6226
Langmuir 2003, 19, 6226-6229
Specific Recognition of Bacteria by Surface-Templated Polymer Films Kanad Das,† Jacques Penelle,†,‡,| Vincent M. Rotello,† and Klaus Nu¨sslein*,§,| Department of Chemistry, Department of Polymer Science and Engineering, and Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003 Received February 12, 2003. In Final Form: May 1, 2003 Real-time selective recognition of cells is an important capability for medicine, molecular biology, and environmental science. We report here a cell-selective polymer film, obtained via polymerization of a thin film of functionalized monomers, in contact with a target cell. Under controlled conditions, the replica maintains exceptional structural memory of both shape and surface functionality of the initial bacterial cell. High selectivity is observed between bacteria featuring different cell surfaces (Gram-positive vs negative), shapes (rods vs spheres), and cell arrangements (single cells vs short chains vs clusters). Interfacing to a quartz crystal microbalance provides real-time, selective detection of bacteria at environmentally relevant concentrations of less than 500 cells/mL, an improvement of 100-fold in response time and 106 fold in sensitivity over current methods.
Introduction Selective and efficient recognition of cells is an important goal in the areas of biomedicine, molecular biology, environmental science, and detection of hazardous biological agents. Bacteria are commonly sensed directly by electrochemical transduction methods, involving the transport of samples to the laboratory and generally taking several minutes to hours.1,2 A chemical and more selective strategy of direct cell detection involves surface techniques, usually self-assembled monolayers (SAMs) or phospholipid bilayers placed on the signal transduction element.3-8 Immunological methods using highly specific antigenantibody reactions have been exploited in the construction of immunosensors by immobilizing the antibody to a suitable transducer.7,9 This methodology is promising; however, the concentrations typically used (106 to 108 cells/ mL) and the sensor response time (several minutes to hours) hinder their applicability in many systems.10-12 To provide a combination of rapid response time and celltype selectivity, we have explored the templation of thin polymer films. Here, we report a thin film polymerization * Address correspondence to this author. E-mail: nusslein@ microbio.umass.edu. † Department of Chemistry. ‡ Department of Polymer Science and Engineering. § Department of Microbiology. | These authors have contributed equally to this work. (1) Pearson, J. E.; Gill, A.; Vadgama, P. Ann. Clin. Biochem. 2000, 37, 119-145. (2) Cavic, B. A.; Hayward, G. I.; Thompson, M. Analyst 1999, 124, 1405-1420. (3) Marx, K. A.; Zhou, T. A.; Montrone, A.; Schutze, H.; Braunhut, S. J. Biosens. Bioelectron. 2001, 16, 773-782. (4) Satki, S. P.; Hauptmann, P.; Zimmerman, B.; Buhlig, F.; Ansorge, C. Sens. Actuators 2001, 78, 257-262. (5) Liebau, M.; Hildebrand, A.; Neubert, R. H. H. Eur. Biophys. J. 2001, 30, 42-52. (6) Vaughan, R. D.; O’Sullivan, C. K.; Guilbault, G. G. Enzyme Microb. Technol. 2001, 29, 635-638. (7) Park, I. S.; Kim, N. Biosens. Bioelectron. 1998, 13, 1091-1097. (8) Blonder, R.; Levi, S.; Tao, G. L.; BenDov, I.; Wilner, I. J. Am. Chem. Soc. 1997, 119, 10467-10478. (9) Uttenthaler, E.; Schraml, M.; Mandel, J.; Drost, S. Biosens. Bioelectron. 2001, 16, 735-43. (10) Dickert, F. L.; Hsyden, O.; Halikias, K. P. Analyst 2001, 126, 766-771. (11) Dickert, F. L.; Hayden, O. Anal. Chem. 2002, 74, 1302-1306. (12) Hayden, O.; Dickert, F. L. Adv. Mater. 2001, 13, 1480-1483.
Figure 1. Schematic representation of the surface-templatedirected process. A layer of monomers 1 and 2 is initially spun coat onto the surface, followed by cells. The monomers are then polymerized and cells are removed by lysis.
strategy that maintains the structural integrity of the target bacterium and leads to template-specific detection at environmentally relevant concentrations. Cell-mediated surface templation was achieved by a fabrication strategy in which monomers (1,5-bis(2-acetylaminoacryloyloxy)pentane 1 and benzyl methacrylate 2, Figure 1) were polymerized in the presence of a specific cell target to create a thin surface templated film that is selective to the target cell type. Diacrylate 1 was chosen for both its excellent adhesive properties to the gold electrode of a quartz crystal microbalance (QCM) chip13 and its hydrogen-bonding moieties. Benzyl methacrylate 2 acted as a reactive diluent during the polymerization process. For templation, a layer of monomers was spincoated directly onto the QCM chip, immediately followed by a spin-coated deposition of a layer of cells. Thermal polymerization (45°C) in air provided the cell-templated thin film. Cells were removed from the polymer layer by a lysis cocktail combining standard lysis techniques (cell disruption with a combination of detergents and hydrolytic (13) Xie, T.; Penelle, J.; Hsu, S. L.; Stolov, A. A. Green Chem. Submitted 2002.
10.1021/la034243r CCC: $25.00 © 2003 American Chemical Society Published on Web 06/26/2003
Specific Recognition of Bacteria by Polymer Films
Langmuir, Vol. 19, No. 15, 2003 6227
enzymes targeting both Gram-positive and Gram-negative cells) followed by mechanical removal of cell debris assisted by methylene chloride (CH2Cl2), which resulted in a thin film that retained specific shape and functional group information templated to the surface.14 Experimental Section Bacterial Strains, Cell Growth Conditions, and Cell Purification. Pure culture cells were grown aerobically on an orbital shaker to mid log phase under standard conditions in rich medium. A measured volume was harvested and washed once by mild centrifugation (4000 × g) in sterile saline solution (0.9% NaCl). Cell motility was inactivated for direct cell counting with Norn’s-Powell solution (phosphate buffered SDS with 5% (vol/vol) of 37% formaldehyde, pH 7.4). Cell concentration was determined by direct counting under phase contrast microscopy in a Petroff-Hauser counting chamber using the average of 25 counts from 16 squares each. Both templation and measuring were done with cell suspensions adjusted to approximately 500 cells/ml in presterilized saline solution. Preparation of Polymer Films. Polymer films were prepared by spin coating 20 µl of a CH2Cl2 (10 mL) solution containing 1 (25.9 mg, 0.08 mmol) and 2 (0.03 mL, 0.02 mmol, 82/18 mol ratio) onto a QCM chip (International Crystal Manufacturing, OK) for 30 s at 7500 rpm. One hundred milliliters of the relevant cell solution was immediately spun coat onto the same QCM chip, which was then heated at 45 °C for 2 h in air to polymerize the monomers. Cells were partially removed from the polymer surface by a lysis cocktail (mixture of the enzymes lysozyme, mutanolysin, and lysostaphin, 10 mg/mL, 25 µg/mL, and 0.5 U/µL final concentration, respectively; 90 min at 37 °C) in a suitable volume of lysis buffer (10 mM Tris HCl (pH 8.5), 5 mM EDTA). The lysis cocktail and cell remnants were washed off briefly with saline solution followed by methylene chloride, and the QCM chip was stored in sterile saline solution at 4 °C in the dark until use. QCM Assays. Surface-templated QCM chips were exposed to the cell solution of interest at dilutions of approximately 500 cells/mL, and the QCM response was measured as a function of time using a home-built QCM recorder and a personal computer. QCM assays were performed in triplicate and results obtained were reproducible to within experimental error.
Results and Discussion Three cell lines were examined to determine sensor selectivity: E. coli (EC) (Gram-negative rods), Staphylococcus aureus (SA) (Gram-positive spheres), and Bacillus megaterium (BM) (Gram-positive rods). These three cell lines allowed for the systematic variation of cell properties (cell wall structure, size, and overall geometry), thereby providing a method to determine selectivity by measuring the frequency response to both the templated and untemplated cell types. Figure 2a-2c shows the measured QCM response to 500 cells/mL saline solutions. Upon exposure to a suspension containing the bacterial cell type that the film was templated to, a decrease in the frequency of the piezoelectric vibration of the QCM chip could be observed because of an increase in mass and shear at the surface.15 Excellent selectivity was evidenced for our films by the large frequency shift from an equilibrium with saline solution upon addition of the templated cell, as opposed to the small shift observed to other cells of varying size, shape, and cell wall composition (Figure 2a-c). A film templated with E. coli, a Gramnegative bacterium, did not appreciably bind either one of the Gram-positive bacteria, SA or BM. The observed frequency shift of ∼ -300 Hz for the nontemplated cell (14) Johnson, J. L. In Methods for General and Molecular Bacteriology, 2nd ed.; Gerhardt, P., Ed.; American Society of Microbiology: Washington, DC, 1993; pp 656-682. (15) O’Sullivan, C. K.; Guilbault, G. G. Biosens. Bioelectron. 1999, 14, 633-670.
Figure 2. Observed frequency shifts obtained from templated surfaces with EC (2a), SA (2b), and BM (2c) to templating and other cell types. Frequency shifts represent a shift from equilibrium with saline solution.
lines can probably be attributed to either nonspecific recognition or a change in the viscosity of the saline solution.16 The same trend in selective cellular binding on the templated surfaces was observed for the other cell lines examined in this study, indicating that the polymerization and lysis process effectively instilled templated “memory” into the polymer film. An appreciable signal was generated in a few seconds, with the maximum frequency shift ∆Fmax observed in about five minutes, making this system an attractive real-time sensor for a variety of applications. A comparison of ∆Fmax (templated)/ ∆Fmax (nontemplated) gives values of 24 (BM), 10 (SA), and 4 (EC), a significant improvement over the current literature.10-12 The concentration of cells used in this study (500 cells/mL) is several orders of magnitude lower than what has been observed so far with “simple” templated surfaces and corresponds to those found in contaminated environmental samples (contaminated groundwater, lakes, food sources, etc.). The femtogram sensitivity and realtime signal provided by a QCM transducer provides distinct advantages in the area of bacterial detection that (16) The apparent change in frequency is due to a viscosity change in the solvent because of the addition of cells (see ref 17). The ionic potential of the buffer system employed is identical to blood or saline solution. We are currently investigating conditions that involve these ionic potentials but media of higher viscosity (blood, plasma, serum, saliva, etc).
6228
Langmuir, Vol. 19, No. 15, 2003
Das et al.
Figure 3. Optical micrographs of the three cells used in this study: EC (A) exists as discrete objects whereas BM (B) can form dimers and trimers. SA (C) tends to form aggregates in solution.
have been previously exploited by several groups but have lacked sensitivity and rapid response time.3,6,9,17-19 Experiments using surface-templated polymer films of different types deposited on a QCM chip have required very high cell concentrations (104 to 108 cells/mL), and exhibited long response times (several minutes to hours) and low selectivity, in sharp contrast to the results obtained in our study.10-12 The shape of the curve obtained in response to each templated cell is directly correlated to the known solution properties of the corresponding cells. EC (Figure 3a) exist as single entities in solution, and a sharp, almost instantaneous, curved signal is obtained. In comparison, BM (Figure 3b) cells are observed to form dimers and trimers in solution. This behavior can explain the shallower slope of the curve to the templated cell, as prebinding equilibria will occur in this case, with deaggregation of the cells required prior to binding to the templated surface. In SA (Figure 3c), several possibilities have to be considered as SA cells strongly aggregate in solution. The obtained two-step QCM curve (Figure 2c) suggests two distinct binding processes are occurring, with the first (I) related to small aggregates or single cells binding and the latter (II) associated to large aggregates of cells binding to the sensor surface. The signal in response to the addition of nontarget cells is the highest in SA. This may be because large aggregates of SA were templated, generating large binding sites and heterogeneity on the surface of the polymer. In repeated experiments, the shape, frequency shift values, and time profile of the SA-SA curve (as well as the BM-BM and EC-EC curves) is reproducible within experimental error. The initial time profile of the SABM and SA-EC curves are not necessarily reproducible, but the frequency shift values and time profile are constant from run to run. The maximum frequency shift (∆Fmax) can also be correlated to cell size. BM cells are much larger than EC cells (Figure 3a), in agreement with ∆Fmax BM much larger than ∆Fmax EC (-4500 to -1500 Hz). Further insight into the nature of the signal generated by the QCM sensor was obtained by removing (by centrifugation) cell debris and proteins that usually “contaminate” the extracellular solution of bacteria. A suspension of washed cells obtained by this technique gave an identical signal to the suspension used for Figure 2ac, confirming that whole bacterial cellssand not extracellular macromolecules or other objects accompanying (17) Janshoff, A.; Galla, H. J.; Steinem, C. Angew. Chem., Int. Ed. 2000, 39, 4004-4031. (18) Janshoff, A.; Wegener, J.; Sieber, M.; Galla, H. J. Eur. Biophys. J. 1996, 25, 93-103. (19) Wegener, J.; Seebach, J.; Jansoff, A.; Galla, H. J. Biophys. J. 2000, 78, 2821-2833.
Figure 4. Observed frequency shifts obtained from templated surfaces with BC (4a) and EC (4b) to templating and other cell types. Frequency shifts represent a shift from equilibrium with saline solution.
the cellssare recognized at the surface. In addition, the frequency response to free proteins and cell debris was minimal compared to the signal obtained from intact cells (less than 400 Hz vs >1500 Hz) as confirmed in control experiments with solutions from the cell lysis or from spent growth medium after removal of intact cells. The nature of the interaction of the templated polymers with cell surface functionality was examined by templating cells of similar shape and size but different cell wall structure. Bacillus cereus (BC) is a Gram-positive rod that exists as a single entity in solution and provides an excellent cell line for comparison to EC, a Gram-negative rod of similar size. Removal of other parameters (shape, size, and solution composition) in the experimental design reduces the variables to cell wall structure only. Figure 4a and 4b shows the response curves obtained from this experiment. Despite the striking resemblance between the two bacterial shapes, a measurable degree of selectivity (∼300 Hz) could be observed between the systems, indicating that the cell wall, coupled with size, shape, and solution properties plays a vital role in the recognition of cells by the templated polymer. Surface features obtained during the templation process were characterized using scanning electron microscopy (SEM).20 Figure 5a-d shows SEM micrographs of the polymer surfaces generated in the absence of cells (Figure 5a), after polymerization in the presence of cells (EC) (Figure 5b), after lysis (Figure 5c), and after the sensing had occurred (after re-uptake of the cell) (Figure 5d), respectively. It can be clearly observed that the thickness of the polymer film (Figure 5b) is much thinner than the cell itself, a probable reason for the extremely fast response time and excellent sensitivity. Figure 5c shows a “divot” left by the cell after lysis occurred. Re-uptake can be observed in Figure 5d. Interestingly, the cell sits at an angle perpendicular to the surface, which suggests that (20) Hyat, M. A. Principals and Techniques of Electron Microscopy, Biological Applications, 4th ed.; Cambridge University Press: New York, 2000.
Specific Recognition of Bacteria by Polymer Films
Figure 5. Scanning electron microscopy (SEM) micrographs of the polymeric surface obtained in the absence of cell template (5a) and of the surface obtained at the three stages of sensor fabrication and testing. 5b corresponds to the surface polymerized in the presence of EC, after the cell has been removed (5c), and after re-uptake of the cell has occurred (5d). Figure 5d also has a few salt crystals resulting from the saline solution drying on the surface during the SEM sample preparation.
the entire cell may not have to bind to trigger a measurable signal, allowing for fast response. Traditionally, detection and recognition of bacterial cell types is dependent on mostly slow, labor-intensive approaches.21 Currently, even the fastest identification in liquid samples is on the order of minutes or hours.22 Here,
Langmuir, Vol. 19, No. 15, 2003 6229
we have shown that templated surfaces can generate a bacterial detection signal with a very short response time, allowing real-time measurements. An additional distinct advantage with this method is the lower detection limit at cell concentrations that match what is found under many environmental conditions (500 cells/mL) with no need for prior sample treatment. Our findings suggest that the replication of structural features of cells and cell clusters on organic surfaces can be applied to selectively detect the original cellular template. Maintaining structural memory by surface templation provides a critical step toward real-time recognition of biological entities, with the potential to provide a general strategy to detect a wide range of solution or air-borne cell types. Acknowledgment. We thank D.A. Callaham for technical assistance with SEM and TEM preparation and imaging. The research was partially supported by a National Science Foundation grant to K.N. from the Life in Extreme Environments Program (NSF 0085495) and by the National Science Foundation-funded University of Massachusetts Materials Research Science & Engineering Center (NSF DMR-9809365). LA034243R (21) Tanner, R. S. Cultivation of Bacteria and Fungi; American Society of Microbiology Press: Washington, D. C., 2001. (22) Wittwer, C. T.; Herman, M. G.; Moss, A. A.; Rasmussen, R. P. BioTechniques 1997, 22, 130-138.
