Ultralow- and Low-Background Surfaces for Single ... - ACS Publications

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Ultralow low-background surfaces for single molecule localization microscopy of multistep biointerfaces for single molecule sensing Manchen Zhao, Philip R. Nicovich, Miro Janco, Qiji Deng, Zhengmin Yang, Yuanqing Ma, Till Boecking, Katharina Gaus, and John Justin Gooding Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01487 • Publication Date (Web): 01 Aug 2018 Downloaded from http://pubs.acs.org on August 2, 2018

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Ultralow low-background surfaces for single molecule localization microscopy of multistep biointerfaces for single molecule sensing Manchen Zhaoabc, Philip R. Nicovichde, Miro Jancode, Qiji Dengde, Zhengmin Yangde, Yuanqing Made, Till Böckingde, Katharina Gausbde* and J. Justin Goodingabc* a. School of Chemistry, University of New South Wales, Sydney 2052, Australia b. Australian Centre for NanoMedicine, University of New South Wales, Sydney 2052, Australia c. ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, University of New South Wales, Sydney 2052, Australia d. EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney 2052, Australia e. ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney 2052, Australia

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ABSTRACT. Single molecule localization microscopy (SMLM) has created the opportunity of pushing fluorescence microscopy from a biological imaging tool to a surface characterization and possibly even a quantitative analytical tool. The latter could be achieved by molecular counting using pointillist SMLM data sets. However, SMLM is especially sensitive to background fluorescent signal, which influences any subsequent analysis. Therefore, fabricating sensing surfaces that resist non-specific adsorption of proteins, even after multiple modification steps, has become paramount. Herein is reported two different ways to modify surfaces: dichlorodimethylsilane-biotinylated BSA-Tween-20 (DbT20) and poly-L-lysine grafted polyethylene glycol (PLL-PEG) mixed with biotinylated PLL-PEG (PLL-PEG/PEGbiotin). The results show the ability to resist non-specific adsorption of DbT20 surfaces deteriorates with an increase in the number of modification steps required after the addition of the DbT20, which limits the applicability of this surface for SMLM. As such a new surface for SMLM that employs PLL-PEG/PEGbiotin, was developed that exhibits ultralow amounts of non-specific protein adsorption even after many modification steps. The utility of the surface was demonstrated for human influenza hemagglutinin (HA)tagged mEos2, which was directly pulled down from cell lysates onto the PLLPEG/PEGbiotin surface. The results strongly indicated the PLL-PEG/PEGbiotin surface satisfies the criteria of SMLM imaging of negligible background signal and negligible nonspecific adsorption.

KEYWORDS. Protein resistant surfaces, bioaffinity surfaces, single molecule localization microscopy, super-resolution microscopy, surface architecture


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INTRODUCTION In the last 10 years, there have been huge advances in light microscopy resulting in the circumvention of diffraction limits to allow the resolution of features down to ~20 nm.1 The suite of techniques that achieve this are collectively referred to as super-resolution fluorescence microscopy. One major family of super-resolution techniques is single molecule localization microscopy (SMLM).2-11 SMLM methods work by exciting a sparse subset of the total fluorophore population in any given image frame and calculating the position of a fluorophore from its intensity profile. In this way it is possible to map and localize the positions of individual molecules.12 Repeated imaging builds a map of many fluorophores in the sample. There is an analogy between the images and the pointillist movement in art so that SMLM data sets are referred to as pointillist data sets. SMLM has predominately been used as an imaging tool in biology; for example, revealing new molecular mechanisms such as for immune cell signaling or proteins structure in neurons.13-14 The power of SMLM, however, goes beyond imaging. Biologically relevant information can be extracted from the mathematical relationships between the positions of the fluorophores in space and time with a subsequent analysis approach.15-21 However, SMLM is especially sensitive to background fluorescent events that contaminate the acquired data. Excessive filtering or segmenting events can deleteriously affect the subsequent analysis. As such, it becomes necessary to acquire SMLM data sets with a minimum number of background events while retaining highly specific detection of desired molecules. However, a continuing methodological challenge is preventing the non-specific binding of biomolecules onto the imaging surface. There is no better example of this challenge than in the use of SMLM to quantify antigen-antibody interactions on a surface.12 Bioaffinity surfaces typically require several modification steps to form the final surface. Hence any surface chemistry to resist non-specific adsorption, depending on when it is deposited in the


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modification process, will be required to maintain the ability to limit background signal even after several further surface modification steps. The purpose of this work is to investigate the potential of two surfaces (Figure 1a and 1b) with the ability to resist background signal and non-specific physisorption even after multiple-step fabrication. Surfaces modified with the well-known protein resistant polymer, poly(ethylene)glycol (PEGylated surfaces), have usually been used as antifouling surfaces to limit non-specific adsorption,22-24 some of which have even been applied in single-molecule studies.25-28

But

the

first

surface

investigated

in

this

paper

was

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dichlorodimethylsilane-biotinylated BSA-Tween-20 (DbT20) surface. This is because, in a recent report by Ha and co-workers, when assessing the antifouling ability to resist nonspecific adsorption of DinB (DNA polymerase IV), the DbT20 surface outperformed eight other surfaces including PEGylated surfaces as determined by having significantly lower non-specific adsorption.29-31 The second surface that was investigated was prepared by electrostatically adsorbing poly-L-lysine grafted PEG (PLL-PEG32) mixed with biotinylated PLL-PEG33-34 onto well-cleaned glass coverslips yielding a PLL-PEG/PEGbiotin surface. Through the biotin-streptavidin interaction, the target molecules were specifically bound to the surface. Surface selectivity, and resistance to non-specific background following subsequent surface modification steps, was tested by using a single-molecule counting assay incorporating total internal reflection microscopy (TIRF) and image analysis to count singlemolecule binding events. Further, the surface performance was tested with SMLM by specifically pulling down35 target molecules (fluorescent protein suitable for photoactivation localization microscopy (PALM)) onto the surface from unprocessed mammalian cell extracts.


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Figure 1. Schematic representation of modification steps for (a) the DbT20 surface and (b) the PLLPEG/PEGbiotin surface. (a) The clean glass coverslip was firstly modified with dichlorodimethylsilane (DDS), followed by incubation with biotinylated BSA, then coated with Tween-20 as the antifouling layer to resist nonspecific adsorption. Biotinylated target molecules can then be tethered to the surface via streptavidin-biotin interactions. (b) PLL-PEG/PEGbiotin was adsorbed onto the glass coverslip and served as both the antifouling layer and a surface anchor for further conjugation via streptavidin.

EXPERIMENTAL SECTION Reagents and Materials are described in SI Experimental Section. FITC-BSA, biotin-Atto-488 and human influenza hemagglutinin-tagged mEos2 (HAmEos2) were used for different purposes in this project. FITC-BSA, as the fluorescent probe, was exposed to a given protein resistant surface to assess if there was any non-specific protein adsorption onto the surface. Biotin-Atto-488 was used to show whether biotinylated molecules would be able to specifically bind to the streptavidin-modified surface and also to probe the amount of streptavidin on the surface. HA-mEos2 was designed for the single-


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molecule pull-down assay for SMLM imaging. mEos2 is a photoactivatable green to red fluorescent protein that widely used in PALM imaging. Modification of the DbT20 surface The glass coverslip was firstly sonicated in ethanol for 10 min, followed by washing with copious Milli-Q water and acetone, respectively. The coverslip was dried with nitrogen and then exposed to an air plasma (700 Torr) inside a plasma cleaning machine (HARRICK PLASMA, Expanded Plasma Cleaner PDC-002) for 3-5 min. Subsequently, 75 mL of hexane was added to a clean beaker followed by the injection of ~0.075 mL of DDS using a 1 mL syringe with needle. DDS was injected quickly with the needle tip under hexane to avoid air contact. The well-cleaned hydrophilic coverslips were immersed in this solution in a beaker under a stream of argon. The beaker containing the surface was immediately covered with a petri dish and sealed with parafilm whereupon the argon stream was removed. The reaction was then protected from light with aluminum foil and gently kept shaking at room temperature for 1.5 h. Once the reaction was stopped, the DDS-coated coverslip was rinsed and then sonicated with fresh hexane for 1 min. This step was repeated 3 times. The contact angle of the DDS-coated coverslips prepared in this way was measured (shown in SI Fig. 1). The DDS-coated hydrophobic coverslip was then incubated and modified with 0.01 nM biotin-BSA in PBS buffer for 5 min and rinsed with PBS buffer. The final DbT20 surface was obtained by being immersed in a given concentration of Tween-20 solution for 10 min (0.01%, 0.05%, 0.10%, 0.20%, 0.50% (v/v), were prepared by mixing Tween-20 with PBS buffer), and referred to as DbT200.01%, DbT200.05%, DbT200.10%, DbT200.20%, DbT200.50%, respectively. Further surface modification with streptavidin and/or antibodies and a series of control surfaces are described in SI Experimental Section.


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Modification of the PLL-PEG/PEGbiotin surface Before the surface modification, a PDMS device was prepared as described in SI Experimental Section. Then, the PDMS device was punctured at the end of each channel with a biopsy needle (1 mm diameter), cleaned from dust by adhesive tape, washed with isopropanol and dried. Meanwhile, the glass coverslip was cleaned with sonication in 100% ethanol for 30 min and rinsed with Milli-Q water. The coverslip was further sonicated in 1 M NaOH for 30 min, rinsed with Milli-Q water and acetone, followed by drying with nitrogen. Finally, the coverslip and PDMS device were cleaned by the plasma cleaning machine (700 Torr) for 3 min. After plasma cleaning, the PDMS device was placed onto the glass coverslip for 1 min to form strong hydrophilic interactions to hold the two components together. Thus, a PDMS device with five channels was finally fabricated. Five polyethylene tubes with a length of 4 cm were connected to the outlets of each channel of the PDMS device allowing solution to flow out of the channels without polluting the other channels nearby. From the inlet hole, 6 µL of 1 mg/mL PLL-PEG/PEGbiotin (mixing ratio depending on the surface density needed in the future experiment) was injected into the channel using pipette. The PLL-PEG/PEGbiotin solution was incubated in the channel for 15 min. The channel was then rinsed with 80 µL of PBS buffer. The PLLPEG/PEGbiotin surface was ready for further modification. Further surface modification with streptavidin and/or antibodies and a series of control surfaces are described in SI Experimental Section. Human influenza hemagglutinin-tagged mEos2 (HA-mEos2) expression and pull down on PLL-PEG/PEGbiotin surfaces HA-mEos2 plasmids were constructed by inserting the HA sequence into pmEOS2-N1 using the NotI restriction sites. The plasmid was transfected into HEK 293T cells for expression of HA-mEos2. Cells were lysed with non-denaturing lysis buffer and cell lysates


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were ready for the HA-mEos2 pull-down assay. Protocols of cells culture, cells transfection and cells lysis are described in SI Fig. 3. The PLL-PEG/PEGbiotin surface was prepared with the incubation of PLL-PEGbiotin solution (the percentage of biotin functionalized PEG = 50%), where the biotin density on the PLL-PEG/PEGbiotin surface was a suitable density of target proteins for imaging (SI Fig. 4). After modification of the surface with streptavidin, 15 µL of 4.4 nM biotin-anti-HA (0.1 mg/mL, 1:150 dilution) in PBS buffer was incubated in the channel for 5 min and washed with 80 µL PBS. Finally, the pull-down assay was carried out by applying 15 µL of cell lysates to the surface for 5 min and washing with 80 µL PBS twice before imaging. Before PALM imaging, the specific binding events of biotin-anti-HA to streptavidin and HA-mEos2 to anti-HA were tested on the surfaces with TIRF imaging, which were the PLL-PEG (no PLL-PEGbiotin) surface incubated with streptavidin, biotin-anti-HA and the cell lysates containing HA-mEos2 (CHA-mEos2), and other control surfaces including the PLLPEG/PEGbiotin surfaces subsequently incubated with CHA-mEos2 only, streptavidin and CHAmEos2,

biotin-anti-HA and CHA-mEos2, and streptavidin and biotin-anti-HA.

Imaging methods and image analysis TIRF image and PALM image sequences were acquired on a total internal reflection fluorescence microscope (ELYRA, Zeiss) with a 100x oil-immersion objective (NA=1.46) and a cooled, electron-multiplying charge-coupled device camera (iXon DU-897; Andor). A TIRF angle between 64° and 67° was used for acquisition. Detailed TIRF and PALM imaging setting are described in SI Experimental Section. Spot counting in each TIRF image was analyzed using a custom code written in MATLAB (available

at

https://github.com/PRNicovich/singleMoleculeSurfaceEvaluation.git).

The

CountSingleMolecule code was used to identify individual fluorescent spots within an TIRF imaging area by bandpass filtering and localizing emitter peaks above a threshold intensity.


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The total number of detected spots in each image is returned by the analysis code. The minimum peak intensity threshold was set to a value between 30 and 60. RESULTS AND DISCUSSION The impact of Tween-20 concentrations on protein adsorption onto the DbT20 surfaces Initially, the ability of the surface to resist non-specific adsorption was tested as a function of the concentration of Tween-20 used to form the DbT20 surfaces. Any background signal from the DbT20 surface, and any non-specific binding of FITC-BSA to this surface, was determined using TIRF microscopy by counting the number of fluorescence spots per imaging area (2500 µm2). As can be seen in Figure 2a, there was significant non-specific adsorption of FITC-BSA in the absence of Tween-20 (Surface A0) but FITC-BSA adsorption on the DbT200.10% surface (0.10% (v/v) of Tween-20 used) was sufficiently low such that the number of spots in the TIRF images was only just above the background levels (Surface A3 and Surface A4). This result agrees well with the report by Ha and co-workers where DbT20 surfaces showed little non-specific adsorption. There are occasions, however, when more modification steps are required after the addition of the antifouling Tween-20. For example, in the formation of bioaffinity surfaces,12 additional surface modifications steps are required. As shown in Figure 2d, the DbT200.10% surface with two additional modification steps (i.e. steps 3 and 4 in the scheme in Figure 1a) after the addition of Tween-20 (Surface B2) was no longer sufficient to limit non-specific adsorption of FITC-BSA despite the success in resisting FITC-BSA adsorption when the addition of Tween-20 is the last step in the surface modification process (Surface A3 in Figure 2a). Thus, the ability of the surface to resist non-specific adsorption deteriorates significantly when additional modification steps are added after the addition of Tween-20. Increasing the Tween-20 concentration during the preparation of the surface to 0.50% (v/v), 9 ACS Paragon Plus Environment

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improved the ability of the DbT20 surface to resist adsorption of FITC-BSA to levels just above background again (Surface B4 and Surface B5). This result indicates higher Tween20 concentrations are required to limit protein adsorption when multiple surface modification steps are required after the application of Tween-20. We believe that this relates to how the Tween-20 has assembled onto the surface. It has previously been shown that protein resistance is highly dependent on the amount of adsorbed surfactant on the surface.36 Taken together, these observations suggest that micelles of surfactants are required to maintain the efficacy of the surface for resisting protein fouling. Such a suggestion is analogous to PEG layers needing to exceed a critical molecular weight range to exhibit low degrees of protein adsorption and cell adhesion.37-38 The critical micelle concentration (CMC) of Tween-20 is 8.04 × 10−5 M and 0.01% (v/v) of Tween-20 is 8.96 × 10−5 M; hence the reason that we used this concentration of Tween-20 or higher is to maintain the concentration above the CMC. When there are more washing and incubation steps after the assembly of the Tween-20 on the surface, there is more chance that some of the surface-bound surfactants desorb from the surface, resulting in the loss of the micelles and decreasing the antifouling ability. Hence, the required concentration of Tween-20 during the preparation of the surface seems to depend on the number of modification steps after the addition of the Tween-20 and this limits the applicability of DbT20 surfaces for SMLM.


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Figure 2. The impact of Tween-20 concentrations on protein adsorption onto DbT20 surfaces. (a) Non-specific adsorption of FITC-BSA with different concentrations of Tween-20 (v/v) on the DbT20 surface where the addition of Tween-20 was the last modification step and (d) on the DbT20 surface where there were two further modification steps after the addition of Tween-20. The non-specific adsorption was measured by the averaged surface spot counts over an imaging area of 2500 µm2. Error bars indicate standard deviation (s.d., n > 5, ‘n’ here and hereinafter refers to the analysis coming out of over 5 regions from different substrates, ~3 regions per substrate). (b)-(c) & (e)-(f) Fluorescence images of non-specifically adsorbed FITC-BSA on the surfaces with 3step (b-c) and 5-step (e-f) modifications (with/without Tween-20).

Investigation of specific binding events on DbT20 surfaces The DbT20 surface was further evaluated (prepared as shown in Figure 1a) by exploring the specific binding of biotin-Atto-488 to the streptavidin-modified DbT200.50% surfaces. It was observed that although the non-protein species biotin-Atto-488 could selectively bind to the streptavidin-DbT200.50% surface, as determined from the comparison of Surface C1 and Surface C3 in Figure 3, the surface itself was still not sufficiently protein resistant to limit


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non-specific adsorption of the protein streptavidin. We arrive at this conclusion by using a surface prepared as in Figure 1a, but where the BSA does not possess a biotin label. In this case, if the surface is effective at resisting non-specific protein adsorption then no streptavidin should bind to the surface. However, there was extensive non-specific adsorption of streptavidin on the surface with non-biotinylated BSA (Surface D1) compared with other control surfaces (Surface D2-D3 and Surface E1-E2). This observation is also consistent with the adsorption of FITC-BSA, shown in Figure 2, when there is insufficient Tween-20. The level of non-specific adsorption of streptavidin on BSA/DbT200.50% (Surface D1) relative to the level of specific streptavidin binding to biotin-BSA/DbT200.50% (Surface C1) varied depending on the incubation time with BSA (compare SI Fig. 2). Despite this variation between samples, these observations show that Surface D1 does not resist protein fouling. Further, Surface D1 exhibits a considerably higher number of biotin-Atto-488 molecules than Surface E1 (which lacks BSA), suggesting that BSA may facilitate nonspecific adsorption of streptavidin. Overall, we conclude that biosensing interfaces prepared with multiple derivatization steps39 require a more robust, reproducible surface passivation procedure. 2000 Number of spots/2500 µm2

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1722

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Figure 3. Specific binding events of biotinylated Atto-488 to streptavidin-modified DbT200.50% surfaces and non-specific adsorption of streptavidin to the DBT200.50% surface where BSA without biotin groups was used in the first modification step. Binding events were measured by the averaged surface spot counts over an imaging area of 2500 µm2. Error bars indicate standard deviation (s.d., n > 5).

Performance of PLL-PEG/PEGbiotin surfaces Next, we asked whether PLL-PEG/PEGbiotin surfaces could be used for SMLM. As shown in Figure 4, specific binding of biotin-Atto-488 to these surfaces via streptavidin (Surface F1) resulted in a high number of fluorescent spots on the surface. In contrast, the number of fluorescent spots on control surfaces prepared without streptavidin (Surface F2) or without PLL-PEGbiotin (Surface F3) was essentially at background levels on surfaces that were not exposed to biotin-Atto-488 (Surface F4). These observations confirmed that streptavidin on PLL-PEG/PEGbiotin surfaces is essentially exclusively bound to the biotin groups on the surface, and hence that the PLL-PEG/PEGbiotin surfaces were effective at limiting nonspecific protein adsorption. The PLL-PEG/PEGbiotin surfaces not only showed low non-specific adsorption of streptavidin but also low FITC-BSA adsorption (Surface G1-Surface G3). This low nonspecific adsorption of FITC-BSA is clearly visible in the representative TIRF images (Figure 4b-e). Surface G3 is notable here as the streptavidin-modified surface was incubated with biotin-anti-mouse CD3ε to replicate an antibody-modified biorecognition surface prior to testing for non-specific adsorption of FITC-BSA. As the antibody was not specific for the BSA, it was encouraging that there was very little FITC-BSA non-specifically adsorbed onto the surface. One final point is the PLL-PEG/PEGbiotin surface showed significantly lower background signal coming from the surface than the DbT20 surface when comparing the fluorescent spots on Surface F4 with Surface C2 (Figure 3). Taken together, the results of Figure 4 show that


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the PLL-PEG/PEGbiotin surface exhibited a persistent resistance to non-specific physisorption even after multiple-step modification, making it a promising interface for SMLM studies.

Figure 4. (a) Specific binding events on PLL-PEG/PEGbiotin surfaces and non-specific adsorption of FITCBSA, measured by the averaged surface spot counts over an imaging area of 2500 µm2. Error bars indicate standard deviation (s.d., n > 5). (b)-(e) Representative TIRF images of biotinylated Atto-488 and FITC-BSA on the indicated surfaces.

PALM imaging of HA-mEos2 pulled down from cell lysates onto PLLPEG/PEGbiotin surfaces The PLL-PEG/PEGbiotin surface was finally tested for its suitability in SMLM experiments. We engineered and expressed a fusion protein (HA-mEos2) containing the common glycoprotein tag human influenza hemagglutinin (HA) and the fluorescent protein


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mEos2. Figure 5a shows the workflow of the pull-down assay, where HA-mEos2 is directly pulled down from cell lysates onto the PLL-PEG/PEGbiotin surface modified with streptavidin and biotin-anti-HA antibodies. To be more specific, the crude cell lysates were applied on the streptavidin/antibody-modified surfaces for 5 min, washed with PBS and then imaged. First TIRF imaging was used to evaluate the binding events of HA-mEos2 and background signal on the PLL-PEG/PEGbiotin surfaces. As shown in Figure 5b, the results confirm the specific binding events of biotin-anti-HA antibodies to streptavidin and HAmEos2 to biotin-anti-HA antibodies. Finally, the data in Figure 5e were collected from PALM imaging, where the number of localization events was recorded after acquisition of 10,000 frames per sample. The results in Figure 5e combined with Figure 5f-g, representative PALM images of the surfaces with control cell lysates (without HA-mEos2) and HA-mEos2-containing lysates, show that the PLL-PEG/PEGbiotin surface could specifically pull-down target molecules, HA-mEos2, from unprocessed mammalian cell extracts with negligible background signal and non-specific adsorption.


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Figure 5. Imaging of HA-mEos2 onto the PLL-PEG/PEGbiotin surface. (a) Schematic workflow of the pulldown assay with the PLL-PEG/PEGbiotin surface. (b) Binding events on PLL-PEF/PEGbiotin surfaces expressed as the averaged surface spot counts in an imaging area of 2500 µm2. Error bars indicate standard deviation (s.d., n > 5). (c-d) Fluorescence TIRF images of HA-mEos2 on the indicated surfaces. (e) Number of localization events per microns comparison between control cell lysates and HA-mEos2-containing lysates obtained with SMLM imaging. Error bars indicate standard deviation (s.d., n = 3, ‘n’ here refers to the analysis coming out of 3 regions on a single substrate). (f-g) PALM images of control cell lysates (without HA-mEos2) and HA-mEos2-containing lysates on the PLL-PEG/PEGbiotin surfaces modified with streptavidin and biotinanti-HA antibodies.


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CONCLUSION SMLM has the potential to characterize antigen-antibody interactions on the biointerface but this requires surfaces that effectively limit non-specific protein adsorption. In this study, the antifouling properties of the DbT20 surface were confirmed for single molecule imaging as reported previously by Ha and co-workers.29 However, the concentration of Tween-20 required to maintain antifouling properties was dependent on the number of modification steps required in forming the biointerface after the addition of the Tween-20. This led us to the conclusion that the DbT20 surface was not ideal for SMLM of interfaces requiring multiple modification steps. As such PLL-PEG/PEGbiotin surfaces were developed for this purpose, which showed excellent resistance to non-specific physisorption of proteins. Additionally, the PLL-PEG/PEGbiotin surface showed negligible background and nonspecific adsorption where target molecules were specifically pulled-down from mammalian cell extracts. This means that PLL-PEG/PEGbiotin surfaces are suitable to study bioaffinity reactions and may even facilitate the quantification of antigen-antibody, protein-protein, drug-receptor interactions with SMLM.


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ASSOCIATED CONTENT Supporting Information SI Experimental Section SI Fig. 1 Contact angle measurement SI Fig. 2 Investigation of specific binding events on DbT20 surfaces SI Fig. 3 HA-mEos2 expression in cells SI Fig. 4 Surface density based on different ratios of PLL-PEG mixed with PLL-PEGbiotin

AUTHOR INFORMATION Corresponding Author J. Justin Gooding email: [email protected] Katharina Gaus email: [email protected]

Present Addresses Philip R. Nicovich present address: Allen Institute for Brain Science, Seattle, Washington, 98106, USA Qiji Deng present address: CSIRO Health and Biosecurity, Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, Victoria, 3220, Australia.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We acknowledge funding from the ARC Centre of Excellence in Convergent Bio-Nano Science and Technology (CE140100036, to J.J.G.), the Australian Research Council for an ARC Laureate Fellowship program (FL150100060 to J.J.G.), the ARC Centre of Excellence in Advanced Molecular Imaging (CE140100011 to K.G.) and the National Health and Medical Research Council of Australia (1091261 to J.J.G. and APP1059278 to K.G.). Ms. Manchen Zhao acknowledges the support received through an “Australian Government Research Training Program Scholarship”.


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