Multiphoton-Polymerized 3D Protein Assay - ACS Applied Materials

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Multiphoton Polymerized 3D Protein Assay Richard Wollhofen, Markus Axmann, Peter Freudenthaler, Christian Gabriel, Clemens Röhrl, Herbert Stangl, Thomas A Klar, and Jaroslaw Jacak ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13183 • Publication Date (Web): 27 Dec 2017 Downloaded from http://pubs.acs.org on December 30, 2017

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ACS Applied Materials & Interfaces

Multiphoton Polymerized 3D Protein Assay Richard

Wollhofen†,

Markus

Axmann§,

Peter

Freudenthaler‡,

Christian

Gabriel#,

Clemens Röhrl§, Herbert Stangl§, Thomas A. Klar†, and Jaroslaw Jacak†,‡,* † Institute of Applied Physics, Johannes Kepler University Linz, 4040 Linz, Austria § Institute of Medical Chemistry, Center for Pathobiochemistry and Genetics, Medical University of Vienna, 1090 Vienna, Austria ‡ Upper Austrian University of Applied Sciences, Campus Linz, 4020 Linz, Austria # Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, 1220 Vienna, Austria

KEYWORDS: two photon lithography, direct laser writing, HDL, immunoassay, functional polymers, confocal microscopy ABSTRACT: Multiphoton polymerization (MPP) enables 3D fabrication of micro- and nanoscale devices with complex geometries. Using MPP, we create a 3D platform for protein assays. Elevating the protein binding sites above the substrate surface allows optically sectioned readout, minimizing the inevitable background signal from non-specific protein adsorption at the substrate surface. Two fluorescence linked immunosorbent assays (FLISA) are demonstrated, the first one relying on streptavidin-biotin recognition and the second one on antibody recognition of apolipoprotein A1, a major constituent of high-density lipoprotein particles. Signal-to-noise ratios exceeding 1000 were achieved. The platform has a high potential for 3D multiplexed recognition assays with increased binding surface for on-chip flow cells.

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INTRODUCTION: Functional proteome analysis holds great promise for a better understanding of diseases at the individual patient level.1 Hence, technologies allowing proteome analysis continuously gain importance. Typically, the proteome is analyzed with protein microarrays. The relative amounts of target proteins in blood sera, saliva, urine etc. indicate function of an organism and disease progression.2,3 The sore spot of many protein microarrays is insufficient passivation of the area surrounding the active spots, which thus limits assay performance.4 In particular, auto-fluorescence of the surface, unspecific binding of non-target proteins and imperfections in the passivation yield background signal.5,6 Thereby, achievable contrast and assay sensitivity are reduced on flat substrates.4,6 Multiphoton polymerization (MPP) with near infrared lasers allows fabrication of complex 3D structures, with lateral and axial feature sizes down to 100 nm and 240 nm, respectively.7,7–11 The maximal size of the whole platform is only limited by the scanning range of the setup and can easily reach several cm.12 Therefore, MPP found many applications in research areas like photonics,13–15 microelectronics,16,17 micromechanics18,19 and biomedicine.20–24 For example, we immobilized streptavidin on MPP fabricated dots and proved that the streptavidin remains capable of biotin binding.25 Nowadays, MPP enables writing of complex structures combining two or more photoresists, with defined contrast of chemical or physical properties.19,21,26–33 Nevertheless, applications such as 3D proteomic sensors have not been established with MPP yet. In this contribution, we apply MPP to establish a 3D fluorescence immunosensor with the potential of being integrated into on-chip flow cells. The 3D platform consists of two types of acrylate polymers. The carrier scaffold is composed of a protein-repellant polymer containing polyethylene-glycol. Binding pins in the nanometer range are written into the carrier scaffold


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with carboxyl-functional acrylates, enabling capture molecule binding.34–36 The binding pins are placed in elevated positions, in order to achieve spatial separation from the substrate surface. Confocal readout separates the fluorescence from the detection antibodies specifically bound to the binding pins from background signal from the surface. So far, optical sectioning has been used for imaging of cells on 3D polymer cell scaffolds.37 RESULTS: The 3D platforms were tested for two recognition assays. First, a streptavidinbiotin interaction is shown. Second, we show antibody recognition of apolipoprotein A1 (ApoA1) in high-density lipoprotein (HDL) particles. HDLs are transport vehicles for lipids and proteins and are responsible for cholesterol transport from tissues to the liver. Screening of the HDL proteome has identified significant protein alterations in a variety of diseases, including cardiovascular diseases.38–42 Up to now, the characterization of the protein profile of HDL occurs with complex and time consuming methods, for example with liquid chromatography–mass spectrometry and size exclusion chromatography followed by reverse phase protein arrays.43 Our method poses a simplified approach for HDL protein profiling. The photoresists used for MPP fabrication were a mixture of the monomers pentaerythritol triacrylate, 2-carboxyethylacrylate and poly(ethylene glycol) diacrylate (see experimental part for detailed information). An SEM image of a representative platform is shown in Figure 1b, with one column of binding pins shaded in blue. The platforms measure 16 µm × 16 µm × 5 µm, having a top square grid with 4 µm periodicity. Figure 1c sketches part of a scaffold (grey) with an attached binding pin (blue), which is covered with immobilized capture proteins. For fluorescence readout, confocal sections were recorded, focused at the position of the binding pins. Fluorescence was excited by a 532 nm CW laser (Verdi-V5, Coherent) and a 660 nm CW laser (opus 660, LaserQuantum).


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Figure 1. Fabrication and readout of a 3D protein assay. (a) Sketch of the setup used for MPP structuring (excitation at 780 nm) and for confocal readout (excitation at 532 nm and 660 nm). (b) SEM image of a 3D platform, with hanging binding pins (5 pins shaded blue). (c) Proteins are immobilized on adhesive binding pins (blue), followed by confocal fluorescence readout. (d) Confocal optical sections show separation of the signal at the binding pins from surface background signal. XZ- and YZ slices were taken along the horizontal and vertical arrows, respectively. (e) For data acquisition, the binding pin signal was determined in 40 regions of interest (ROI). For each ROI (6x6 pixels, 200 nm pixel size), the collected average photon number was determined. (f) 25 ROIs were used to determine the background signal originating from the carrier scaffold. Representative confocal slices of a platform incubated with HDL are shown in Figure 1d. The binding pin signal in the XY slice (4 µm above the surface) is well separated from the surface background signal, visible in the XZ- and YZ-slices. For fluorescence analysis of each platform, a mask of regions of interest (ROIs) that resembles the geometry of the 40 binding pins was overlaid on each image; see Figure 1e. To correct for non-specific protein adsorption to the scaffold, a mask of 25 ROIs is placed on the image (Figure 1f). Within these ROIs, the fluorescence signal from a platform was analyzed.


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Figure 2. Streptavidin-based FLISA on a 3D platform. (a) First, Alexa555-streptavidin is immobilized on the binding pins. A biotinylated mouse IgG antibody represents the antigen. Detection is performed with an Atto655-labeled secondary anti-(mouse-IgG) antibody. (b) Confocal XY-scan of Alexa555-streptavidin (excitation 532 nm). (c) Confocal XY-scan of Atto655-anti-(mouse-IgG) (excitation 660 nm). Linear color scales are shown in the insets. (d) Logarithmic bar chart of Alexa555 (green) and Atto655 (red) fluorescence signals from two experiments, showing the positive assay (left) and the control sample testing for unspecific binding of the secondary antibody (right, without biotinylated mouse IgG antibody). The specific Atto655 signal is ~165 times higher than the unspecific signal. (e) Logarithmic profiles of confocal fluorescence images along the white dashed lines in (b) and (c), showing a signal to noise ratio of ~10³. The first fluorescence linked immunosorbent assay (FLISA) relies on streptavidin-biotin interaction, see Figure 2a. First, Alexa555-labeled streptavidin was immobilized on the binding pins, followed by passivation of remaining binding sites with fetal bovine serum. In the next step, the platform has been incubated with a biotinylated mouse-IgG antibody as antigen. As a probe, a secondary Atto655-labeled anti-(mouse-IgG) antibody was used. Figure 2b depicts a confocal microscopy image of a platform incubated with Alexa555-labeled streptavidin; Figure 2c shows the corresponding Atto655-labeled anti-(mouse-IgG) antibody signal. To estimate the number of proteins per binding pin, single molecule signals were recorded. The fluorescence of individual molecules, sparsely distributed on glass, was collected. For individual


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Alexa555-streptavidin molecules, 84±52 fluorescence photons were collected. For single Atto655-anti-(mouse-IgG) antibodies, an average fluorescence photon count of 62±43 was measured. The reported single pin signals show a relatively large standard deviation. This strong pin-topin fluctuation is mainly caused by the stochastic chemical labeling process of the proteins and lipoproteins.44,45 Typically, labeling as well as protein adhesion to the pins are stochastic processes. Furthermore, proteins as well as lipoproteins may form clusters during storage.44,45 Hence, a broad (Poissonian-like) distribution of the signals of the pins is expected. For the intensity analysis of a whole array, we averaged the signal of all 40 pins. The calculated standard error of this average is typically below 15%. We calculated the ratio of (binding pin fluorescence) and (individual molecule fluorescence) to give an estimate for the number of molecules per binding pin (neglecting different photon emission probabilities at the interfaces of glass, polymer and buffer). The calculated average numbers of molecules per binding pin are ~3140 streptavidin molecules and ~950 secondary antibodies. Figure 2d displays the average fluorescence signal obtained from a positive assay and from a control sample, where the primary biotinylated mouse IgG antibody was left out. The specific antibody signal is 165 times higher, compared to the control sample. Figure 2e shows logarithmic profiles of the fluorescence images in Figures 2b and 2c, with a signal to noise ratio exceeding three orders of magnitude. The selective recognition of the biotinylated antibody on streptavidin coated binding pins proves the functionality of the immobilized proteins as well as the applicability of the platform for immunoassays.


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Figure 3. HDL-ApoA1 lipoprotein assay on a 3D platform. (a) Alexa647-labeled HDL is immobilized on the binding pin. Chicken anti-ApoA1 antibody binds to apolipoprotein A1. A secondary Alexa555-labeled anti-(chicken-IgY) antibody is used for detection. (b) Confocal XYscan of Alexa647-HDL (excitation 660 nm). (c) Confocal XY-scan of Alexa555-labeled anti(chicken-IgY) antibody (excitation 532 nm). Linear color scales are shown in the insets. (d) Logarithmic bar chart of a positive assay (left) and two negative controls (middle, right) to test for unspecific binding of primary antibody (1st ab) and secondary antibody (2nd ab). Middle: no HDL was immobilized, but both antibodies were applied. Right: HDL was immobilized, no primary antibody was used, but the secondary Alexa555-labeled anti-(chicken-IgY) antibody was applied. (e) Logarithmic profiles of confocal fluorescence images, along the white dashed lines in (b) and (c). The signal to noise ratio exceeds 10³. A medically more relevant application of the new platform was tested with a FLISA of apolipoprotein A1 (ApoA1), the main constituent of the HDL particle; see Figure 3a. First, HDL was immobilized on the structure. Passivation of remaining adhesive surfaces was performed with bovine serum albumin in this case, since fetal bovine serum contains (bovine) HDL particles. The passivated platform was incubated with chicken anti-ApoA1 antibody. For visualization of the binding, an Alexa555-labeled anti-(chicken-IgY) antibody was used. Figure 3b shows a confocal image of a platform incubated with Alexa647-labeled HDL. Figure 3c depicts the corresponding Alexa555-anti-(chicken-IgY) fluorescence signal. To determine the number of molecules per binding pin, single molecule signals were measured. The


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collected average fluorescence photon counts of Alexa647-HDL and Alexa555-anti-(chickenIgY) antibody were 278±211 and 85±71, respectively. By calculating the ratio of (binding pin fluorescence) and (individual molecule fluorescence) we get an estimated average number of ~500 HDL molecules and ~1420 secondary antibodies per binding pin. These numbers indicate that on average three Alexa555-labeled antibodies were attached to one HDL particle via chicken anti-ApoA1 antibodies. The selectivity of the binding was tested in control experiments without incubation with HDL or without chicken anti-ApoA1 antibodies. A comparison of a positive assay versus two control samples is shown in a logarithmic bar chart in Figure 3d. The detected signals of Alexa647-HDL and Alexa555-labeled anti-(chicken-IgY) antibody are comparable for the positive assay (left). For the first control experiment (middle, unspec. binding 1st ab), HDL particles were omitted to determine the non-specific chicken antiApoA1 antibody adsorption, which was detected with the Alexa555-anti-(chicken-IgY) antibody. The Alexa555-signal was lower by three orders of magnitude compared to the positive assay. For the second control (right, unspec. binding 2nd ab), the primary chicken antibody was not added to the HDL carrying binding pins. Adding the Alexa555-anti-(chicken-IgY) antibody for detection, a more than 300 times lower signal compared to the positive assay was observed. Figure 3e depicts logarithmic profiles of the Alexa647-HDL (red) and Alexa555-labeled anti-(chickenIgY) antibody (green) fluorescence signals along the dashed lines in Figures 3b and 3c. The selective recognition of ApoA1 proves the applicability of our platforms for HDL profiling.


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DISCUSSION: An obvious advantage of a confocally read-out assay, elevated 4 µm above the substrate, is the suppression of background fluorescence from unspecific binding to the substrate. Optical sectioning provided by confocal or multi-photon microscopy enables to observe a single focal plane with shallow depth of field. Out-of-focus signal is suppressed, which enables higher contrast and sensitivity.46,47 Numerous standard biochip readers use confocal detection for 2D assays, without taking advantage of optical sectioning. Nevertheless, an existing standard reader would allow straightforward integration of 3D assays into existing workflows. Moreover, there is another big advantage of the proposed 3D assay: the increase in reactive surface area. In the current case, the proteins were attached to hanging binding pins as shown in Figures 1b and 1c. The binding pin dimensions were ~450 nm in diameter and ~1 µm in height. The calculated surface was therefore ~1.41 µm² (assuming a cylinder with a half sphere on top), which is ~9 times larger compared to a two dimensional spot with the same diameter (0.16 µm²). Therefore, an almost tenfold higher number of capture molecules can be presented on the same footprint by the additional extent along the axial direction. This number could easily be further improved, for example by stacking such structures or writing of longer pins. Especially, in case of low NA objective lenses (NA = 0.5, λ = 650 nm), which are typically used in microarray scanners,48 the binding surface in the readout volume could be increased more than 100 times by using sub-diffraction structuring, as described in more detail in the Supporting Information. Consequently, on the same footprint, the steric accessibility, the amount of binding sites and the functional density could also be increased.49,50 A higher functional density allows fabrication of even smaller devices, enabling higher throughput and lower sample consumption.5,6,51–54 Particularly, microfluidics would benefit from the increased binding surface, since channels of ~100 µm width limit the available footprint. MPP can be readily combined with microfluidics to


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overcome diffusive limitations: higher velocities of the Poiseuille flow can be reached for elevated reactive binding pins.55–57 To improve the biocompatibility, the platforms could be fabricated out of unsensitized photoresists, avoiding addition of potentially fluorescent or toxic photoinitiators.58 So far, only micro-bead assays,59,60 a 2.5D polymer living graft polymerization assay61 and stacked 2D FLISA microarrays46,49,62 have been realized and introduced as 3D assays. On the one hand, since the stacked 2D FLISA consists of planar assays,49 background signal is present in each section. On the other hand, micro-bead assays have limitations relating to microfluidics, since the micro-bead colloids often clot and disturb the flow and mixing.63–65 In our experiments, the lipoproteins have been purified and 5 ng of HDL has been used for incubation. If an average weight of ~210 kD is assumed,66 24 fmol of HDL molecules per platform have been used for prototyping (lower concentration have not been tested). So far, our unique methodology presented here shows the detection of HDL over three orders of magnitude of dynamic range via antibody-binding to Apo-A1 using a three-dimensional structure. This limit arises thereby from the biochemical interaction/binding selectivity and not from the physical signal detection as shown previously using another system, likewise with single-molecule sensitivity.5,6 CONCLUSION AND OUTLOOK: In summary, we have shown the applicability of 3D MPP platforms for proteomics. A two-component structure consisting of a protein-repellant scaffold and sub-micrometer sized protein-adhesive binding pins was used as 3D protein recognition platform. We tested the 3D platforms for two FLISA immunoassays. The first FLISA was a streptavidin-biotin dummy system; the second FLISA was designed for protein profiling of HDL


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particles. Using a confocal readout, we obtained a signal to noise ratio of over three orders of magnitude for the optical signal in both cases. In principle, the design of the 3D platform is flexible, enabling versatile applicability for molecular recognition assays, tissue engineering and fluid dynamic analysis. The three-dimensional nature of our system increases the surface area with the detection volume and thereby the sensitivity and the magnitude of the dynamic range. Typically, the dynamic range is on the one hand limited by the analyte concentration and on the other hand by the number of immobilized probe molecules (specific signal) and local background. In case of small dot surfaces, the reaction area limits the number of probe molecules on the surface and hence the maximally obtained signal. Multiplexing has not been tested within one microarray. Multichannel microfluidics might circumvent this limitation.67 A multiplexed analysis may also be achieved by fabricating binding pins with different chemical functionality.26,68,69 Using orthogonal chemistry would then allow placement and covalent coupling of different capture molecules. Binding pins with functional polymers could be used to immobilize gold nanoparticles, using them as anchors for label free plasmonic sensing.70,71 Last but not least, the enhancement of the reactive surface due to the 3D geometry and the possibility of optical sectioning provides a completely new access to protein chip design. EXPERIMENTAL: The photoresists used for MPP fabrication were a mixture of the monomers pentaerythritol triacrylate (PETA, Sigma Aldrich), 2-carboxyethylacrylate (CEA, Sigma Aldrich) and poly(ethylene glycol) diacrylate (PEGDA, average Mn 575, Sigma Aldrich). The photoresist used for the protein-repellant scaffold consists of a monomer mixture of 4:1 PEGDA-PETA with 1% wt. IRGACURE819© (BASF Schweiz AG) as photoinitiator. The binding pins were structured with a photoresist composed of a 9:1 PETA-CEA monomer mixture with 1% wt. IRGACURE819© as photoinitiator. Figure 1a shows a sketch of the setup used for


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MPP lithography and confocal fluorescence microscopy. Acrylate polymerization was initiated via multi-photon absorption of 780 nm ultra-short laser pulses (50 MHz repetition rate, 100 fs pulse duration, FemtoRay780, Menlo Systems GmbH), focused with an oil immersion objective lens (Zeiss α-plan Apochromat, 100 × , numerical aperture NA = 1.46). First, the carrier scaffold was fabricated using the protein-repellant PEGDA-PETA photoresist. Next, the binding pins were written using the protein-adhesive PETA-CEA photoresist. Detailed descriptions of the lithography setup and the fabrication process are given in the Supporting Information. ASSOCIATED CONTENT Supporting Information The HDL preparation and labeling procedure, a detailed setup description, fabrication protocol and protein assay protocols, data acquisition protocols and an estimation of surface enhancement by 3D MPP structuring are given in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author *Email: [email protected] Funding Sources: The work was funded by the Austrian Science Fund (FWF) projects P 26461 (T.A.K., C.G.), P 29110-B21 (H.S., M.A.) and P 25763-B13 (C.R.) Additionally, the work was funded by the Interreg European Fund project ATCZ14 (J.J.) and the Upper Austrian University of Applied Sciences basic funding BF-PolFunk (J.J.). Notes


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The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENT We would like to cordially thank Heidi Piglmayer-Brezina for taking the SEM images and Habed Habibzadeh, Bernhard Fragner, and Alfred Nimmervoll for technical support. REFERENCES (1) Lee, J.-R.; Magee, D. M.; Gaster, R. S.; LaBaer, J.; Wang, S. X. Emerging Protein Array Technologies for Proteomics. Expert Rev. Proteomics 2013, 10, 65–75. (2) Hartmann, M.; Roeraade, J.; Stoll, D.; Templin, M. F.; Joos, T. O. Protein Microarrays for Diagnostic Assays. Anal. Bioanal. Chem. 2009, 393, 1407–1416. (3) Mitchell, P. A Perspective on Protein Microarrays. Nat. Biotechnol. 2002, 20, 225–229. (4) Jonkheijm, P.; Weinrich, D.; Schröder, H.; Niemeyer, C. M.; Waldmann, H. Chemical Strategies for Generating Protein Biochips. Angew. Chem. Int. Edit. 2008, 47, 9618–9647. (5) Hesse, J.; Jacak, J.; Kasper, M.; Regl, G.; Eichberger, T.; Winklmayr, M.; Aberger, F.; Sonnleitner, M.; Schlapak, R.; Howorka, S.; Muresan, L.; Frischauf, A.-M.; Schütz, G. J. RNA Expression Profiling at the Single Molecule Level. Genome Res. 2006, 16, 1041–1045. (6) Schmidt, R.; Jacak, J.; Schirwitz, C.; Stadler, V.; Michel, G.; Marmé, N.; Schütz, G. J.; Hoheisel, J. D.; Knemeyer, J.-P. Single-Molecule Detection on a Protein-Array Assay Platform for the Exposure of a Tuberculosis Antigen. J. Proteome Res. 2011, 10, 1316–1322. (7) Maruo, S.; Nakamura, O.; Kawata, S. Three-Dimensional Microfabrication with TwoPhoton-Absorbed Photopolymerization. Opt. Lett. 1997, 22, 132–134. (8) Farsari, M.; Vamvakaki, M.; Chichkov, B. N. Multiphoton Polymerization of Hybrid Materials. J. Opt. 2010, 12, 124001–124016. (9) Li, L.; Fourkas, J. T. Multiphoton Polymerization. Mater. Today 2007, 10, 30–37. (10) Fischer, J.; Wegener, M. Three‐Dimensional Optical Laser Lithography Beyond the Diffraction Limit. Laser Photon. Rev. 2013, 7, 22–44. (11) DeVoe, R. J.; Kalweit, H. W.; Leatherdale, C. A.; Williams, T. R. Voxel Shapes in TwoPhoton Microfabrication. Proc. SPIE 4797: Multiphoton Absorption and Nonlinear Transmission; 310–316. (12) Bückmann, T.; Stenger, N.; Kadic, M.; Kaschke, J.; Frölich, A.; Kennerknecht, T.; Eberl, C.; Thiel, M.; Wegener, M. Tailored 3D Mechanical Metamaterials Made by Dip-In DirectLaser-Writing Optical Lithography. Adv. Mater. 2012, 24, 2710–2714. (13) Sherwood, T.; Young, A. C.; Takayesu, J.; Jen, A.; Dalton, L. R.; Chen, A. Microring Resonators on Side-Polished Optical Fiber. IEEE Photon. Technol. Lett. 2005, 17, 2107– 2109. (14) Yokoyama, S.; Nakahama, T.; Miki, H.; Mashiko, S. Fabrication of Three-Dimensional Microstructure in Optical-Gain Medium Using Two-Photon-Induced Photopolymerization Technique. Thin Solid Films 2003, 438, 452–456.


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