Optimizing Bacteriophage Surface Densities for Bacterial Capture and

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Optimizing Bacteriophage Surface Densities for Bacterial Capture and Sensing in QCM-D Adam L.J. Olsson, Andreas Wargenau, and Nathalie Tufenkji ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b02227 • Publication Date (Web): 12 May 2016 Downloaded from http://pubs.acs.org on May 16, 2016

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ACS Applied Materials & Interfaces

Optimizing Bacteriophage Surface Densities for Bacterial Capture and Sensing in QCM-D

ADAM L. J. OLSSON, ANDREAS WARGENAU and NATHALIE TUFENKJI*

Department of Chemical Engineering, McGill University, Montreal, Quebec, H3A 0C5, Canada

*

Corresponding Author. Phone: (514) 398-2999; Fax: (514) 398-6678; E-mail: [email protected]

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Abstract Surface immobilized bacteriophages (phages) are increasingly used as biorecognition elements on bacterial biosensors (e.g., on acoustical, electrical, or optical platforms). The phage surface density is a critical factor determining a sensor’s bacterial binding efficiencies; in fact, phage surface densities that are too low or too high can result in significantly reduced bacterial binding capacities. Identifying an optimum phage surface density is thus crucial when exploiting the bacteriophages’ bacterial capture capabilities in biosensing applications. Herein, we investigated surface immobilization of the Pseudomonas aeruginosa specific E79 (tailed) phage and the Salmonella Typhimurium specific PRD1 (non-tailed) phage and their subsequent bacterial capture abilities using quartz crystal microbalance with dissipation monitoring (QCM-D). The QCM-D was used in two experimental set-ups, (i) a conventional set-up, and (ii) a combined setup with ellipsometry. Both setups were exploited to link the phages’ immobilization behaviors to their bacterium capture efficiency. While E79 displayed characteristic optima in both the mechanical (QCM-D) and the optical (ellipsometry) data that coincided with its specific bacterial capture optimum, no optima were observed during PRD1 immobilization. The characteristic optima suggests that the E79 phage undergoes a surface rearrangement event that changes the hydration state of the phage film, thereby impairing the E79 phage’s ability to capture bacteria. However, the absence of such optima during deposition of the non-tailed PRD1 phage suggests that other mechanisms may also lead to reduced bacterial capture by surface immobilized bacteriophages. Keywords: Bacteriophage, Biosensing, QCM-D, Ellipsometry, Bacteria

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1. Introduction Bacteriophages (phages) are viruses that infect bacteria.1,2 Although the details of phages’ life-cycles vary among the different classes of phage, the fundamental principles of phage infection remain the same. Infection always begins when the phage virion attaches to specific receptors present on the host bacterium surface whereby, by different mechanisms, the bacteriophage’s genetic material enters the bacterium. Once entered, the host bacterium functions as a factory for the propagation of new phages. In many cases, these daughter phages eventually escape the host bacterium by lysis.3 Given these properties, surface immobilized bacteriophages can be used as a biorecognition element on acoustic, electrical or optical bacterial biosensor platforms or even as a host-specific antimicrobial surface modification.4–6 Immobilizing bacteriophages onto surfaces to obtain optimum bioactivity is challenging. On the one hand, a high phage surface density is desirable as it increases the number of bacterial binding domains present on the surface. On the other hand, a too high surface density can lead to partial inactivation of the phage film, a phenomenon that was recently explained by phage tailfiber entanglement within phage clusters formed on the surface,7,8 or by phage bundling in the case of filamentous phage.9 It appears that phage films can lose significant activity within a narrow range of phage surface densities; for instance, an approximate 10-fold reduction in the number of captured E. coli K12 bacteria was observed as the T4 phage surface density increased from 19 to 28 phages µm-2.7 The notion that phages display a narrow window of practical phage surface densities poses a serious limitation for biosensor technologies utilizing phage-based biorecognition as it means that small differences in phage surface density can significantly reduce the sensor 3 ACS Paragon Plus Environment


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performance. For instance, Arya et al. observed a 30% signal reduction for E. coli K12 capture on a surface plasmon resonance sensor platform when increasing T4 phage immobilization concentration from 1.5×1011 to 5×1011 pfu mL-1.8 Hence, to optimize the bioactivity of phagemodified surfaces, the bacterium binding efficiency must be evaluated over a range of phage surface densities. The purpose of this work was to determine the phage-mediated bacterium capture efficiency over a range of phage surface densities of Pseudomonas aeruginosa infecting E79 phage and Salmonella Typhimurium infecting PRD1 phage using an acoustic sensor platform; namely, the quartz crystal microbalance with dissipation monitoring (QCM-D). The QCM-D measures mass adsorption by means of changes in the resonance frequency (∆f) of an oscillating quartz crystal sensor. Additionally, the sensor oscillation decay rate provides information about binding-induced dissipative energy losses (dissipation, ∆D). When the sensor is modified with a biorecognition element, both ∆f and ∆D can be used as transduction signals in biosensing.10,11 As an additional feature to be exploited herein, the combination of ∆f and ∆D facilitates further analysis of properties of surface-deposited nanostructures, such as phages. During nanostructure attachment, most dissipation originates from the particle-liquid boundary as the sensor oscillation moves the particle through the bulk liquid.12,13 The magnitude of the dissipation is essentially determined by the amount of liquid associated with the particles (referred to as hydration coat) which, in turn, depends on the particles’ size,14,15 shape,16 coupling with the surface12,13,15,17,18 and possibly orientation.15,19 The dissipation response in relation to the frequency shift (i.e., the so-called ∆D/∆f-ratio) has also been used to study protein surface clustering.20–22 Phages were selected based on their geometries; while E79 serves as a model of the common tailed-type geometry, PRD1 serves as a model of the less common non-tailed 4 ACS Paragon Plus Environment

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icosahedral geometry. We first monitored phage surface deposition onto the sensor surface (i.e., phage immobilization) and, subsequently, tested the bacteriophage-modified sensor surface’s ability to capture host bacteria over a range of phage surface densities. The bacterium capture efficiencies were further corroborated by determining the number of captured bacteria on the sensor surface from images obtained using fluorescence microscopy. In addition to the conventional QCM-D experiments, in-situ combined ellipsometry and QCM-D experiments were performed to test for complementary changes in the optical film properties during the course of phage deposition. As an optical method that measures the phase shift and the amplitude component of the complex reflectance ratio (∆ and tan(Ψ), respectively), ellipsometry is well-suited to determine the thickness or density of a growing surface film. However, quantitative analysis of ∆ and Ψ critically relies on knowledge of the optical properties of the growing film and the underlying substrate – a requirement that is difficult to meet when depositing anisotropic particles (such as phage) on a QCM-D crystal. Nonetheless, the complementary optical data can indicate surface alterations during the deposition process that are not accompanied by significant mechanical changes detected by the QCM-D. In addition, ellipsometry can be performed at local regions on the QCM-D surface and thus has the potential to resolve fast processes that cannot be resolved by the surface-averaged QCM-D data (e.g., due to laterally inhomogeneous deposition).

2. Materials and methods 2.1. Bacterial strains and culture conditions



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Salmonella Typhimurium and Pseudomonas aeruginosa PA01 were prepared for bacterial capture assays and phage propagation. An inoculum from frozen glycerol stock (kept at −80 °C) was streaked onto a lysogeny broth (LB) agar plate, incubated overnight at 37 °C and kept, for further use, at 8 ºC for no longer than 2 weeks. Bacterial cultures were prepared from a single colony inoculated into 5 mL of LB-broth and incubated overnight at 37 °C at 130 rpm. The overnight bacterial cultures were harvested by centrifugation at 3000g for 5 minutes and resuspended in 5 mL of SM-buffer (5.8 g/L NaCl, 120 mg/L MgSO4, 50 mL of 1 M Tris-HCl, pH 8), a procedure that was performed 3 times to remove any remnants of LB medium. For bacterial capture experiments, harvested bacterial cultures were diluted to a final optical density (OD600) of 0.6 corresponding to bacterial concentration of 109 CFU/mL.

2.2. Phage propagation & characterization The following phages were used in this study: (a) Pseudomonas aeruginosa infecting E79-phage, which is a tailed phage consisting of a 70 nm head and 150 nm long tail,24 and (b) Salmonella Typhimurium infecting PRD1 phage, which is a non-tailed phage with bacterium binding domains located symmetrically on all vertexes and with a reported approximate “dry” diameter of 65 nm25 and an hydrodynamic diameter of 105 nm.6 For the phage propagation, two overnight cultures of the bacterial host were prepared as described above. One of the overnight cultures (5 mL) was inoculated into 200 mL LB broth and incubated at 37 °C for 6 h at 130 rpm. The second overnight culture (5 mL) was thoroughly mixed with 100 µL phage stock (106-1010 PFU/mL) by vortex and kept at room temperature for 15 min (PFU = plaque-forming units). The phage-bacterium mixture was then added to the 200 6 ACS Paragon Plus Environment

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mL culture which was allowed to grow overnight at the aforementioned conditions. The lysate was subsequently centrifuged at 10,000 g (RC-6, Sorvall) for 10 min to remove bacterial debris and purified using the PEG8000/NaCl aqueous two-phase method.23 Unassembled proteins were removed using Ultracel 100K (Amicon Ultra centrifugal filters, Millipore). The concentrated phages were suspended in 5 mL SM buffer and finally passed through 0.45 µm and 0.22 µm pore-sized syringe filters (Millex®, Merck Millipore Ltd., Ireland) to remove any potential bacterial contamination. This method resulted in a phage titer (i.e., concentration) of 1011−1012 PFU/mL, which was determined as follows: 10-fold serial dilutions of each phage suspension were prepared and 100 µL of each of these dilutions was mixed with 100 µL overnight culture of host bacteria. These mixtures were kept at room temperature for 15 min before they were thoroughly mixed (by vortex) into 5 mL of molten soft agar (LB containing 0.7% agar, kept at 45ºC) which was then poured onto LB plates. Plaques formed on the LB plates were counted after overnight static incubation at 37 °C to determine the phage titer. The stock phage suspensions were diluted to the desired titer immediately prior to use in QCM-D experiments. The phages’ hydrodynamic diameter (i.e., size of the phages) was determined by dynamic light scattering (DLS, ZetaSizer Nano, Malvern) measurements using 1 mL phage samples that were prepared by diluting phage stocks 1000× in SM buffer. The presented hydrodynamic diameters refer to Z-average data.

2.3. Phage immobilization in QCM-D



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QCM-D experiments were performed in an E4 chamber (Q-Sense, Sweden), which allows for simultaneous measurements on four QCM-D sensors mounted in separate liquid flow modules. The temperature inside the flow modules was maintained at 22°C. The measurements were performed using SiO2 coated QCM-D sensors (QSX 303, Q-Sense). The sensors were cleaned prior to use by 15 min sonication in 2% Hellmanex solution and thorough rinsing in de-ionized water (DI) followed by drying in high purity nitrogen gas and 15 min UV/ozone treatment. To form a favorable surface for phage deposition, the SiO2 QCM-D sensors were pre-coated with the positively charged polyelectrolyte poly-L-lysine (PLL) (0.01%, Sigma Aldrich) prior to phage immobilization. The sensors were coated with PLL while mounted in the QCM-D by flowing 0.001% PLL-solution (i.e., 10× diluted in SM-buffer) at a flow rate of 10 µL min-1, reaching equilibrium at a final frequency shift of -5 Hz. This step was followed by SMbuffer rinse until both frequency and dissipation remained stable. The phage surface immobilization was achieved by flowing phage suspension over the sensor surface at 10 µL min1

for a fixed time of 30 min, followed by a 30 min SM-buffer rinse to remove any unbound

phage. The phage surface density was varied by using suspensions of different phage concentrations. Surface-averaged mass of deposited phage was calculated according to the Sauerbrey relation 26:

∆m = −

C ∆f n

(1)

where ∆m denotes the change in mass density at the crystal surface, ∆f is the observed frequency shift at the nth harmonic and C is the mass sensitivity constant (C = 17.7 ng cm-2 Hz-1).


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2.4. Phage sizing in QCM-D The acoustic thickness (i.e., size) of the phages was determined by the change in the dissipationfrequency ratio with phage surface coverage. The analysis involves plotting ∆D/∆f as a function of ∆f and extrapolating a linear regression fit to identify the intercept value of ∆f. The intercept ∆f-value corresponds to a hypothetical full phage surface coverage where dissipation approaches zero and the Sauerbrey equation applies.14 Because the Sauerbrey-derived mass is surface averaged (ng cm-2), dividing the Sauerbrey-derived mass at the hypothetical full surface coverage with the phage density (1.08 g cm-3)27 yields the hydrated layer thickness that corresponds to the average phage diameter.14–16

2.5. Bacterial capture in QCM-D To reduce non-specific attachment of host bacteria, the phage modified sensor surfaces were rinsed before each bacterial capture experiment with 1 mg/mL bovine serum albumin (BSA) solution (dissolved in SM-buffer) for 60 min to block any exposed PLL-coating (i.e., phage-free surface), followed by SM-buffer rinse until the frequency and dissipation were again stable. Suspensions of host bacteria were then flowed into the QCM-D modules for 60 min to allow the bacteria to interact with the sensor surface. When Pseudomonas aeruginosa PAO1 capture by E79 phage was investigated, experiments were performed at a flow rate of 50 µL min-1. PRD1 was found less efficient in capturing S. Typhimurium and the flow rate was therefore adjusted to 10 µL min-1 for that set of experiments. As the final step of the QCM-D measurements, the sensor surfaces were rinsed with SM-buffer at maintained flow rates to remove loosely (nonspecifically) bound bacteria. 9 ACS Paragon Plus Environment


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2.6. Determining the number of captured bacteria After each QCM-D experiment, the sensor surfaces were removed from the QCM-D flow modules and the captured bacteria were stained for 15 min with 100 µL of fluorescent nucleic acid stain SYTO9 (Life Technologies, USA) solution (prepared by diluting one vial in 10 mL SM-buffer). Excess stain was removed by carefully replacing the staining solution twice with 100 µL SM-buffer. Excess liquid was gently removed by brief contact between the edge of the sensor and a Kimwipe tissue, before mounting the sensor-surface face-down on a drop of glycerol on top of a clean microscope slide. The stained bacteria were viewed at a magnification of 100× (Olympus IX71 inverted fluorescence microscope, Japan) with a filter set capable of illuminating SYTO9-stained bacteria (excitation (EX)/emission (EM) 490 nm/520 nm). 30 images of randomly chosen surface locations were captured with an Evolution VF cooled monochrome CCD camera (1392×1040 resolution with 2×2 binning) and the number of bacteria on each image was determined using ImageJ particle analysis tool.

2.7. Combined ellipsometry and QCM-D Combined ellipsometry and QCM-D experiments were performed using an EP3 imaging ellipsometer (Accurion, Göttingen, Germany) and a Q-Sense E1 system equipped with an ellipsometry module, which allows for simultaneous optical measurements at a fixed reflection angle of 65°. The measurements were conducted as described in section 2.3., except that the flow rate was set to 0.10 mL min-1 and a lower PLL concentration was used for the initial PLL deposition.


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The ellipsometer, equipped with a Xenon lamp and a 2× objective, was set up to record Ψ and ∆ values (ellipsometric angles) based on a one-zone nulling procedure. Within this procedure, the polarizer angle was varied by ± 20° and the analyzer angle was varied by ± 15°, while the compensator angle was kept at 45°. The ellipsometric angles were determined for different regions on the crystal surface, each of which covering a surface area of 0.08 mm2. The wavelength of the light beam was chosen to be 551 nm.

3. Results 3.1. Bacteriophage deposition Bacteriophage were deposited onto the PLL-coated SiO2 sensor surfaces for a fixed period of 30 min. To achieve different end-point surface densities, the concentration of the phage suspension was systematically varied. Figure 1a and b presents representative frequency and dissipation shifts during E79 and PRD1 deposition onto PLL-coated SiO2 QCM-D sensors for three different phage concentrations. During deposition of both bacteriophages, the sensor resonance frequency shifted in the negative direction. The non-tailed PRD1 phage behaved similarly to spherical particles reported in literature,14,15 i.e., showing an initial linear decrease in f and a linear increase in D that gradually levels off to a plateau as the surface becomes saturated. The tailed E79 phage exhibited a noticeably different behavior; instead of a gradual change of f and D towards constant values, both quantities reached an optimum value once a specific phage surface density was reached. In the representative experiments shown, the reversal in the signal slopes can be observed for the highest phage concentration (9.4 × 1010 pfu mL-1). Control experiments with another tailed-type phage, the Escherichia coli specific T4 phage, displayed similar trends of the



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QCM-D signals during deposition at high phage concentration (supplementary data, Figure S1), suggesting that this particular behavior is linked to the phages’ geometry. Figure 1c and d present the average ∆f and ∆D values obtained after 30 min of phage deposition and module rinse steps at different phage concentrations (i.e., at t = 70 min in Figure 1a and b for E79 and PRD1, respectively). This data presentation reflects the different deposition behaviors of the two phages. In agreement with Figure 1a and b, end-point frequency and dissipation of nontailed PRD1 decrease and increase, respectively, over the entire range of phage immobilization concentrations, whereas end-point frequency and dissipation of tailed E79 both reach an optimum at a phage immobilization concentration of 7 × 109 pfu mL-1.

3.2. Bacteriophage sizing The immobilized bacteriophage sizing analysis for the E79 and PRD1 phages is presented in Figure 2a and b, respectively. Converting the frequency shift at the intercept of the x-axis (850 Hz and 575 Hz for E79 and PRD1, respectively) to surface mass density results in corresponding phage layer thicknesses of 139 nm for E79 and 94 nm for PRD1 (see Materials and Methods section for details). These calculated hydrated phage layer thicknesses are in good agreement with the corresponding hydrodynamic diameters obtained by dynamic light scattering (Z-average). The values, found to be 121 nm and 105 nm, respectively, validate that the extrapolated x-intercept provides an adequate estimation of the hypothetical frequency shift at full sensor surface coverage. Note that the frequency values obtained after the minimum in ∆f (closed symbols in Figure 2a) did not follow the expected linear behavior and were therefore not


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included in the sizing analysis. An explanation for this behavior is provided in the Discussion section. 3.3. Bacterial capture efficiency of phage modified surfaces After each phage deposition experiment (as presented in Figure 1), the host bacterium capture efficiency of the different phage-modified surfaces was evaluated using QCM-D and microscopy. Figure 3a and b presents the measured ∆f after exposing the phage-modified surface to a suspension of host bacteria for 60 min. In this particular instance, we have chosen to analyze the shifts of the fundamental resonance frequency (i.e., 5 MHz). These shifts were found to be larger than the frequency shifts of any available harmonic frequency. No artifacts were observed on the fundamental resonance frequency that could have influenced the interpretation of results. During exposure to bacterial suspension, all phage-modified surfaces gave rise to positive ∆f values. For the E79 phage, ∆f (induced by Pseudomonas capture) increased with phage surface density, but only up to the concentration that corresponds to the immobilization optima in ∆f and ∆D (Figure 1c). PRD1-mediated Salmonella capture displayed an overall lower ∆f response (about one order of magnitude) than E79-mediated Pseudomonas capture. An optimum in bacterial capture is also observed for PRD1, despite the absence of minima in ∆f and maxima in ∆D during PRD1 immobilization (Figure 1d). However, the capture optimum, which occurred at an intermediate phage concentration, was less pronounced. To ensure that the measured ∆f was induced by bacterial capture, the number of captured bacteria was also determined from images obtained by fluorescence microscopy after each experiment (Figure 3c and d). The number of captured bacteria displayed larger variability than the corresponding QCM-D data which is an influence of the sample preparation prior to imaging (i.e., removal of the sensor, bacterial staining, rinsing, etc). The average number of captured bacteria was nonetheless highest at 13 ACS Paragon Plus Environment


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intermediate phage immobilization concentrations that coincide with the optima in bacterialcapture induced ∆f, validating that the transduction signal was induced by bacterial capture (Figure 3).

3.4. Combined ellipsometry and QCM-D The results of the combined ellipsometry and QCM-D measurements are shown in Figure 4. The QCM-D data (upper diagrams), which correspond to concentrations of 2.6 × 1010 mL-1 for PRD1 and 7.1 × 109 mL-1 for E79, are directly comparable to the respective frequency and dissipation shifts shown in Figure 1. For PRD1, a continuous decrease in frequency and a continuous increase in dissipation was observed, which is in agreement with the deposition results observed in the conventional QCM-D experiments. The optical data complements this behavior. Consistent with observations made for the deposition of spherical 115 nm polystyrene nanoparticles (data shown in the Supporting Information, see Figure S2), both Ψ and ∆ increased during the course of phage immobilization. Interestingly, Ψ leveled off at a point in time where the mechanical data (f and D) still indicated phage deposition. However, as ∆ continued to increase, this behavior appears to be due to a reduced sensitivity of Ψ at high surface coverage, rather than being a result of an actual change in the intrinsic properties of the phage film. In the case of E79 deposition, by contrast, the optical data revealed temporal changes in the film properties that cannot be explained by the continuous deposition of the phage alone. Although Ψ increased initially as in the case of PRD1, a comparably sharp drop was observed after approximately 10 min of deposition (~ 0.05° in 2 min). After the occurrence of the Ψ optimum, further increase of this quantity was observed. A similarly sharp maximum was also observed in ∆, which was otherwise decreasing over the course of E79 deposition. 14 ACS Paragon Plus Environment

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Both optical optima occurred at a time point that corresponded with the broader optima in the QCM-D data. However, when comparing the time points of different maxima in Ψ or ∆ that were obtained for different regions on the QCM-D crystal (data not shown), significant variations can be observed in the temporal occurrences of their peak values. These variations, together with the short time scale of the Ψ and ∆ changes at approximately 10 min of phage deposition, indicate that the event that results in the change of the mechanical properties of the phage film is much faster on a local level than the broader optima of the surface-averaged QCM-D data suggest. Despite the qualitative agreement of the QCM-D data from the conventional QCM-D experiments and those conducted using the ellipsometry module (i.e., the observation of optima in the resonance frequency and the dissipation), it should be noted that no optimum was reached within the first 30 min of E79 deposition in the conventional QCM-D set-up when using the same phage concentration as in the combined QCM-D/ellipsometry experiment (7.1×109 mL-1). For this reason, the frequency and dissipation shifts indicated in Figure 1a are significantly greater than the corresponding values shown in Figure 4a. As noted above, the time point at which the optima occur is a function of the phage surface density. However, the QCM-D data of the combined experiments further indicates a secondary influence of the flow conditions. In fact, the combined experiments, which required a 10× greater flow rate, led to significantly earlier optima with the lowest absolute frequency and dissipation shifts observed in this study. Control experiments in which E79 was deposited at different flow rates in the conventional E4 QCM-D setup showed that increased flow rates led to faster phage deposition kinetics and an earlier occurrence (both in terms of time and magnitude of mass deposition) of the observed minima and maxima in ∆f and ∆D (see supporting information, Figure S3a). Similarly, the observed



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minima and maxima in ∆f and ∆D occurred earlier in control experiments where the phage deposition kinetics were accelerated by increasing the phage concentration (Figure S3b).

4. Discussion We investigated the deposition of phages onto PLL-coated QCM-D sensors and their subsequent ability to capture host bacteria over a range of phage surface densities with the aim to determine optimum phage surface densities for two different bacteriophages. Both investigated bacteriophages displayed an optimum phage surface density. The tailed E79 phage displayed the highest bacterial capture ability when a phage concentration of 7.1 × 109 pfu/mL was used for the phage immobilization (Figure 3a and c), corresponding to a measured frequency shift of -122 ± 5 Hz (Figure 1c) and Sauerbrey derived mass of 2.2 ± 0.1 µg/cm2. For the non-tailed PRD1 phage, a phage immobilization concentration of 8.6 × 109 pfu/mL generated the most efficient bacterial capture (Figure 3b and d), corresponding to a measured frequency shift of -93 ± 7 Hz (Figure 1) and Sauerbrey derived mass of 1.6 ± 0.1 µg/cm2. With the predicted ∆f for full phage surface coverage (i.e., extrapolated intercept in Figure 2a and b), the frequency shifts at optimum bacterial capture transform into estimated phage areal surface densities of 14% for E79 and 16% for PRD1. Interestingly, during tailed-type phage deposition (E79 and T4), both ∆f and ∆D passed through an optimum, while such optima were absent during the icosahedral PRD1 phage deposition. Resonance frequency and dissipation optima are usually linked to a reduction in the amount of surface-associated water that is sensed by the QCM-D (i.e., a mass loss); for instance, due to the release of captured water28 (e.g., liposome rupture) or due to changes in the hydration state of surface adsorbed molecules (e.g., protein denaturation).29 Bacteriophages are 16 ACS Paragon Plus Environment

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mechanically rigid.30 Therefore, a mass reduction by the release of water captured inside the phages, similar to that of liposome rupture, appears unlikely. If the optima are caused by a deposition-triggered change in the molecular structure of the phage (e.g., through the denaturation of surface proteins), the optima would occur independently of the phage surface density, and thus, observed at all bacteriophage concentrations in Figure 1a. A clue to the origin of the QCM-D optima may lie in the fact that the phage deposition optima coincide with the bacterial capture optima. As shown by Evoy and coworkers, surface deposited tailed-type bacteriophages tend to cluster, and the clustering leads to phage inactivation due to entanglement of bacterium-binding domains (present at the tip of the tail).7,8 Phage clustering appears likely as a possible mechanism, if the phage deposition data is interpreted within the context of nanoparticle deposition in QCM-D. Nanoparticles that deposit in the QCM-D not only add their mass to the sensor, but also increase the sensor surface area, leading to an increased liquid interfacial area, which is sensed by the QCM-D as an increased amount of hydrodynamically coupled water (referred to as hydration coat).12,13 However, neighboring particles share hydrodynamically coupled water through an overlap of their hydration coats, and the amount of hydrodynamically coupled water being shared depends on the particle-particle separation distance. If the particle-particle separation distance decreases because of the addition of new particles, the loss in mass of hydrodynamically coupled water can be nonetheless outweighed by the added particle mass, leading to a net negative frequency shift. If the particle-particle distance decreases by means other than the addition of new particles, e.g., by clustering of already deposited particles, the reduction in fractional amount of water of the particle film could lead to a measurable loss in hydrodynamically coupled water (i.e., a positive frequency shift). This may explain the non-linear behavior of the E79 phage in the ∆D/∆f



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nanoparticle sizing analysis (Figure 2) as it relies on the assumption that the reduction in the ∆D/∆f-ratio is caused by a gradual overlapping of particle hydration coats due to the accumulation of particles on the sensor surface. Further indication for the possible surface clustering of E79 is found in the ellipsometry data. In contrast to PRD1, Ψ and ∆ showed obvious peaks during E79 deposition, which coincided well with the simultaneously measured optima in ∆f and ∆D. While QCM-D is sensitive to the mechanical properties of a deposited film, ellipsometry complements the respective data with information on its optical properties. Due to the heterogeneous and intrinsically anisotropic character of the phage film, Ψ and ∆ have the potential to vary with the surface mass density (film thickness and surface coverage), the lateral distribution of the phages, and/or their individual alignment. In fact, the comparatively short time scale of the changes in Ψ and ∆ immediately after their maxima (compared to the observed QCM-D optima) suggests that the ongoing deposition of E79 phage (i.e., the increase in surface mass density) cannot have been the only governing factor for the occurrence of the optical maxima. Rather, this observation points to a phage surface clustering event that occurs at a specific phage surface density. The notion of a “surface density triggered” phage surface clustering finds support in the literature. Naidoo et al. found that phage attachment onto a dithiobis(succinimidyl propionate) SAM layered gold surface can be described by a Brouers-Sotolongo isotherm.7 The BrouersSotolongo isotherm assumes sorption on an energetically heterogeneous surface, whereby the authors suggested that deposited phages provided lower-energy sites for subsequent phage attachment, instigating extensive phage clustering at high phage immobilization concentrations. Such a “surface density triggered” clustering event could involve alterations in the lateral distribution of the phages and/or in their individual alignment. 18 ACS Paragon Plus Environment

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Another interesting observation in this study is that the characteristic optima in f and D occurred at different time points and at different frequency shifts when depositing E79 phage at different flow rates and phage concentrations (control experiments, Figure S3a and b in Supporting Information). This suggests that the optimum phage surface density is a function of the phage deposition rate and the flow conditions in the immediate environment of the phage film. Hence, the optimum phage surface densities determined herein cannot be considered universal, but must be considered specific for these particular experimental settings. Unraveling the kinetic dependencies would require further investigation that falls outside the scope of this study. Nonetheless, it is demonstrated that various phage immobilization conditions must be carefully optimized for the preparation of a highly bioactive phage functionalized surface. The surface clustering of tailed phages7,8 may be one mechanism for reducing the bioactivity of surface-immobilized phage. However, the fact that the non-tailed PRD1 phage also displays an optimum in bacterial capture at a particular surface density, despite showing no optima in either the QCM-D or ellipsometry phage deposition data, suggests that other mechanisms could also affect optimum phage surface density. Importantly, the two phages investigated in this study bind to different types of receptors; while E79 binds to lipopolysaccharides (LPS),31 PRD1 has been suggested to bind to a membrane bound nonelongating P-type pilus protein complex in the bacterial cell wall.32 The location and density of these receptors on the bacterium surface are factors that influence the likelihood of the phages binding to their host bacterium. Receptors that are surface appendages (e.g., LPS) are essentially more likely to come into contact with the surface-immobilized phages’ bacterial binding domains, as compared to a membrane bound receptor protein which can be more difficult to access due to the outermost LPS layer of the host.4 Even though Kotilainen et al. did not find



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significant differences in PRD1 propagation efficiency when S. Typhimerium mutant strains expressing LPSs of different lengths were infected,32 the situation is very different for surfaceimmobilized PRD1. While suspended phages approach bacteria by diffusion, surfaceimmobilized phages are restricted in their alignment abilities towards an approaching bacterium. For this reason, it is possible that over a certain threshold phage surface density, neighboring phages become a steric barrier hampering PRD1-receptor interaction to occur. The optimum phage surface density is unarguably important for the performance of a phage-based biosensor as it determines the maximum amount of bacteria that can be captured by the immobilized phages and therefore directly affects the bacterial detection limit. However, the detection limit of a biosensor also depends on the transduction signal strength (herein the QCMD signal generated per captured bacterium). The transduction signals in the QCM-D are the frequency and dissipation shifts. In this study, the phage mediated bacterial capture induced positive shifts in both the resonance frequency and the energy dissipation of all overtones indicating elastic coupling of the bacteria to the surface.33 This is in agreement with the work of Olsen et al.,34 where bacteria captured by QCM-sensor immobilized filamentous phage gave rise to a positive frequency shift. In the case of elastic coupling, the frequency and dissipation shifts are, to a large extent, determined by the mechanical properties (stiffness) of the mass-surface coupling. This contrasts with the case of inertial loading where the frequency shifts can be fully ascribed to the mass of the deposited film.35–37 Accordingly, in the case of the elastically coupled bacteria, the mechanical properties of the bacterium-surface coupling influences the sensitivity of the sensor. The frequency and dissipation responses to bacterial capture thus depend on a number of properties including, among others, the host-receptor type, the host-receptor density, the phage’s geometry, its orientation, and its surface density. As an example for the influence of the


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phage properties on the transduction signal strength, it has been shown that chloroform treatment of sensor-immobilized phage, which resulted in a contracted yet fully functional phage film, improved the QCM response for bacterial capture.38 When combining QCM-D with microscopy, as conducted herein, the frequency based transduction signal strength can be assessed by dividing the number of captured bacteria (Figure 3c and d) by the corresponding bacterium induced ∆f at the optimum phage surface densities (Figure 3a and b). Based on such a calculation, it can be stated that Pseudomonas capture by E79 resulted in an approximately 7 fold stronger transduction signal (1.5×105 bacteria cm-2 Hz-1) than Salmonella capture by PRD1 (9.6×105 bacteria cm-2 Hz-1).

5. Conclusions QCM-D was shown to be a valuable technique to study phage deposition behavior and to determine the optimum phage surface density for bacterial capture. The two bacteriophages investigated herein (E79 and PRD1) both displayed optimum phage surface densities, but they demonstrated distinctly different deposition behaviors. The anisotropic E79 phage deposition displayed optima in the QCM-D data (f and D) and the ellipsometric quantities (Ψ and ∆), and these optima coincided with the phage’s bacterial capturing optimum. Deposition optima are commonly the result of a reduction in the amount of water associated with the sample, suggesting that the E79 phage undergoes a surface rearrangement event that changes the hydration state of the phage film, and that this event impairs the E79 phage’s ability to capture bacteria. The surface density at which E79 QCM-D optima occurred was found to depend on phage immobilization kinetics, indicating that E79 phage immobilization conditions must be carefully optimized for the preparation of highly bioactive E79 phage functionalized surfaces.



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Icosahedral PRD1 deposition displayed no optima in either f and D or Ψ and ∆, suggesting that a different mechanism impedes bacterial capture by this non-tailed phage. Phage mediated bacterial capture in QCM-D shifted both the sensor’s resonance frequency and its energy dissipation in the positive direction. Hence, elastic coupling of the bacteria is considered to be the main factor that determines the transduction signal strength. With respect to biosensor performance, the E79 phage surface modification demonstrated an approximately 7 times more potent transduction signal than the PRD1 phage surface modification during their respective host bacterium capture. Supporting Information Figures showing: QCM-D experiments of tailed T4 bacteriophage, combined QCM-D and ellipsometry experiments of polystyrene nanoparticles, and tailed E79 bacteriophage QCM-D experiments performed using different concentrations and flow rates. Acknowledgements The authors acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada (SENTINEL Research Network), the CRC program, and the Ministère du Développement économique, Innovation et Exportation (MDEIE) PSR-SIIRI program. References (1)

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Figure 1. Upper row panels: representative frequency (closed blue symbols) and dissipation (open red symbols) shifts of the 3rd overtone as a function of time during 30 min phage deposition onto PLL-coated SiO2 QCM-D sensors and subsequent 30 min SM-buffer rinse at three different phage concentration for E79 (a) and PRD1 (b). Lower row panels: frequency (blue closed circles) and dissipation (red open squares) shifts of the 3rd overtone after 30 min of phage deposition (corresponds to t = 70 min in upper row panels) as a function of phage suspension concentration for E79 (c) and PRD1 (d).


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Figure 2. D/f-ratio of the third overtone as a function of the resonance frequency for E79 (a) and PRD1 (b). Each data point corresponds to the average frequency and dissipation shifts obtained with different phage concentration (c.f. Figure 1c and d). The reduction in ∆D/∆f as a function of ∆f is caused by overlapping phage hydration coats as the phage surface density increases leading to a gradual reduction in fractional amount of water associated with the phage sample. The xintercept of the extrapolated linear regression is equivalent to ∆f at full phage surface coverage (i.e., 100 % surface coverage). Closed symbols in panel a) correspond to ∆D and ∆f values measured after the optima in ∆D and ∆f during E79 deposition and were found to deviate from the expected linear behavior (these data points were excluded from the phage sizing analysis). .



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Figure 3. Upper row panels: average frequency shifts of the fundamental frequency, induced by host bacterium capture onto phage-modified QCM-D sensor surfaces as a function of the phage deposition concentration for E79 (a) and PRD1 (b). Lower row panels: average number of captured bacteria on the phage modified QCM-D sensor surfaces as a function of the phage deposition concentration for E79 (c) and PRD1 (d). Averages and error bars were determined from three independent experiments using separately grown cultures of respective host bacteria. Optimum phage densities determined from frequency shifts (upper row panels) coincide with optimum phage densities determined from imaging (lower row panels) for both phages (highlighted by light green shaded areas) validating that the measured frequency shifts were induced by bacterial capture. The line represents the average number of captured bacteria on the


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control surface (i.e. only BSA) and is added as a visual aid for easier comparison with the phagemodified surfaces.



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Figure 4: Combined QCM-D and ellipsometry of phage deposition on a PLL coated SiO2 QCMD sensor. QCM-D resonance frequency and dissipation shifts of the 3rd overtone during PRD1 (a) and E79 (b) deposition are shown together with the corresponding, simultaneously recorded, ellipsometric angles Ψ and ∆ (c and d). Concentrations of the PRD1 and the E79 phage suspensions were 2.6 × 1010 mL-1 and 7.1 × 109 mL-1, respectively.


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TOC Graphic


Optimizing Bacteriophage Surface Densities for Bacterial Capture and

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