection was conducted simultaneously. 3.4.2. Method B For the DEP-assisted agglutination process, cell suspension containing antibody (0.1 mg/ml final concentration) was continuously introduced into the chamber with
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needle electrodes (Fig. 5). DEP trapping was conducted with ac voltage of 100 kHz and 100 V for 30 min. In order to realize DEP cell trapping, the needle electrode system had to be energized with higher electrode potential than a microelectrode system because of the larger size. A preliminary experiment revealed that bacteria were not damaged by the high electrical potential. After the DEP process, the ac voltage was switched off and liquid flow rate was increased so that all bacteria were flushed away from the electrode chamber. These procedures were repeated four times in order to increase the number of agglutination products. The outcoming suspension liquid was stored and used for the following DEPIM inspection. The stored cell suspension liquid was supplied to a microelectrode at a flow rate of 0.9 ml/ min. DEPIM was conducted under almost the same conditions as Method A. However, ac voltage amplitude was decreased to 1 V in order to selectively capture agglutinated E. coli that became larger than nontarget one.
4. Results and discussion 4.1. Method A As a preliminary test, a pure suspension of E. coli (108 CFU/ml) was inspected by Method A. Photographs of the DEP collection of E. coli are shown in Fig. 6. At first, bacteria were trapped around the electrode corner due to positive DEP (Fig. 6a). Without addition of E. coli-specific antibody, trapped cells were washed away by increasing liquid flow rate when the electrode potential and the resultant DEP force were decreased (Fig. 6b). On the other hand, some bacteria were left in the electrode gap even after the washing process by adding antibody (Fig. 6c). Comparing Figs. 6a and c, it was noticed that bacteria formed pellet-like products possibly as a result of agglutination. Results of DEPIM measurements simultaneously conducted with these optical observations are summarized in Fig. 7. The electrode conductance increased during the first DEP trapping process. Without antibody addition, the conductance sharply decreased to the initial value right away after the washing process. From Fig. 6b, it is obvious that this conductance drop is due to elimination of DEP-trapped bacteria by washing liquid flow. Contrary to this, the electrode conductance was almost kept constant even after the washing when agglutinated bacteria were left on the electrode. It was also noticed that the conductance slightly decreased just after the antibody addition. It was found that the decrease became greater when the antibody concentration was increased. Although the reason was not clear at this point, it might be possible that the electrical conductivity of the bacterial membrane decreased by the antibody binding. Hereafter, the electrode conductance measured after the washing process is defined as ‘‘residual conductance’’. Similar experiments were conducted with pure Serratia suspension. As expected, Serratia was not agglutinated by E. coli-specific antibody and was not left after the washing process. DEPIM results were not influenced by E. coli-specific antibody addition. These results prove that Method A is at least applicable for identifying unknown pure bacteria.
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Electrode
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Trapped E. coli
(b)
(a)
Agglutinated E. coli
(c) Fig. 6. Photographs of DEP collection process of pure E. coli in Method A. (a) Preliminary DEP trapping. Cells were trapped around the electrode corners where the electric field became stronger. (b) Without adding antibody, almost bacteria were washed away from the electrode gap by streaming liquid flow (washing process). (c) By adding E. coli-specific antibody, E. coli cells agglutinated in the electrode gap. Contrary to (b), the agglutinated bacteria could be left in the electrode gap even after hydrodynamic washing process.
Similar experiments were further conducted with mix suspension of E. coli and Serratia at various mixing ratios. Photographs of the DEP collection of the mixture are summarized in Fig. 8. Agglutination of E. coli was observed even for the mix suspension. However, the number of agglutinated E. coli decreased as the relative ratio of Serratia increased (Figs. 6c, 8a and b). Especially, little agglutination was observed when Serratia population was more than that of E. coli (Fig. 8c). DEPIM
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Antibody addition
Washing
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Conductance, GT (µS)
Without antibody (control) With antibody (agglutination)
200 150 100 Residual conductance
50 0 0
1200 2400 Time, t(s)
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Fig. 7. DEPIM results of pure E. coli obtained by Method A. Measurements were conducted simultaneously with visual observation shown in Fig. 6. As a control, conventional DEPIM result without antibody addition is also depicted. Agglutination could be electrically detected as residual conductance increase after washing process.
results for the mix suspension are shown in Fig. 9. The residual conductance measured after the washing process was influenced by the mixing ratio. As summarized in Fig. 10, the residual conductance decreased with increasing the relative ratio of Serratia. No residual conductance was detected when Serratia population was more than that of E. coli and little agglutination of E. coli was formed as shown in Fig. 8c. According to a theoretical DEPIM model proposed by the authors, the conductance increase was proportional to the number of bacteria trapped to a microelectrode [1]. It was rather difficult to precisely count the number of agglutination in a microscope photograph as shown in Fig. 8. However, Figs. 8 and 10 qualitatively showed that the residual conductance increased when more agglutination products were retained on the microelectrode. 4.2. Method B Fig. 11 is a photograph showing E. coli trapped around two needle tips by positive DEP (107 CFU/ml concentration). Many cells were trapped and formed pearl-chains in the high electric field region. After the DEP trapping, it was observed that these cells agglutinated as a result of antigen–antibody reaction. These agglutinated cells were released from needle tips to the streaming suspension liquid by switching off the electrode potential. Fig. 12 shows agglutinated E. coli which were recovered by the washing process. They were larger than single cells and their size was typically in the range from 5 to 10 mm. As shown in Fig. 13, more agglutination products were formed by increasing antibody concentration. The agglutination of E. coli was obtained also for mix suspension with Serratia. Fig. 14 shows a photograph of E. coli
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Electrode
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Agglutinated E. coli
(b)
(a)
(c) Fig. 8. Photographs showing effects of mixing ratio on DEP collection process of mix suspension of E. coli and Serratia after washing process of Method A. (a) E. coli:Serratia=108:106 CFU/ml. (b) E. coli:Serratia=108:108 CFU/ml. (c) E. coli:Serratia=108:109 CFU/ml. In (a) and (b), agglutination products of E. coli were left in the electrode gap, while little agglutination was observed in (c).
agglutination trapped by positive DEP at the final DEPIM stage in Method B for the mix suspension of E. coli and Serratia (both 107 CFU/ml). It should be noted that large agglutination products of E. coli were selectively trapped because the electrode potential was set to be rather low so that smaller single bacteria (not only Serratia but also E. coli that happened not to agglutinate) could not be dielectrophoretically captured due to dominant drag force exerted by liquid flow. On the other hand, both types of bacteria were not agglutinated nor captured in the microelectrode gap in a control experiment that was conducted without antibody addition in the preliminary DEP cell trapping. DEPIM results obtained by Method B are depicted in Fig. 15.
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Antibody addition
Washing
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6 E.coli:Serratia 10 8 :10 CFU/mL E.coli:Serratia 10 8 :107 CFU/mL 8 E.coli:Serratia 10 8 :10 CFU/mL 8 9 E.coli:Serratia 10 :10 CFU/mL
100 50 0 0
1200 2400 Time, t (s)
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Fig. 9. Effects of mixing ratio on DEPIM results of mix suspension of E. coli and Serratia obtained by Method A.
Normalized residual conductance by pure E. coli
1 0.8 0.6 0.4 0.2 0 10-2
10-1 100 Serratia / E. coli mixing ratio
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Fig. 10. Effects of mixing ratio on the residual conductance measured after washing process of Method A. The residual conductance drastically decreased when Serratia increased beyond E. coli.
Without antibody, the electrode conductance was almost constant because no cells were trapped. By adding antibody to pure E. coli suspension or to the mixture with Serratia, the electrode conductance increased with time due to DEP trapping of the agglutinated E. coli as shown in Fig. 14. The conductance increase for the mix suspension was smaller than for pure E. coli suspension even though E. coli density was identical. When Serratia population was more than that of E. coli, the conductance increase was not observed even with antibody addition.
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100 µm
Trappedand agglutinated E.coli Needle tip Fig. 11. A photograph of preliminary DEP collection process (for 30 min) of pure E. coli in Method B. A large number of cells were trapped and agglutinated around the needle tips.
Agglutinated E. coli
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Single E. coli
Fig. 12. Agglutinated E. coli cells which were formed in a needle electrode system and then recovered by washing process in Method B. Agglutinated cells were larger than single cells.
4.3. Effects of mixing ratio In both methods of A and B, it was found that selective detection of target bacteria (E. coli) was considerably influenced by mixing ratio of nontarget bacteria (Serratia). As shown in Fig. 8, agglutinated E. coli decreased in the number as well as in the size as relative ratio of Serratia increased. As expected from Fig. 2, E. coli and Serratia are equally trapped because their dielectric properties are similar. This means that they are jumbled together as they are enriched and trapped by positive DEP. Serratia cells may prevent direct contact and resultant agglutination of E. coli cells, especially when their number is increased beyond that of E. coli. As depicted in Figs. 10 and 15, proposed two methods enabled selective DEPIM detection of E. coli as long as their population was more than that of Serratia. The required selectivity for practical
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4
3
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0 0 0.1 0.2 0.3 0.4 0.5 0.6 Concentration of antibody (mg/mL)
Fig. 13. Influence of antibody concentration on the number of agglutination products recovered by washing process in Method B. Agglutination was enhanced by increasing the final concentration of antibody added to pure E. coli suspension (107 CFU/ml concentration).
Electrode
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Trapped Agglutination of E.coli
Fig. 14. A photograph of E. coli agglutination trapped by positive DEP at the final DEPIM stage in Method B for the mix suspension of E. coli and Serratia.
bacteria inspection may vary according to the application field. The proposed selective DEPIM may be useful in some areas that do not need extremely high selectivity.
5. Conclusions In order to add selectivity to bacteria inspection by DEPIM, an antigen–antibody reaction was introduced in two different ways. As a result of agglutination caused by
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7
Mix without antibody (E.coli 10 +Serratia 107 CFU/mL) Mix with antibody (E.coli 107 +Serratia 107 CFU/mL) Pure E.coli with antibody (E.coli 107 CFU/mL)
Conductance, GT (µS)
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1
0
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Fig. 15. Results of the final DEPIM inspection in Method B. Conductance increase was observed only when antibody was added in the preliminary DEP process to form E. coli agglutination. Conductance increase for the mix suspension of E. coli and Serratia was lower than that for pure E. coli.
the antigen–antibody reaction, target bacteria became larger than nontarget one and could selectively captured or hold under dominant dielectrophoretic force to hydrodynamic drag force. In one method, all species of bacteria were firstly captured and then agglutinated target cells were selectively left by washing process. In the other method, agglutination products were produced in advance of DEPIM and selectively trapped and detected. It was experimentally confirmed that proposed two methods could selectively detect E. coli from mix suspension with Serratia as long as E. coli population was more than that of Serratia. The latter method seemed to provide higher sensitivity because the number of agglutination products could be increased by repeating preliminary agglutination process enhanced by positive dielectrophoresis. This needs to be clarified by future studies. Acknowledgements The authors wish to thank Mr. Ryo Hamada and Mr. Fumihiro Kusaba for their help in experimental works. Special thanks go to Mr. Ryuichi Yatsunami and Mr. Iwao Yamada for their valuable discussion. This work was partly supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 14550421). References [1] J. Suehiro, R. Yatsunami, R. Hamada, M. Hara, J. Phys. D 32 (1999) 2814–2820. [2] K.R. Milner, A.P. Brown, D.W.E. Allsopp, W.B. Betts, Electron. Lett. 34 (1998) 66–68.
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[3] J.E. Ehiri, G.P. Morris, J. McEwen, Food Control 6 (1995) 341–345. [4] J. Suehiro, R. Hamada, D. Noutomi, M. Shutou, M. Hara, J. Electrostat. 57 (2003) 157–168. [5] J. Suehiro, D. Noutomi, R. Hamada, M. Hara, Proceedings of the IEEE/IAS Annual Meeting, Chicago, Illinois, 2001. [6] P. Tijssen, Practice and Theory of Enzyme Immunoassays, Elsevier, Amsterdam, 1985. [7] T.B. Jones, Electromechanics of Particles, Cambridge University Press, Cambridge, 1995. [8] G.H. Markx, Y. Huang, X-F. Zhou, R. Pethig, Microbiology 140 (1994) 585–591. [9] H. Matsuoka, E. Tamiya, I. Karube, Anal. Chem. 57 (1985) 1998–2002.
Selective detection of specific bacteria using dielectrophoretic impedance measurement method combined with an antigen–antibody reaction Junya Suehiro*, Daisuke Noutomi, Masanori Shutou, Masanori Hara Department of Electrical and Electronic Systems Engineering, Graduate School of Information Science and Electrical Engineering, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan Received 21 October 2002; received in revised form 7 March 2003; accepted 11 March 2003
Abstract This paper describes a new selective detection method for specific bacteria by using a dielectrophoretic impedance measurement method combined with an antigen–antibody reaction. The authors have previously proposed a bacteria detection technique called dielectrophoretic impedance measurement (DEPIM) using positive dielectrophoretic force to capture bacteria in suspension onto an interdigitated microelectrode array. In this paper, the authors propose a selective bacteria detection method using DEPIM combined with an antigen–antibody reaction. A suspension containing Escherichia coli (E. coli) and Serratia marcescens (Serratia) was tested as a sample specimen. Those two bacteria were captured onto a microelectrode by positive dielectrophoresis in almost equal amounts. Agglutination of target bacteria caused by an antigen–antibody reaction was combined with conventional DEPIM in two different ways. As a result of agglutination, target bacteria became larger than nontarget ones and could experience higher dielectrophoretic force. In one method, E. coli and Serratia were trapped together under positive dielectrophoresis and then agglutinated E. coli was selectively left in the electrode gap by washing process. In the other method, agglutination products, which had been produced in advance, were selectively trapped and detected by DEPIM. It was experimentally confirmed that proposed two
*Corresponding author. Tel.: +81-92-642-3912; fax: +81-92-642-3964. E-mail address: [email protected] (J. Suehiro). 0304-3886/03/$ - see front matter r 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0304-3886(03)00062-7
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methods could selectively detect E. coli from the mix suspension as long as E. coli population was more than that of Serratia. r 2003 Elsevier Science B.V. All rights reserved. Keywords: Dielectrophoresis; Impedance; ‘‘DEPIM’’; Bacteria inspection; Antigen–antibody reaction; Agglutination
1. Introduction Recently, bacteria inspection is becoming more important in diverse fields such as bioscience research, medical diagnosis and hazard analysis in food industry. Conventional bacteria inspection methods, such as a colony counting technique, are well established and reliable. However, they require rather long time (typically a few days) and therefore cannot provide a fast diagnosis in case of emergency. The authors have previously proposed a new detection technique for biological cells by using dielectrophoresis (DEP) [1]. The technique called dielectrophoretic impedance measurement (DEPIM) utilizes the positive dielectrophoretic force to trap suspended biological cells onto an interdigitated microelectrode array in the form of pearlchains. Higher cell concentrations result in faster development of the pearl-chains, which are electrically connected in parallel to the electrode gap and hence increase the conductance and the capacitance between the electrodes. By monitoring the temporal variation of the electrode impedance or admittance, the cell population can be quantitatively evaluated. A similar impedance technique for measuring dielectrophoretic collection of biological cells has been proposed by Milner et al. [2]. DEPIM can realize fast and simple bacteria inspection by using only electrical phenomena and instruments. Thus DEPIM is suitable for an automated bacteria inspection system, which is necessary in a food safety system based on Hazard Analysis and Critical Control Point (HACCP) regulation [3]. In bacteria inspection, there are many cases in which a specific bacteria is to be detected according to the species or its physiological state. The authors have previously demonstrated a selective DEPIM technique according to cell viability [4]. It was found that dielectrophoresis of heat-treated bacteria showed strong dependency on viability when applied field frequency was as high as 1 MHz. As a result, viable bacteria could be exclusively collected by positive dielectrophoresis and selectively detected by the DEPIM technique from a suspension also containing heat-treated nonviable cells. However, the selective DEPIM based on this principle is limited to the case where dielectric properties of target bacteria are distinctively different from those of other bacteria in the mixture. The authors have already proposed the other type of selective DEPIM technique, which utilized an antigen–antibody reaction to realize selective detection of bacteria of a certain cell strain [5]. The antigen–antibody reaction was utilized in DEPIM measurements in two different ways. In the first method, antibody was added to the cell suspension after dielectrophoretic trapping of bacteria in order to cause agglutination of the antibody-specific bacteria. The DEP force exerted on agglutinated bacteria, whose apparent size increased, became more dominant than
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hydrodynamic drag force exerted by a liquid flow. In the second method, antibody molecules were immobilized onto the microelectrode surfaces before the DEP cell capture so that only antibody-specific bacteria would be bound to the immobilized antibody. These two methods provided the means whereby specific bacteria could be selectively left between the electrode gap when the electric field was reduced after the preliminary DEP cell collection procedure. However, these methods had been tested only with suspension of one bacteria species (E. coli) and not yet applied to a truly selective bacteria detection from mix suspension of various types of cells. In this work, selective DEPIM inspection of E. coli from a mix suspension of E. coli and Serratia marcescens (Serratia) was demonstrated using agglutination phenomena caused by E. coli-specific antibodies. Two different protocols were proposed and tested. In one method, antibody was directly added to a microelectrode chamber of DEPIM. The other method employed an additional electrode system that served as an agglutination reactor assisted by dielectrophoresis. It was experimentally confirmed that agglutinated E. coli was selectively detected by both methods.
2. Principles Fig. 1 depicts a basic diagram of DEPIM apparatus, which consists of three major parts: a microelectrode, a voltage source and impedance measurement equipment. By energizing the microelectrode with ac signals of appropriate amplitude and frequency, positive dielectrophoresis is achieved near the electrode arrays. As a result, suspended cells are trapped in the electrode gap. If trapped cells possess
Bacteria Dielectrophoresis
Microelectrode
Ac voltage source Impedance analyzer (Lock-in amplifier) Fig. 1. A schematic diagram of DEPIM for bacteria inspection.
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higher electrical admittance than the suspension medium, total admittance of the microelectrode increases as the number of dielectrophoretically trapped cells increases with elapsed time. By analyzing increment rate of admittance, cell concentration in suspension liquid can be quantitatively estimated [1]. Typical DEPIM results are shown in Fig. 2, in which four different bacteria (E. coli, Klebsiella pneumoniae, Serratia marcescens and Pseudomonas aeruginosa), which are similar in their size and shape, were individually measured at the same order of concentration (108 CFU/ml, CFU means colony forming units). In the figure, only a conductance component of the measured admittance increment is shown because a susceptance component increases in the same manner. As explained in the authors’ previous work [1], the conductance increase is due to the presence of bacteria that are trapped and enriched between the electrode gap under positive DEP. It was found that the conductance increase rate at t ¼ 0 was directly proportional to the cell population that was to be quantitatively evaluated by the DEPIM method. Temporal increase of conductance showed similar characteristics and hardly depended on bacteria species. Similar results were obtained with various electric field frequencies ranging from 1 kHz to 1 MHz although the relative order of conductance increase was altered between these bacteria. This result implies that dielectric properties of those bacteria are so similar that their experiencing dielectrophoretic force and electrical impedance do not show distinctive features, which are necessary to realize a selective DEPIM inspection [4].
200 E.coli Klebsiella Pseudomonas Serratia
Conductance, GT (µS)
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100
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Time, t (s) Fig. 2. An example of DEPIM detection results of four different bacteria (E. coli, Klebsiella pneumoniae, Serratia marcescens and Pseudomonas aeruginosa) which are similar in their size and shape. Electric field frequency was 1 MHz.
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One possible way to overcome this limitation and to add selectivity to the conventional DEPIM technique is additional introduction of a biochemical reaction. Especially, an antigen–antibody reaction with high selectivity is possibly suitable for improving DEPIM selectivity. Biological cell membranes contain protein molecules (antigen), which are specific to the cell species or strain. An antibody is a molecule that can be bound with a particular type of antigen. The binding reaction between antigen and antibody is highly selective on a molecular basis, and has been widely employed as a means of identifying unknown microorganism in clinical diagnosis [6]. In this study, agglutination of bacteria caused by an antigen–antibody reaction was utilized to realize a selective DEPIM technique. Agglutination is a binding reaction between many particles or biological cells covered with antigen. Antibody serves as a kind of ‘‘glue’’ that clumps these particles together by a biochemical reaction. Usually, the agglutination products can become large enough to be recognized with the naked eyes. This is a reason why the agglutination has become one of the most widely used immunoassay techniques. Protocols of selective DEPIM utilizing agglutination are schematically illustrated in Figs. 3 and 4. In this study, the following two different methods were proposed and tested.
Nontarget cell
Target cell
Antibody
DEP force Electrode Glass substrate
(a)
(b) Liquid flow
Agglutinated target cells
(c) Fig. 3. A schematic diagram outlining the principle of a selective DEPIM method utilizing agglutination phenomena caused by antigen–antibody reaction (Method A). (a) Preliminary DEP cell trapping to a microelectrode without selectivity. (b) Adding antibody specific to target bacteria in order to cause their agglutination. (c) Washing process to eliminate nontarget bacteria that do not cause agglutination and kept the original size.
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Target cell Antibody Liquid flow DEP force Agglutinated target cells To reservoir or DEPIM chamber Needle electrode
Nontarget cell
(a)
(b) Agglutinated target cells Liquid flow
DEP force
Electrode Glass substrate (c) Fig. 4. A schematic diagram outlining the principle of a selective DEPIM method utilizing agglutination phenomena caused by antigen–antibody reaction (Method B). (a) Preliminary DEP cell trapping to needle electrode tips without selectivity. Antibody specific to target bacteria has been added to the cell suspension in advance so that target bacteria cause agglutination enhanced by positive dielectrophoresis. (b) Washing process. The agglutinated target bacteria and nontarget ones are washed away together by liquid flow and sent to a reservoir for the following DEPIM inspection. (c) Selective DEPIM inspection of agglutinated target bacteria. DEP force and drag force are adjusted so that only agglutination products of target bacteria can be selectively trapped to a microelectrode.
2.1. Method A (Fig. 3) Firstly, suspended bacteria are trapped by positive dielectrophoresis (Fig. 3a). When several different species of bacteria with similar dielectric properties are mixed in the suspension, they will be equally trapped as expected from Fig. 2. After the preliminary DEP trapping, antibody that is specific to the target bacteria is added to
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cause agglutination (Fig. 3b). The apparent size of target bacteria can be increased as a result of agglutination. By adding antibody after DEP trapping of bacteria, agglutination can be secured because trapped cells have previously contacted with each other in the electrode gap. Finally, the electric field strength is decreased and liquid flow is introduced into the chamber so that nontarget cells, which keep their intact shape and size, are removed from the electrode gap by drag force exceeding the DEP force (Fig. 3c, washing process). On the other hand, agglutinated target cells can be left on the electrode even after decreasing electric field strength in liquid flow. For a spherical particle, drag force is proportional to the particle radius a; while DEP force increases with the particle volume or a3 [7]. As a result, relative DEP force to drag force is proportional to a2 : Thus the agglutinated target bacteria can be selectively retained under a dominant DEP force. Markx et al. have proposed a similar separation method of biological cells based on imbalance between DEP and drag forces although the force imbalance was realized by DEP force dependence on their dielectric properties not on their size [8].
2.2. Method B (Fig. 4) In Method A, antibody is added to a microelectrode in which bacteria have been already trapped and enriched by positive dielectrophoresis. One advantage of this method is that one microelectrode can be used for the DEP enhanced agglutination as well as the following DEPIM inspection. However, the DEP trap region achieved in a microelectrode is limited to a rather small area near the thin electrode surface. This implies that the number of agglutinated bacteria may be limited and that resultant impedance change may not be large enough to realize highly sensitive bacteria detection. To overcome this drawback, Method B employs two individual electrode systems as shown in Fig. 4. The first electrode system serves only as an agglutination reactor assisted by dielectrophoresis (Fig. 4a). Contrary to the Method A, antibody is added to suspension before DEP cell trapping. It is expected that agglutination is enhanced by DEP because cells can collide with each other more frequently [9]. In order to increase the number of agglutinated bacteria, the electrode system preferably has a larger configuration such as a needle-to-needle electrode system so that the DEP trap region is enlarged. After the DEP capturing, the electrode potential is switched off so that the agglutinated target bacteria and nontarget ones are washed away together by streaming liquid and sent to a reservoir for the following DEPIM inspection (Fig. 4b). By repeating these procedures, more agglutination products can be obtained and stored. Finally, suspension of these bacteria mixture is introduced to the second microelectrode system, which is used only for DEPIM inspection (Fig. 4c). Agglutinated target cells can be selectively captured by positive dielectrophoresis under a condition that DEP force exceeding the drag force effectively acts only on larger agglutinated target bacteria according to the same principle as Method A.
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3. Materials and methods 3.1. Cells and antibody E. coli strain K12 and Serratia marcescens (Serratia) bacteria were incubated on agar plates. The size of both bacteria was almost 1 mm. Right before the measurements, cells were harvested from agar and suspended in 0.1 M mannitol solution. After several washing by centrifugation, they were individually resuspended in 0.1 M mannitol solution at desired concentrations as determined by a Neubauer haemacytometer or colony counting method. The electrical conductivity of suspension was adjusted to 0.2 mS/m. These cell suspensions were finally mixed in various ratios. E. coli-specific antibody (specific to all ‘‘K’’ and ‘‘O’’ antigenic serotypes, 5 mg/ml concentration) was purchased from Cosmo Bio Co. (Japan) and dialyzed against distilled water for 12 h before use. 3.2. Electrodes Two types of electrode system were used. An interdigitated microelectrode of chrome thin film, which was employed both in Methods A and B, was patterned on a glass substrate by photolithography technique. The electrode had a castle-wall pattern in order to form high and low electric field regions periodically [4]. Each electrode finger had 5 mm length and 5 mm the minimum clearance. The castellations were squares with sides of 50 mm. The 20 electrode fingers formed 19 castellated gaps. The castle-wall electrode was surrounded by a silicon rubber spacer to form a chamber in which cell suspension liquid of 15 ml was stored. In Method B, a multiple needle electrode system was additionally employed for the first DEP-enhanced agglutination process (Fig. 4a). As shown in Fig. 5, four pairs of stainless steel needles (tip radius 10 mm, diameter 1 mm) were placed facing each other so that facing two needle tips were placed with a clearance of 300 mm. The needle electrodes were inserted into a silicon rubber chamber (200 ml volume). The lateral spacing of the needles was 5 mm. 3.3. DEPIM equipment Details of the DEPIM principle and apparatus have been described elsewhere [1,4,5]. Sinusoidal ac voltage was generated by a function generator (FG110, Yokogawa, Japan) and applied to the electrode system. If necessary, the output signal was amplified by a high-speed bipolar amplifier (Model 4055, NF Corporation, Japan). Electrode impedance measurements were carried out using a DSP lock-in amplifier (Model 7280, Perkin-Elmer Instruments, USA). The impedance measurement apparatus was controlled by a personal computer, which also served as a data recorder and analyzer. Visual observation of dielectrophoresis and antigen–antibody reaction was conducted by using an inverted microscope
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Fig. 5. Construction of a needle electrode system used for preliminary DEP trapping in Method B.
(Diaphoto, Nikon, Japan) and a CCD digital camera (COOLPIX 990, Nikon, Japan). 3.4. Experimental procedures 3.4.1. Method A Cell suspension liquid was continuously fed into the microelectrode chamber from a reservoir by a peristaltic pump at a flow rate of 0.9 ml/min. DEP cell trapping to the microelectrode was performed with ac voltage of 100 kHz frequency and 7 V amplitude (peak-to-peak value) for 20 min. Then 200 ml of antibody solution was added to cell suspension in the reservoir (1.8 ml volume) and introduced into the microelectrode chamber to cause agglutination reaction. The final concentration of antibody was 0.5 mg/ml. Twenty minutes later, ac voltage amplitude was decreased to 0.5 V and liquid flow rate was increased by 6 times. During these processes, DEPIM inspection was conducted simultaneously. 3.4.2. Method B For the DEP-assisted agglutination process, cell suspension containing antibody (0.1 mg/ml final concentration) was continuously introduced into the chamber with
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needle electrodes (Fig. 5). DEP trapping was conducted with ac voltage of 100 kHz and 100 V for 30 min. In order to realize DEP cell trapping, the needle electrode system had to be energized with higher electrode potential than a microelectrode system because of the larger size. A preliminary experiment revealed that bacteria were not damaged by the high electrical potential. After the DEP process, the ac voltage was switched off and liquid flow rate was increased so that all bacteria were flushed away from the electrode chamber. These procedures were repeated four times in order to increase the number of agglutination products. The outcoming suspension liquid was stored and used for the following DEPIM inspection. The stored cell suspension liquid was supplied to a microelectrode at a flow rate of 0.9 ml/ min. DEPIM was conducted under almost the same conditions as Method A. However, ac voltage amplitude was decreased to 1 V in order to selectively capture agglutinated E. coli that became larger than nontarget one.
4. Results and discussion 4.1. Method A As a preliminary test, a pure suspension of E. coli (108 CFU/ml) was inspected by Method A. Photographs of the DEP collection of E. coli are shown in Fig. 6. At first, bacteria were trapped around the electrode corner due to positive DEP (Fig. 6a). Without addition of E. coli-specific antibody, trapped cells were washed away by increasing liquid flow rate when the electrode potential and the resultant DEP force were decreased (Fig. 6b). On the other hand, some bacteria were left in the electrode gap even after the washing process by adding antibody (Fig. 6c). Comparing Figs. 6a and c, it was noticed that bacteria formed pellet-like products possibly as a result of agglutination. Results of DEPIM measurements simultaneously conducted with these optical observations are summarized in Fig. 7. The electrode conductance increased during the first DEP trapping process. Without antibody addition, the conductance sharply decreased to the initial value right away after the washing process. From Fig. 6b, it is obvious that this conductance drop is due to elimination of DEP-trapped bacteria by washing liquid flow. Contrary to this, the electrode conductance was almost kept constant even after the washing when agglutinated bacteria were left on the electrode. It was also noticed that the conductance slightly decreased just after the antibody addition. It was found that the decrease became greater when the antibody concentration was increased. Although the reason was not clear at this point, it might be possible that the electrical conductivity of the bacterial membrane decreased by the antibody binding. Hereafter, the electrode conductance measured after the washing process is defined as ‘‘residual conductance’’. Similar experiments were conducted with pure Serratia suspension. As expected, Serratia was not agglutinated by E. coli-specific antibody and was not left after the washing process. DEPIM results were not influenced by E. coli-specific antibody addition. These results prove that Method A is at least applicable for identifying unknown pure bacteria.
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Electrode
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Trapped E. coli
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Agglutinated E. coli
(c) Fig. 6. Photographs of DEP collection process of pure E. coli in Method A. (a) Preliminary DEP trapping. Cells were trapped around the electrode corners where the electric field became stronger. (b) Without adding antibody, almost bacteria were washed away from the electrode gap by streaming liquid flow (washing process). (c) By adding E. coli-specific antibody, E. coli cells agglutinated in the electrode gap. Contrary to (b), the agglutinated bacteria could be left in the electrode gap even after hydrodynamic washing process.
Similar experiments were further conducted with mix suspension of E. coli and Serratia at various mixing ratios. Photographs of the DEP collection of the mixture are summarized in Fig. 8. Agglutination of E. coli was observed even for the mix suspension. However, the number of agglutinated E. coli decreased as the relative ratio of Serratia increased (Figs. 6c, 8a and b). Especially, little agglutination was observed when Serratia population was more than that of E. coli (Fig. 8c). DEPIM
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Antibody addition
Washing
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Conductance, GT (µS)
Without antibody (control) With antibody (agglutination)
200 150 100 Residual conductance
50 0 0
1200 2400 Time, t(s)
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Fig. 7. DEPIM results of pure E. coli obtained by Method A. Measurements were conducted simultaneously with visual observation shown in Fig. 6. As a control, conventional DEPIM result without antibody addition is also depicted. Agglutination could be electrically detected as residual conductance increase after washing process.
results for the mix suspension are shown in Fig. 9. The residual conductance measured after the washing process was influenced by the mixing ratio. As summarized in Fig. 10, the residual conductance decreased with increasing the relative ratio of Serratia. No residual conductance was detected when Serratia population was more than that of E. coli and little agglutination of E. coli was formed as shown in Fig. 8c. According to a theoretical DEPIM model proposed by the authors, the conductance increase was proportional to the number of bacteria trapped to a microelectrode [1]. It was rather difficult to precisely count the number of agglutination in a microscope photograph as shown in Fig. 8. However, Figs. 8 and 10 qualitatively showed that the residual conductance increased when more agglutination products were retained on the microelectrode. 4.2. Method B Fig. 11 is a photograph showing E. coli trapped around two needle tips by positive DEP (107 CFU/ml concentration). Many cells were trapped and formed pearl-chains in the high electric field region. After the DEP trapping, it was observed that these cells agglutinated as a result of antigen–antibody reaction. These agglutinated cells were released from needle tips to the streaming suspension liquid by switching off the electrode potential. Fig. 12 shows agglutinated E. coli which were recovered by the washing process. They were larger than single cells and their size was typically in the range from 5 to 10 mm. As shown in Fig. 13, more agglutination products were formed by increasing antibody concentration. The agglutination of E. coli was obtained also for mix suspension with Serratia. Fig. 14 shows a photograph of E. coli
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Electrode
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Agglutinated E. coli
(b)
(a)
(c) Fig. 8. Photographs showing effects of mixing ratio on DEP collection process of mix suspension of E. coli and Serratia after washing process of Method A. (a) E. coli:Serratia=108:106 CFU/ml. (b) E. coli:Serratia=108:108 CFU/ml. (c) E. coli:Serratia=108:109 CFU/ml. In (a) and (b), agglutination products of E. coli were left in the electrode gap, while little agglutination was observed in (c).
agglutination trapped by positive DEP at the final DEPIM stage in Method B for the mix suspension of E. coli and Serratia (both 107 CFU/ml). It should be noted that large agglutination products of E. coli were selectively trapped because the electrode potential was set to be rather low so that smaller single bacteria (not only Serratia but also E. coli that happened not to agglutinate) could not be dielectrophoretically captured due to dominant drag force exerted by liquid flow. On the other hand, both types of bacteria were not agglutinated nor captured in the microelectrode gap in a control experiment that was conducted without antibody addition in the preliminary DEP cell trapping. DEPIM results obtained by Method B are depicted in Fig. 15.
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Antibody addition
Washing
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6 E.coli:Serratia 10 8 :10 CFU/mL E.coli:Serratia 10 8 :107 CFU/mL 8 E.coli:Serratia 10 8 :10 CFU/mL 8 9 E.coli:Serratia 10 :10 CFU/mL
100 50 0 0
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Fig. 9. Effects of mixing ratio on DEPIM results of mix suspension of E. coli and Serratia obtained by Method A.
Normalized residual conductance by pure E. coli
1 0.8 0.6 0.4 0.2 0 10-2
10-1 100 Serratia / E. coli mixing ratio
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Fig. 10. Effects of mixing ratio on the residual conductance measured after washing process of Method A. The residual conductance drastically decreased when Serratia increased beyond E. coli.
Without antibody, the electrode conductance was almost constant because no cells were trapped. By adding antibody to pure E. coli suspension or to the mixture with Serratia, the electrode conductance increased with time due to DEP trapping of the agglutinated E. coli as shown in Fig. 14. The conductance increase for the mix suspension was smaller than for pure E. coli suspension even though E. coli density was identical. When Serratia population was more than that of E. coli, the conductance increase was not observed even with antibody addition.
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Trappedand agglutinated E.coli Needle tip Fig. 11. A photograph of preliminary DEP collection process (for 30 min) of pure E. coli in Method B. A large number of cells were trapped and agglutinated around the needle tips.
Agglutinated E. coli
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Single E. coli
Fig. 12. Agglutinated E. coli cells which were formed in a needle electrode system and then recovered by washing process in Method B. Agglutinated cells were larger than single cells.
4.3. Effects of mixing ratio In both methods of A and B, it was found that selective detection of target bacteria (E. coli) was considerably influenced by mixing ratio of nontarget bacteria (Serratia). As shown in Fig. 8, agglutinated E. coli decreased in the number as well as in the size as relative ratio of Serratia increased. As expected from Fig. 2, E. coli and Serratia are equally trapped because their dielectric properties are similar. This means that they are jumbled together as they are enriched and trapped by positive DEP. Serratia cells may prevent direct contact and resultant agglutination of E. coli cells, especially when their number is increased beyond that of E. coli. As depicted in Figs. 10 and 15, proposed two methods enabled selective DEPIM detection of E. coli as long as their population was more than that of Serratia. The required selectivity for practical
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4
3
2
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0 0 0.1 0.2 0.3 0.4 0.5 0.6 Concentration of antibody (mg/mL)
Fig. 13. Influence of antibody concentration on the number of agglutination products recovered by washing process in Method B. Agglutination was enhanced by increasing the final concentration of antibody added to pure E. coli suspension (107 CFU/ml concentration).
Electrode
10 µm
Trapped Agglutination of E.coli
Fig. 14. A photograph of E. coli agglutination trapped by positive DEP at the final DEPIM stage in Method B for the mix suspension of E. coli and Serratia.
bacteria inspection may vary according to the application field. The proposed selective DEPIM may be useful in some areas that do not need extremely high selectivity.
5. Conclusions In order to add selectivity to bacteria inspection by DEPIM, an antigen–antibody reaction was introduced in two different ways. As a result of agglutination caused by
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7
Mix without antibody (E.coli 10 +Serratia 107 CFU/mL) Mix with antibody (E.coli 107 +Serratia 107 CFU/mL) Pure E.coli with antibody (E.coli 107 CFU/mL)
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3
2
1
0
0
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Fig. 15. Results of the final DEPIM inspection in Method B. Conductance increase was observed only when antibody was added in the preliminary DEP process to form E. coli agglutination. Conductance increase for the mix suspension of E. coli and Serratia was lower than that for pure E. coli.
the antigen–antibody reaction, target bacteria became larger than nontarget one and could selectively captured or hold under dominant dielectrophoretic force to hydrodynamic drag force. In one method, all species of bacteria were firstly captured and then agglutinated target cells were selectively left by washing process. In the other method, agglutination products were produced in advance of DEPIM and selectively trapped and detected. It was experimentally confirmed that proposed two methods could selectively detect E. coli from mix suspension with Serratia as long as E. coli population was more than that of Serratia. The latter method seemed to provide higher sensitivity because the number of agglutination products could be increased by repeating preliminary agglutination process enhanced by positive dielectrophoresis. This needs to be clarified by future studies. Acknowledgements The authors wish to thank Mr. Ryo Hamada and Mr. Fumihiro Kusaba for their help in experimental works. Special thanks go to Mr. Ryuichi Yatsunami and Mr. Iwao Yamada for their valuable discussion. This work was partly supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 14550421). References [1] J. Suehiro, R. Yatsunami, R. Hamada, M. Hara, J. Phys. D 32 (1999) 2814–2820. [2] K.R. Milner, A.P. Brown, D.W.E. Allsopp, W.B. Betts, Electron. Lett. 34 (1998) 66–68.
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[3] J.E. Ehiri, G.P. Morris, J. McEwen, Food Control 6 (1995) 341–345. [4] J. Suehiro, R. Hamada, D. Noutomi, M. Shutou, M. Hara, J. Electrostat. 57 (2003) 157–168. [5] J. Suehiro, D. Noutomi, R. Hamada, M. Hara, Proceedings of the IEEE/IAS Annual Meeting, Chicago, Illinois, 2001. [6] P. Tijssen, Practice and Theory of Enzyme Immunoassays, Elsevier, Amsterdam, 1985. [7] T.B. Jones, Electromechanics of Particles, Cambridge University Press, Cambridge, 1995. [8] G.H. Markx, Y. Huang, X-F. Zhou, R. Pethig, Microbiology 140 (1994) 585–591. [9] H. Matsuoka, E. Tamiya, I. Karube, Anal. Chem. 57 (1985) 1998–2002.