Monitoring the Switching of Single BSA-ATTO 488 Molecules

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Monitoring the switching of single BSA-ATTO 488 molecules covalently end-attached to a pH responsive PAA brush Namik Akkilic, Robert Molenaar, Mireille M. A. E. Claessens, Christian Blum, and Wiebe M. de Vos Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b01064 • Publication Date (Web): 15 Aug 2016 Downloaded from http://pubs.acs.org on August 16, 2016

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Monitoring the switching of single BSA-ATTO 488 molecules covalently end-attached to a pH responsive PAA brush Namik Akkilic1, Robert Molenaar2, Mireille M.A.E. Claessens2, Christian Blum2, Wiebe M. de Vos1* 1

Membrane Science and Technology, Mesa+ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands 2 Nanobiophysics, MESA+ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands KEYWORDS poly(acrylic acid) brush, fluorescence, single-molecule, bovine serum albumin, switchable sensor

ABSTRACT: We describe a novel combination of a responsive polymer brush and a fluorescently labeled biomolecule, where the position of the bio-molecule can be switched from inside to outside of the brush and vice versa by a change in pH. For this, we grafted ultra-thin, amino-terminated poly(acrylic acid) brushes to glass and silicon substrates. Individual bovine serum albumin (BSA) molecules labeled with the fluorophore ATTO 488 were covalently end-attached to the polymers in this brush using a Bis-Nsuccinimidyl-(pentaethylene glycol) linker. We investigated the dry layer properties of the brush-protein ensemble and it’s swelling behavior using spectroscopic ellipsometry. Total internal reflection fluorescence (TIRF) microscopy enabled us to study the distance-dependent switching of the fluorescently labeled protein molecules. The fluorescence emission from the labeled proteins ceased (out-state) when the polymer chains stretched away from the interface at basic pH conditions, and fluorescence recurred (instate) when the chains collapsed at acidic conditions. Moreover, TIRF allowed us to study the fluorescence switching behavior of fluorescently labeled BSA molecules down to the single molecule level and we demonstrate that this switching is fast, but that the exact intensity during the in-state is the result of a more random process. Control experiments verify that the switching behavior is directly correlated to the responsive behavior of the polymer brush. We propose this system as a platform for switchable sensor applications, but also as a method to study the swelling and collapse of individual polymer chains in a responsive polymer brush.

Ultrathin polymer brushes that are responsive to chemical/biochemical, thermal, electric or optical stimuli are of great interest for various advanced applications including data storage, microelectromechanical systems, optical devices and textiles.1-3 These smart layers undergo a strong conformational change with external triggers (e.g. pH, temperature, voltage), and are well known for their ability to allow tuning of the surface properties. By combining responsive polymer brushes with biological macromolecules or inorganic nanoparticles,4 these systems become even more promising to be employed in applications such as controlled drug delivery and release systems,5, 6 biomedical/tissue engineering7 and biosensor/actuator systems.8, 9 Numerous experimental10-16 and theoretical17-20 work has clearly shown that adsorption of biomacromolecules to responsive brushes is a very simple method to combine the properties of both. Here, the polymer brush serves as a smart material surface to which proteins can randomly and unspecifically bind (by e.g., electrostatic interactions), while maintaining their biological activity.21 The adsorption energy of the protein to

the brush polymers needs to be rather high as penetration of a protein molecule into a brush comes at a significant entropic penalty due to excluded volume interactions. The adsorption of proteins to polymer chains has been investigated both in bulk and on a solid surface (brushes) by several methods including spectroscopic ellipsometry,11, 13, 22 quartz crystal microbalance,10, 13, 23, 24 X-ray photoelectron spectroscopy,10, 25 neutron reflectometry,12 TIRF spectroscopy,26 ATR-FTIR spectroscopy,12 reflectometry,27 surface plasmon resonance spectroscopy,28 and chromatography29, 30. However, in all of these measurements, the brush-protein interactions were studied by looking at the average behavior of a very large number of molecules. A key challenge would be to look at the local differences or even the behavior of individual (biomacro)molecules attached to brush chains at a well-defined position. This would require a strong bonding between the individual molecule and polymer chain to prevent detachment of molecules when exposed to environmental stimulus. This work is based on an optical method with the ability to work down to the single molecule level, well beyond what is

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feasible by conventional detection methods. The ability to monitor single biomolecules is scientifically and commercially of great importance for the development of, for instance very sensitive biomedical devices31, 32 and can be achieved with total internal reflection fluorescence (TIRF) microscopy.33, 34 TIRF microscopy uses the evanescent field that is generated on the low refractive index side of an interface of a high and low refractive medium when light is totally internally refracted in the high refractive index medium. The evanescent field extending into the low refractive index medium decays exponentially and penetrates the low refractive index medium only roughly 100 nm. Thus only fluorophores very close to the interface can be excited by the evanescent field. TIRF microscopy hence discriminates between fluorophores very close to the interface (bright) and fluorophores further away from the interface (dim/dark). TIRF microscopy has for example been used to investigate adsorption behavior of various proteins on solid interfaces based on the exponentially decaying of evanescent field away from the interface that allows the discrimi-

Figure 1: Illustration of fluorescence switching of BSA/ATTO 488 molecules covalently end-attached to a pH responsive poly(acrylic acid) brush. The incident light reflects at the interface between the high refractive index glass substrate and the low refractive index aqueous environment, resulting the evanescent field in the aqueous environment. The evanescent field decays exponentially from the interface, resulting in the excitation of fluorophores only when the brush is collapsed and hence confined close to the interface. When the brush is stretched the fluorophores are too far away from the interface to be excited by the evanescent field.

nation between adsorbed and non-adsorbed fluorescently labeled proteins.15, 16, 26 In this study, we have used poly(acrylic acid) (PAA) to make a brush via the “grafting-to” methodology.35 This approach does not require bio-incompatible metal ions as in the “graftingfrom” approach utilizing atom transfer radical polymerization (ATRP), nor does it produce the unstable tethering as in “physisorption”.36 As a weak polyelectrolyte, PAA has a carboxylic (-COOH) group on each repeating unit. Because the degree of ionization is dependent on the bulk pH,37 the presence of charges provides a dramatic transition in brush height under different solution conditions.38 The chains of this polyelectrolyte form compact structures in acidic pH whereas they will stretch away from the grafting interface under basic solution conditions. Still, engineering of a responsive PAA brush via

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the grafting-to method is rather difficult because it may create loops on the surface due to free –COOH groups that can also be grafted to a reactive interface. This problem can be avoided with a macromolecule anchoring layer, poly(glycidyl methacrylate) (PGMA),39 which significantly increases the grafting density,40, 41 and leads to strongly stretched and standing polyelectrolyte chains.38 Previously, it has been shown that poly(ethylene glycol) (PEG) grafted to a PGMA-modified silicon surface has a much higher grafting density than the PEG chains grafted to an untreated silicon surface.40, 41 Here, we have covalently attached individual bovine serum albumin (BSA) molecules labeled with the fluorophore ATTO 488, to the end-points of a PAA brush (Fig. 1). We then observed reversible fluorescence switching of the ATTO 488BSA attached PAA conformation, when switching between a high and a low pH. In the TIRF measurements the pH dependent conformational change of the PAA is visible as a decrease in the fluorescence intensity of the ATTO 488 attached to BSA molecules (out-state) when the brush is strongly stretched and the fluorophore moves out of the evanescent field at the basic pH conditions. On the other hand, the fluorescence intensity increases again (in-state) at acidic solutions because the brush is in a more collapsed state bringing the fluorophores into the evanescent field exciting the fluorophores. The change in the fluorescent intensity is thus a direct result of movement of the PAA attached labeled protein molecules moving closer and further away from the interface. Control experiments verify that this change in fluorescence directly correlates with the responsive behavior of the PAA brush and not due to the change in pH. We confirmed the TIRF data with the in situ spectroscopic ellipsometry. The pH dependent switching of individual PAA chains and the concomitant change in the position of the end-attached BSA molecules resulted, using fluorescence of the labelled BSA excited in the evanescent field as a readout, in an 80% fluorescence intensity switching between the stretched and collapsed conformations of PAA. Furthermore, we show that switching behavior can be studied laterally resolved approaching the single molecule level. We followed the proximity to the surface of 158 isolated positions, potentially representing single fluorescently labelled protein molecules, for the first five pH-switch cycles. Results showed a direct observation of the location of protein molecules when brush chains are collapsed and stretched as a result of the pH dependent switching behavior of the polymer brush. Furthermore, we demonstrated that individual molecules can switch in a very different fashion than the ensemble. Materials and Methods Sample preparation Protein labeling was performed using a slightly modified version of a previously described protocol.42, 43 Bovine serum albumin (BSA) was incubated in a molar ratio of 1:1 with the NHS-ester of the fluorescent label ATTO 488 (ATTO-TEC, Germany) in 20 mM HEPES buffer at pH 8.3, for 2 hours as recommended by the manufacturers for N-terminal labeling. Illustration of BSA labeling with NHS-ester of the fluorescent label ATTO 488 was shown in Fig. S1. The unreacted label was then removed using a Centrispin–10 size exclusion column with a 5–kDa cut–off (Princeton separations, Adelphia, NJ, USA).


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In previous work,43 MALDI analysis demonstrated that the connection of a Cy5 fluorescent group, via an NHS ester, mainly occurred via reaction with the N-terminus of the protein. Due to strong similarities with our system (the main difference being the type of fluorescent dye) we assume the same for our ATTO488-NHS-ester, although NHS esters have also been found to link via lysine residues.44 In the second step, Bis-N-succinimidyl-(pentaethylene glycol) ester (Bis(NHS)PEG5, Thermo Scientific) was covalently bound to primary amine groups of BSA labeled with ATTO488, forming amide bonds. The reaction releases Nhydroxysuccinimide (NHS). As a large protein, each BSA molecule consists of α-amine group present on the N-terminus and ε-amine group of 59 lysine residues, 30 to 35 of which have primary amines that are capable of reacting (accessible) with Bis(NHS)PEG5. The length of the later is about 2 nm. This is much smaller than the size of the protein ((N (30 × 80 Å) → F (40 × 129 Å) transition with decreasing pH)46-51. Thus having a large protein molecule with quiet abundant accessible lysine residues and a short PEG linker reduces the possibility of loop formation. Nevertheless, homobifunctinal amine reactive crosslinkers has successfully been used under suitable precautions,52 by adjusting the buffer conditions and concentration. According to the instructions from the manufacturer, Bis(NHS)PEG5 linker was added to a 100–fold excess of the protein in 20 mM HEPES buffer at pH 8.3. After 1 hour, the excess of the linker was then removed using a Centrispin–10 size exclusion column with a 5–kDa cut–off. Hence, we expect deactivation of the positively charged groups (mainly the lysines) after the reaction of amine groups with the linker. This will reduce the possibility of unspecific binding of the BSA molecules at the low pH conditions (pH 3). Thus when the chains are collapsed at pH 3, we also reduce the electrostatic interactions between the BSA and PAA due to the less positively charged protein molecules. Surface functionalization In this work, we have used silicon wafers and glass slides as substrates for grafting PAA and they underwent the same procedures throughout the whole brush fabricating process. As glass and silicon are known to have the same silica outer surface, and as they were cleaned and coated in the same way, it is reasonable to expect that the polymer brushes on the silicon wafers and glass slides have the same structure and grafting density. Highly polished silicon wafers () orientation were cut into sizes of 6 mm × 8 mm. Glass slides were MENZEL GLÄSER Nr. 1.5 (Gerhard Menzel GmbH, Germany). All the wafers and slides were cleaned by covering them with a freshly prepared Piranha solution (1:3 mixture of 30% H2O2:conc. H2SO4) for 30 minutes (note that this solution is highly corrosive and should be handled with considerable care!). The substrates were then rinsed with copious amounts of MilliQ water and blown dry with nitrogen. The surface of the cleaned substrates was covered by spin-coating (2000 rpm for 30 s) with an anchoring layer of Poly(glycidyl methacrylate)39-41 (1 mg/ml PGMA, Polymer Source Inc., Quebec, Canada) with Mn = 17500 g/mol. The PGMA covered surface, was then annealed in a vacuum oven at 110 °C for 1 hour to enable the covalent attachment on the glass surface.39-41 During the annealing, PGMA epoxy groups are reacted with the silanol groups of a silicon substrate to establish an anchoring

layer covalently bound via ether bondings to the substrate. Because of its loop-tail structure these anchoring layers provide an increased amount of epoxy groups for the grafting of brush polymers compared to self-assembled functionalized monolayers.39-41 Unbound PGMA was rinsed off in THF solution. Afterwards, 50 mg/ml NH2-terminated poly(acrylic acid) (pKa ≈ 4.8) (PAA, Polymer Source Inc., Quebec, Canada) with Mn = 61000 g/mol was dissolved in ethanol and immediately spin coated at 2000 rpm for 30 s.38 The PAA covered surface was immediately annealed in a vacuum oven at 110 °C for 1 hour.38 Hence, keeping the annealing temperature just above the glass transition temperature (Tg) of PAA and short annealing time, we reduced the possibility of tail-loop formation. It has been shown that COOH groups react with epoxy much faster than the amino-groups. In a study by Orr et al.53 the rates of nine melt coupling reactions were measured by reacting terminally functional polymer chains. It has been shown that second-order reaction constants for COOH-epoxy and NH2epoxy are 2.1 and 0.34, respectively.53 This means COOH groups reacts with the epoxy groups about 6 times higher than the amino groups. Moreover, Mijovic et al.54 measured the primary amine-epoxy reaction rate 10 times lower than the reaction rate in polymer melt shown by Orr et al.53. In our case, this rate should be much higher because we have several COOH groups on the PAA chain, and only one NH2 group engineered at the end-group of each PAA chain. Ungrafted PAA chains were rinsed off with ethanol solution and the substrates were then rinsed with copious amounts of milliQ water, blown dry with nitrogen. All the polymers were used as received. Dry layer thicknesses of SiO2, PGMA and PAA were measured throughout the fabrication process for full characterization. Finally, 10 nM of Bis(NHS)PEG5 modified BSA–ATTO488 in 20 mM HEPES buffer pH 8.3 was incubated on a PAA grafted glass slide overnight at 4°C. In this way we enabled a covalent attachment between the free N-hydroxysuccinimde (NHS) ester of the linker and the amino-terminated PAA chains forming amide bonds. As shown by Wittemann et al,45 and others14 BSA only adsorbs to PAA brushes below a pH of around 7, above this pH all BSA would dissociate because of electrostatic repulsion between the strongly negative brush and protein. Therefore, rinsing our sample several times with a 20 mM HEPES buffer solution (pH 8.3) and subsequently MilliQ water allowed us to remove any non-covalently bound BSA. This described approach gave reproducible results of specifically immobilized individual BSA molecules on the PAA grafted glass surface. As a final test we performed the procedure described above without the presence of the NHS linker. Without the NHS linker no covalent bonds could be formed between the protein and the NH2 terminated brush chains, and as expected no single protein molecules were observed on the surface after rinsing with our buffer solution. This experiment demonstrates that the protein indeed binds as the results of a covalent bond and not as the result of non-specific adsorption. Absorption and Fluorescence Spectroscopy Absorption spectra were measured using a UV-Visible spectrophotometer (Varian, Carry 300) with a slit width equivalent to a bandwidth of 2 nm (Fig. S2). Fluorescence spectra and


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time courses in bulk were measured with a Fluoromax-4 (HORIBA Jobin Yvon) spectrofluorometer. Excitation and emissions slits were set to 2 nm band pass. ATTO 488 fluorescence was excited at 488 nm, and the fluorescence emission at 524 nm was collected. To exclude that fluorescence intensity changes resulted from changes in the pH of the solution, fluorescence intensity was measured using a 5x5 mm quartz cuvette (Perkin Elmer) in MilliQ water upon changing the pH. The ATTO 488 concentration was 100 nM. The control experiments were performed by adding 1 M of HCl or NaOH from freshly prepared concentrated stock solutions (1–10 M) directly into the cuvette. Spectroscopic ellipsometry Dry film thickness and in situ swelling experiments of the PAA brushes were performed by means of spectroscopic ellipsometry (M2000, J.A. Woollam Co., Inc.). The liquid cell (TLC-100-17.01, J.A. Woollam Co., Inc.) made of stainless steel, PEEK polymer has 5 mL of liquid capacity and consists of a 2 mm thick × 12.5 mm diameter fused silica windows. The cellʼs nominal angle of incidence is 75°. Ellipsometric data was determined upon reflection of white light on the brush coated silicon wafers in dry state as well as in situ resulting in both a relative phase shift, ∆, and relative amplitude ratio, tan Ψ. Dry film measurements were performed at angles of incidence of 65°, 70°, and 75° whereas for the in situ ellipsometry incident light was set to an angle of 75º in the wavelength range of 370-900 nm and 632.8 nm, respectively.55 In order to improve the measurement accuracy, particularly with respect to refractive index determination of both the dry and swollen films, a calibration procedure was run before the measurement of each sample. It involved the determination of in-plane Delta parameter offsets from a measurement on a 25 nm SiO2 on Si wafer prior to an actual measurement. These parameters were used to correct the in situ measurement of both dry and wet samples. A three-layer model consisting of silicon substrate, silicon oxide layer and polymer layer was used to simulate experimental data.55 For the dry layer thickness of polymer films the refractive indices were taken from literature. The refractive index (n) for the PAA was set to 1.5556 and for PGMA to 1.525.57 According to the supplier, the bulk densities of the polymers used are 1.08 g/cm3 for PGMA and 1.4 g/cm3 for PAA.58 Initially, we have measured the dry layer thickness of the first layer (PGMA) and then the dry layer thickness of the second layer (PAA) which is covered at the top of the PGMA layer using the given n values. Dynamic swelling experiments were performed in different aqueous solutions either at pH 3 or pH 10. The pH of the solution was adjusted by adding 1 M of HCl or NaOH from freshly prepared concentrated stock solutions. Before replacing with the next solution, the solution in the liquid cell was removed by purging air with a 10 mL syringe and then washed off with about 30 mL MilliQ water between each pH cycle.

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APO 100x 1.45) illuminated by a 488 nm laser (Melles Griot, IMA101040ALS) laterally focused on the back focal plane. The laser power at back focal plane was 0.318 mW. The ATTO488 fluorescent signal passes through a 505 nm longpass filter and is imaged on an EMCCD camera (Andor, ixon DU-885). Results Surface Modification To ensure protein attachment at a well-defined position on the PAA chains, we have covalently attached BSA molecules to the NH2-terminated PAA brush. Here, Bis(NHS)PEG5 linker enabled covalent attachment between the brush and the protein molecule illustrated in Scheme 1. We have avoided electrostatic coupling between the Bis(NHS)PEG5 modified BSA molecule and the PAA brush by incubating the protein at pH 8.3. It is well known that such an interaction starts at pH ≤ 6.13, 27-29 Thus, using this immobilization scheme we were able to obtain reproducible, individual BSA molecules that are covalently attached to PAA brush end groups, as will be clearly shown in the next sections. Characterization of dry polymer brushes and their swelling behavior To characterize the dry layer thickness and swelling behavior of thin brush films we used spectroscopic ellipsometry. In fact, ellipsometry gives us the ensemble behavior of brush chains, i.e. the average length of the brush chains when exposed to different pH conditions. Several parameters have been evaluated to reveal the dry layer properties. The surface coverage (grafting amount), Γ (mg/m2), was calculated from the thickness of the dry layer, d (nm), by the following equation:59 (mg/m2) (1) Γ = ∙ where ρ is the polymer density. The grafting density (σ) is given by:59 ∙ ∙ (chains/nm2) (2) =

where NA is the Avogadros constant and Mn is the average molecular weight of the polymer chains. The average distance between grafting sites, D (nm), and the area per polymer chain (A) were calculated using the following equations, respectively:59 (nm) (3) = 4⁄⁄ = 4⁄

(nm2/chains)

(4)

Table 1. Dry layer thickness (d) and calculated polymer brush parameters (surface coverage (Γ), grafting density (σ), grafting distance (D) and area per polymer chain (A)) for PGMA and PAA. Kuhn length of PAA chain 0.64 nm.60

TIRF microscopy

Polymer

d

Γ

σi

Di

Ai

Fluorescent experiments were performed on an objective based Total Internal Reflection Fluorescence (TIRF) microscope (Nikon, Eclipse Ti with TIRF module). The microscope was equipped with a high NA objective (Olympus, PLANO-

PGMA

3 ±0.2

3.24

-

-

-

PAA

13.5±0.6

18.9

0.19

2.6

6.8

i

Note: PGMA is not considered as a brush, as it is a cross-


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linked layer.

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Scheme 1. Surface functionalization with PAA-NH2 brush.

Table 2. Swelling of PAA brush and BSA covalently attached to PAA brush pH

dPAA (nm)

SDPAA

dPAA/BSA (nm)

SDPAA/BSA

3 10

30

1

22

1.6

90

10

66

2

Table 1 shows the dry layer thickness of PGMA and of a PAA brush fitted according to the described parameters and further parameterized using equations 1-4. The PGMA thickness was found to be in the range of ref.38 (2.65 nm). We have obtained, on top of the PGMA layer, a PAA brush layer with a thickness of 13.5 ±0.6 nm and a grafting density of 0.19 chains/nm2 that are in the range of common experimental values (σ=0.01-0.3 polymer chains/nm2).61, 62 The amount of PAA at the surface in our case is 18.9 mg/m2. Note that, because PGMA is an anchoring layer and forms crosslinks on the surface it is not considered a brush. We have performed in situ ellipsometry in order to determine the swelling properties of PAA and BSA attached PAA films, which are summarized in Table 2. The PAA brush alone can stretch to 90 ±10 nm at pH 10 and at pH 3 conditions the brush collapses to 30 ±1 nm due to the deprotonation of the polymer carboxyl groups. However, attaching BSA molecules to the end-group of the brush reduced the thickness to 66 ±2 nm and 22 ±1.6 nm for swollen and collapsed forms (Fig. 2), respectively. When protein molecules are attached to the chains, we observed lower thickness values in both stretched and collapsed states in in situ measurements. Similar effects have been observed for biotin immobilization on PAA brushes.63 This lower degree of swelling in response to pH changes was ascribed to the interactions of -COOH groups with the protein. Chu and co-workers64 reported that the thickness of the PAA layer in PBS (pH 7.4) as determined by optical waveguide after immobilization of the galactose ligand was lower than that of the original PAA layer and explained this as a result of suppressed swellability. To illustrate, the average swollen thickness for PAA and BSA attached PAA brushes are depicted in Figure S3.

Figure 2. Ellipsometry data on swollen brush thickness (red curve) and refractive indices (blue curve) of a BSA attached PAA brush at λ = 631.5 nm. The pH was altered from 10 (NaOH) to 3 (HCl) at each cycle. Dry layer thickness is 13.8 nm.

It is noticeable that, in Figure 2 we have seen slower kinetics at the swelling conditions, compared to the collapsed state. This could be due to different diffusion kinetics of NaOH and/or HCl in the solution: Protonation occurs first at the outer sphere of the chains because protons can reach the outer brush much faster than the interior brush.47 It is clear that ellipsometry gives an indication of the ''ensemble'' thickness for the swelling behavior of very thin films. However, the method is not so sensitive for the detection of the exact brush height or density profile, especially when the refractive index approaches very close to the refractive index of water (n ≈ 1.33) which naturally occurs at the high swelling conditions (pH 10 in Fig. 2) for a polyacid brush. Furthermore with ellipsometry we can only measure the ensemble thickness of the brush coupled to proteins. In our case, to stretch also means that the attractive interaction between BSA and PAA chains that is present at low pH needs to be broken, something that would require additional time. We believe the reason of the slow kinetics at pH 10 could be due to the entanglements of the polymer chains which need more time to stretch than collapsing.65-68 Opposite behavior was observed by Cheesman et al. for polybasic poly(2-(diethylamino)ethyl methacrylate) brushes where


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a slower solvent expulsion (collapse) was measured when cycling the pH from 4 to 9.67 Monitoring pH dependent PAA switch using a fluorescent reporter molecule TIRF microscopy was used to monitor pH-based switching of BSA molecules that are covalently end-attached to PAA chains as illustrated in Figure 3. The excitation in TIRF is the result of an evanescent field generated at the interface between a high-refractive-index and a low-refractive-index medium. The intensity of the evanescent wave has the exponential decay relationship as follows: Iz =I0 ·e-z⁄d

(5)

where Iz is the intensity of the evanescent field at distance z from the interface, I0 is the intensity at the interface and d is the penetrating depth.15 For the applied conditions, the penetrating depth of the evanescent field as a function of distance from the interface is about 75 nm(Fig. S4). Large intensity gradient from the evanescent field enables us to discriminate between stretched and collapsed brushes. Thus, as shown in Table 2, the penetration depth of the evanescent wave is slightly larger than the thickness of the BSA attached PAA brush film. This penetration depth should allow us to observe the location of protein molecules that are end-attached to pHresponsive PAA chains as A

B

C

D

Figure 3. TIRF microscopy images of BSA-ATTO 488 molecules (A and C) and corresponding illustration of the PAA switching (B and D) at pH 10 (out-state) and pH 3 (in-state) solution conditions, respectively. Image size is 15x15 µm2. A full video can be seen in the supporting information. Intensity profiles of 4 molecules (red line in Figure 3C) were shown in Figure S5. The switching ratio from swollen to collapsed a shown in B and D follows directly from experimental observations (figure 2).

also confirmed by spectroscopic ellipsometry results (Table 2 and Figure 2). In order to ascertain the distance-dependent switching of fluorescently labeled BSA molecules immobilized on a PAA grafted glass substrate, we changed the solution pH from pH 3

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to pH 10 and back as illustrated in Figure 1. We show the effect of the pH on the fluorescence excited by the evanescent field in Figure 3: The fluorescent intensity of individual BSAATTO 488 molecules was diminished at high pH (Fig. 3A) whereas it reappeared at the low pH (Fig. 3C), corresponding to a swollen and a collapsed state of the PAA brush as illustrated in Figures 3B and 3D, respectively. Intensity profiles confirmed that most of the single spots monitored on Figure 3C are in the range of diffraction limited spot size (300-350 nm). In Figure S5, we show the intensity profile over multiple spots (red line in Fig. 3C) and corresponding Gaussian fittings. The spots show some variation in size, smaller spots are diffraction limited and are hence likely to represent single, potentially multiple fluorescently labeled, BSA proteins. Bigger spots are believed to belong to multiple BSA proteins in close proximity that cannot be resolved optically. Laterally resolved fluorescence switching down to individual BSA molecules pH-dependent conformational switching of individual PAA chains and the resulting change in position in the evanescent field of the coupled BSA-ATTO 488 molecules (diffraction limited) is clearly visible when monitoring the fluorescence intensity as a function of time. The fluorescence time traces in Figure 4 follows an in-out switching behavior, which depends on the solution pH. For instance, upon addition of NaOH, the fluorescence intensity drops due to the diminishing of excitation by the evanescent field when the polymer chains are stretched as a result of deprotonation of polymer carboxyl groups (out-state, see Figures 3A-B). Subsequent addition of HCl produces an increase in the fluorescence intensity because

Figure 4. pH-dependent fluorescence in-out switching of four individual positions representing single or few labeled BSA molecules attached to a PAA brush. Fluorescence is low at basic pH (out-state), yet it recovers (in-state) at acidic pH conditions. The pH was altered from 10 (NaOH) to 3 (HCl) at each cycle.

of collapsing of brush chains (in-state, see Figures 3C-D) and the fluorophores moving close to the interface where they are efficiently excited. In principle, this pH-dependent cycle can be repeated several times as shown in Supporting Movie 1. We show the typical time traces for four single-BSA molecules in Figures 4A-D. The time profiles obtained from 5 repeating cycles show a pronounced fluorescence switching


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behavior, and appears to be dominated by two discrete intensity levels, corresponding to the collapsed (in) and stretched (out) state of PAA. A fast switching response is observed (Fig. 4) compared to the results obtained from in situ spectroscopic ellipsometry (Fig. 2). Furthermore, to exclude major contributions of the pH on ATTO 488 fluorescence, the fluorescence intensity of BSA labeled with ATTO 488 was measured as a function of the pH in a control experiment (Fig. S6). We observed about 4% change in fluorescence intensity in bulk under the same conditions, which can be attributed to the photophysical behavior of fluorescent probes.69 At each position, the single BSA-ATTO 488 molecules attached to brush chains in Fig. 4 shows a different characteristic collapsing and stretching movement. It is clearly visible from these traces that the movement of single attached molecules is very dynamic, especially the intensity fluctuations in in-states are large. Several factors may contribute to these large fluctuations including microenvironmental variations such as a different distance to the surface and a different orientation of the BSA-ATTO 488 molecules once more gives different distances to interface.47, 70 Because of the large intensity gradient from the evanescent field (Fig. S4) the size of the protein may have the impact on the intensity fluctuations. Hence, a change in the local electrostatic features on the protein, such as the pH, can change the protein structure from heart-shaped to extended structure (N (30 × 80 Å) → F (40 × 129 Å) transition with decreasing pH).46-51. Based on the observation of the different intensity fluctuations in in-states at Figure 4, we propose the position of BSA molecules when the PAA chains are in the collapsed state i.e. at the solid-liquid interface as illustrated in Figure 5. The different intensity levels thus correspond to different locations and orientations of the protein while complexed with the brush PAA chains. This is in agreement with the previous works which showed the complex formation of PAA and BSA both in bulk28, 29 and uptake of BSA in PAA brushes27 as a result of the electrostatic interaction of BSA molecules with PAA chains at pH ≤ 5. Furthermore, we have calculated the fluorescence switching ratio of isolated BSA–ATTO488 molecules attached to PAA brush. The fluorescence switching ratio (SR) is defined as follows:

for a larger population of 158 (~5x30) isolated spots representing BSA-ATTO 488 molecules attached to PAA chains and 5 in and out cycles in figure S12. The different switching ratios are the result of a difference in distance between the

Figure 6. Switching ratio histograms of 34 isolated BSA-ATTO 488 molecules attached to a PAA brush for 5 in and out cycles. Molecules are tracked from the 3rd in-state (170. s). Switching ratios at (A) 1st cycle (B) 2nd cycle (C) 3rd cycle (D) 4th cycle and (E) 5th cycle.

fluorophore and the surface after collapse. The switching histograms can thus be seen as a distribution of possible distances between protein molecules and surface after chain collapse. In Figures 6 and S7-S11 it can also be observed that the distribution in SR of the selected ~5x30 isolated spots varied significantly for the various cycles. Moreover, an observation of very bright spots in one cycle (high SR, such as (7) seen in Fig. 6C), did not provide any predictive information for the SR in the previous (Fig. 6B) or next cycle (Fig. 6D). The switching ratio’s for the various spots were thus found to be quite random in nature, in line with the traces observed in Figure 4. We conclude that the exact position of the protein in the collapsed brush is very much a matter of chance. = 1

Figure 5. Schematic representation of the BSA-ATTO488 molecules when the PAA chains are in the collapsed state. Right image is the zoomed in image of Figure 3D.

In Figure 6 A-E (also shown in Fig. S9), we show the distribution of switching ratios of 34 isolated spots, tracked from the 3rd in-state (170. s), representing BSA-ATTO 488 molecules attached to PAA chains as calculated from 5 in and out cycles using Equation 7. This figure is a clear illustration of the varying degrees of switching already demonstrated in Figure 4, but shows more clearly the broadness of the distribution. The broadness of the distribution in switching ratios is illustrated

Conclusions In this work, we have covalently attached individual BSAATTO 488 molecules to the ultra-thin PAA brushes. The results from in situ ellipsometry measurements showed that PAA-BSA modified surfaces can stretch and collapse to 66 nm and 22 nm upon pH change, respectively. Subsequently, we have demonstrated that TIRF microscopy is a powerful tool to monitor the distance-dependent switching of single (or very few labeled) BSA molecules that are end-attached to a responsive polymer brush chain. By tuning the pH conditions in solution, we have obtained a reversible and fast in-out dis-


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tance-dependent switching that is monitored through TIRF microscopy: Fluorescence intensity diminished by 80% upon increasing the pH while the recovery of the intensity was observed when decreasing the pH. Moreover, we have looked at the movement of single (or few) BSA molecules covalently end-attached to a switching PAA brush, which was not possible to observe with the ensemble techniques used up to date. We have made a quantitative assessment of the displacement for 158 BSA molecules and showed the dynamics of this complex system. With our TIRF approach, it would also be possible to attach a single fluorescent probe directly to the chain end to follow the chain dynamics in a polymer brush system. However, we propose this pH-responsive polymer brush/protein platform to be used for biosensing applications (e.g. aptamer or glucose sensor) where a biomolecule is hidden deep inside a protective brush layer (in-state) can capture the target molecules only when it is exposed to the solution (out-state)9 at the enhanced sensitivity and selectivity, but here it provides fundamental insight in the swelling and collapse of responsive polymer brushes. We plan to extend this platform to a mixed brush system in the near future.

ASSOCIATED CONTENT Supporting Information. Absorbance spectra of BSAATTO488, penetration and intensity depths of the evanescent wave, intensity profiles of 4 molecules, control experiments in bulk, switching ratio histograms of 5 cycles. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

Author Contributions All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT This work was supported by the Netherlands Organisation for Scientific Research (NWO) under programme VENI 722.012.008.

ABBREVIATIONS PAA, poly(acrylic acid); BSA, bovine serum albumin; TIRF, total internal reflection fluorescence; ATRP, atom transfer radical polymerization; PGMA, poly(glycidyl methacrylate); PEG, poly(ethylene glycol); Bis(NHS)PEG5, Bis-N-succinimidyl(pentaethylene glycol) ester; SD, standard deviation; SR, switching ratio.

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Monitoring the Switching of Single BSA-ATTO 488 Molecules

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