Localized Visualization and Autonomous Detection of Cell Surface

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Localized Visualization and Autonomous Detection of Cell Surface Receptor Clusters Using DNA Proximity Circuit Yan Shan Ang, Jia’En Jasmine Li, Pei-Jou Chua, Cheng Teng Ng, Boon-Huat Bay, and Lin-Yue Lanry Yung Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00722 • Publication Date (Web): 02 Apr 2018 Downloaded from http://pubs.acs.org on April 3, 2018

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Analytical Chemistry

Localized Visualization and Autonomous Detection of Cell Surface Receptor Clusters Using DNA Proximity Circuit Yan Shan Ang,1 Jia’En Jasmine Li,1 Pei-Jou Chua,2 Cheng-Teng Ng,2 Boon-Huat Bay2 and Lin-Yue Lanry Yung1,* 1

Department of Chemical & Biomolecular Engineering, National University of Singapore, Singapore 117585, Singapore 2 Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore

Supporting Information Placeholder ing than other dimer combinations.8 The current understanding is that high HER2 expression level is correlated with aggressive cell proliferation and tumour development in breast cancer, and is recognized as an important biomarker for the prognosis of breast cancer.9 However, a significant proportion of patients show drug resistance against antibody-based therapy for HER2 target.10 It is believed that alternative signaling pathways are involved in the drug resistance mechanism, particularly via HER2:HER3 heterodimerization.11 On the clinical level, HER2:HER3 heterodimer is increasingly gaining attention as a potential biomarker for the prognosis of breast cancer.12-15 In this work, we will focus specifically on the detection and visualization of HER2:HER2 homodimers and HER2:HER3 heterodimers. Conventional methods of assessing the patient HER status include immunofluorescence and in situ hybridization which involve multiple manipulation steps and hours to days of processing to complete.16 Advanced microscopy techniques such as stimulated emission depletion (STED) microscopy17 and stochastic optical reconstruction microscopy (STORM),18 are used for the fundamental study of receptor clustering dynamics with nanometer precision and millisecond temporal resolution.19 However, they involve the use of sophisticated instruments and specialized operation capability, and are not suitable tools for day-to-day analysis and clinical diagnostics. DNA circuit offers a simple molecular toolbox for the autonomous evaluation of cell receptor clustering due to well established Watson-Crick base pairing and good sequence programmability. We present the design and application of a split proximity circuit (SPC) to transduce the recognition of biomolecule(s) in close proximity into DNA-based readout signal based on an improved association toehold design.20The original association toehold design was previously used to detect DNA and individual protein molecules by Chen,20 and Le and co-workers21-23 respectively. However, the detection of single molecular targets did not leverage on the key strength of association toehold in generating specific outputs based on

ABSTRACT: Cell surface receptor plays an important role in mediating cell communication and is used as disease biomarkers and therapeutic targets. We present a one-pot molecular toolbox, which we term the split proximity circuit (SPC), for the autonomous detection and visualization of cell surface receptor clusters. Detection was powered by antibody recognition and a series of autonomous DNA hybridization to achieve localized, enzyme-free signal amplification. The system under study was the human epidermal growth factor receptor (HER) family, i.e. HER2:HER2 homodimer and HER2:HER3 heterodimer, both in cell lysate and in situ on fixed whole cells. The detection and imaging of receptors were carried out using standard microplate scans and confocal microscopy respectively. The circuit operated specifically with minimal leakages and successfully captured the receptor expression profiles on three cell types without any intermediate washing steps.

Introduction Cell surface receptor plays an important role in cellular signal transduction processes, such as from the extracellular matrix to the cell and cell-to-cell communication. A classic example is the T cell signaling pathway involved in immune response.1 Receptor clustering also serve as important biomarkers for disease diagnosis2,3 and as therapeutics targets.4 The spatial distribution of the receptors on the cell membrane regulates the clustering pattern and hence signaling process.5 In particular, receptor dimerization and oligomerization often follows from ligand binding and is the general mechanism for activating receptors with single transmembrane domain.6 An interesting receptor system is the human epidermal growth factor receptor (HER) family responsible for the control of cell growth and proliferation.7 The receptors can exist either as monomers and dimers, of which the HER2 heterodimers are known to trigger much stronger intracellular signal-

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different combinations of inputs recognized, which we will

demonstrate here for detecting various HER dimers.

Figure 1. Schematics of SPC for the localized detection and visualization of cell surface receptor clustering. In absence of target(s), the initiator strands (I1 and I2) exist as two separate entities. Upon recognition of a proximity event, I1 and I2 associates at domain a to trigger hybridization chain reaction (HCR) between the hairpin monomers (HP1 and HP2). Another in situ, DNA probe-based detection method called proximity ligation assay (PLA) is a commonly used toolbox in recently years. PLA is a DNA-based assay where a pair of proximity probes bind to the target and hybridize with a connector oligonucleotide for enzymatic ligation,24 to trigger downstream readouts, e.g. rolling circle amplification (RCA).25 However, the amplified signals tend to saturate easily, partly due to the depletion of substrates, and so this method is more suitable for analyzing targets of low abundance. 26 Interestingly, an enzyme-free variant was recently developed based on the proximity-dependent initiation of hybridization chain reaction (HCR).27 Still, the execution involved a series of probe addition and washing steps, and necessitate low probe concentrations; without which, high target-independent background noise may result.

and stored at 4 °C for up to a year, except for Cy3/Cy5modified DNAs which were stored at – 20 °C and protected from light. The following chemicals were used as received: sodium chloride (NaCl, ≥ 99.5%), magnesium chloride (MgCl2, ≥ 98%), paraformaldehyde powder, recombinant human epidermal growth factor (EGF), insulin solution, dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), streptavidin from Streptomyces avidinii and biotin were purchased from Sigma Aldrich. 10X phosphate buffered saline (PBS, pH 7.4) and 1X tris-ETDA (TE, pH 8.0) was purchased from 1st BASE. Tween20 was purchased from Sinopharm. RPMI-1640 medium (+2.05 mM L-glutamine), DME/F12 1:1 (+ 2.5 mM Lglutamine), fetal bovine serum (FBS) and trypsin EDTA were purchased from GE Healthcare. For western blot, SuperSignal West Pico was purchased from Thermo Fisher and Clarity Chemiluminescent Substrate System was purchased from BioRad. Biotinylated anti-ErbB2 affibody® (ab31890) was purchased from Abcam. Biotinylated anti-human erbB3/HER-3 (AB_756158) was purchased from Biolegend. HER2/ErbB2 antibody (2242) and HER3/ErbB3 antibody (12708) were purchased from Cell Signaling Technology. Milli-Q water with resistance >18.2 MP/cm was used throughout the experiment.

Our circuit design comprised of two initiator strands (I1 and I2) conjugated to specific recognition antibodies. They existed as separate entities in absence of the target receptor dimers (Figure 1). Upon binding to a pair of target receptors in close proximity, the local concentration of I1 and I2 increased, which promoted association at domain a to assemble the complete trigger strand. Our modified association toehold design has reduced circuit leakage, faster association rate and improved signal-to-noise ratio compared to the original association toehold design.28 The complete trigger strand then activates the HCR to generate localized, amplified fluorescent signal in the form of long DNA chains tagged with Cy3-Cy5 FRET pair which we reported previously (Figure S1).29 HCR was chosen as the signal amplification method due to its fast kinetics, isothermal and enzyme-free nature. The circuit was designed to operate autonomously in a self-contained format for improved hybridization kinetics.30

Preparation of Split Proximity Circuit Components Streptavidin was used as the linker molecule to link biotinylated antibody to the biotinylated initiators. Equivolume amount of 2.5 µM of biotinylated DNA and 2.5 µM of streptavidin were mixed in 1X PBS for 1 h at 37 °C before cooling down to room temperature for 30 min. Equivolume amount of 1.25 µM of biotinylated antibody was then added to the reaction mixture and heated to 37 °C again for 1 h before cooling down to room temperature for 30 min. The final probes were stored in 4 °C for up to 2 weeks. Unreacted streptavidin sites were blocked using 100 µM biotin in 1X PBS. The hybridization chain reaction (HCR) hairpins were prepared just before the experiments. 1 µM of hairpin 1 (HP1) tagged with Cy5 and hairpin 2 (HP2) tagged with Cy3 was heated separately in 1X PBS and 10 mM MgCl2 to 95 °C for 5 min and cooled to room temperature over 30 min.

Experimental Section Materials All DNA oligonucleotides used in this study were purchased from Integrated DNA Technologies (IDT), and HPLC purified by IDT unless otherwise stated. The DNA sequences are shown in Table S1. The lyophilized DNA was reconstituted in 1X Tris-EDTA buffer (1X TE, pH 8.0) to give ca. 100 µM stock

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Analytical Chemistry Thereafter, the circuit components were mixed in the following sequences to the stated reaction concentrations: 0.2 µg/mL anti-HER2-I1, 20 nM HP2, 40 nM HP1 and 0.2 µg/mL anti-HER2-I2 or 2.0 µg/mL anti-HER3-I2.

Cells were seeded at a density of ca. 1.5 × 105 cells / cm2 in a 96-well microplate overnight to achieve monolayer coverage. The cells were then fixed with 4% paraformaldehyde for 15 min and blocked with 1% BSA and 0.05% Tween-20 in 1X PBS for 2 h. Permeabilization step was previously carried out and found to be not necessary as the target proteins were presented on the cell surface. The pre-mixed split proximity circuit in a reaction buffer consisting of 1% BSA, 0.05% Tween-20 and 10 mM MgCl2 in 1X PBS was added one-pot to the fixed cells. The kinetics of HCR FRET signal generation was monitored on a microplate reader (Tecan M200) using an excitation wavelength of 530 nm and emission wavelengths of 576 nm (Cy3) and 670 nm (Cy5).

Cell Culture AU565 breast cancer cells (ATCC CRL-2351) and MDA-MB231 triple negative breast cancer cells (ATCC HTB-26) were cultured in RPMI 1640 media supplemented with 10% FBS. MCF10A normal breast epithelial cells (ATCC CRL-10317) were cultured in DMEM F12 (with L-glutamine) supplemented with 5% FBS, 20 ng/mL EGF, 100 ng/mL cholera toxin, 0.01 mg/mL insulin and 500 ng/mL hydrocortisone. Cell cultures were maintained in an environment of 5% CO2 and 37 °C.

In Situ Visualization Under Confocal Microscopy Cell Lysate Analysis

The cells were seeded overnight at a density of 0.5 × 105 cells/cm2 in an 8-well chambered coverglass. Similar to cell ELISA preparation, the cells were washed, fixed with 4% paraformaldehyde for 15 min and blocked with 1% BSA and 0.05% Tween-20 in 1X PBS for 2 h. Thereafter, the blocking buffer was removed and the chamber was washed once with 1X PBS. Next, the cells were incubated with 1 µg/mL of Hoechst dye (diluted with 1X PBS from a working concentration of 1 mg/mL immediately before use) for 5 min, followed by washing thrice with 1X PBS. The circuit reaction mixture (prepared in the same way as in one-pot cell ELISA) was added to the cells in one-pot and imaged directly under Nikon A1 confocal microscope for kinetics study. Otherwise, the reaction proceeded for 30 min before pipetting the reaction mixture from the chamber and replacing with the reaction buffer. This effectively quenched the circuit reaction at the 30 min mark to ensure comparable reaction time across all chambers. The Nikon A1 laser scanning confocal system was used for FRET, time-lapse and z-stack imaging. To achieve FRET, the donor (Cy3) was excited using 488 nm Argon gas laser and the acceptor (Cy5) emission was collected using Cy5 filter wheel. Cell nucleus stained with Hoechst dye was excited with 405 nm diode laser and the emission collected with DAPI filter wheel. Most images were captured using 40X objective to survey a more representative cell population. Close-up images at 100X oil objective were taken to confirm structural details. The gain and laser power were kept constant for all confocal imaging. The captured images were analysed using ImageJ with the same brightness and contrast adjustment.31

The membrane and cytosol proteins were extracted from the cell lines in separate fractions as per manufacturer’s protocol (Mem-Per plus membrane protein extraction kit, Thermo Scientific). The respective expression levels were confirmed using western blot. 30 μg of the extracted protein was loaded to a 4-12% gradient polyacrylamide gel for separation and transferred to PVDF membrane at 15V for 50 min. The PVDF membrane was blocked for 2 h, followed by incubation at 4°C overnight with BSA diluted rabbit primary antibodies for HER2, HER3 and β actin mouse primary antibody which act as the internal loading control. The following day, membrane was washed thrice with Tris-buffered saline and 0.1% Tween-20 (TBST) for 10 min per wash before incubating with anti-rabbit secondary antibodies and anti-mouse secondary antibody for 1h at room temperature. The membrane was then rinsed twice with TBST, followed by once with TBS for 10 min per wash. SuperSignal West Pico and Clarity Chemiluminescent Substrate System was used to detect the proteins of interest at specific protein sizes. The cell lysate fractions were quantified using Nanodrop measurement and adjusted to 100 µg/mL across all fractions to ensure fairness of comparison. 100 µg/mL was ranged within 50 – 500 µg/mL and involved at least five times dilution, as recommended for commercially available cell lysate assays. No further purification of the cell lysate was carried out. The pre-mixed split proximity circuit in a reaction buffer of 1X PBS (pH 7.4) and 10 mM MgCl2 was added one-pot to the cell lysate for detection of HER2:HER2 homodimers and HER2:HER3 heterodimers. Fluorescence measurement was taken at 30 min interval in Tecan Spark M200 reader to monitor the kinetics of signal evolution. The FRET ratio was determined by taking the ratio of Cy3 (λem = 576 nm) to Cy5 emission (λem = 670 nm) for a given excitation wavelength (λexc = 530 nm). The background emission was subtracted at the respective wavelengths by exciting a blank well containing the same volume of reaction buffer without any DNA species.

Results and Discussion Dimer Detection in Cell Lysate We first confirmed that our SPC could detect the receptor dimers at the protein level in cell lysate. Three cell lines were used for this study – AU565 breast cancer cell line which overexpresses HER2 and HER3,32 MDA-MB-231 triple negative breast cancer cell line with no HER2 and HER3 expression,33 and MCF10A normal breast epithelial cell line with basal-to-no expression of HER2 and HER3.34 Their respective expression levels were characterized using western blot (Figure S2). The antibody-conjugated initiators and HCR hairpins were pre-mixed and added one-pot to the cell lysate extract without intermediate washing steps. Obvious HCR FRET sig-

One-Pot Cell Enzyme-Linked Immunosorbent Assay (ELISA)

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nals developed for both HER2:HER2 homodimers and HER2:HER3 heterodimers in the membrane faction of the positive AU565 cell line (Figure 2). In contrast, the negative MDA-MB-231 and normal MCF10A cells were indistinguishable from the negative control where no target was added. Negligible amount of FRET signal was generated in the cytosolic fraction across all cell lines and negative controls, in line with our western blot characterization (Figure S2). This demonstrates the specificity of our circuit in detecting protein complexes even under a complex reaction environment consisting of lysis buffer and a cocktail of extracted proteins.

HER3-I2 confirmed that the circuit was triggered only when two biomolecular recognition events occurred.

Figure 3. The FRET ratio of the test and control set-ups was analyzed after 30 minutes of reaction. (a) Different control set-ups were tested on the positive AU565 cell line. (b) We probed for HER2:HER2 homodimer and HER2:HER3 heterodimer against the negative control for all three cell lines. Onesided t-test was performed for test samples against the negative control where no antibody was conjugated to the initiator strands (No Ab). Data is shown as mean ± standard deviation (n = 3). *** refers to p < 0.005. The SPC returned a statistically significant positive result only for AU565 and not MDA-MB-231 or MCF10A cells, thereby demonstrating the specificity of circuit recognition (Figure 3b). We noted the higher FRET ratio for the negative control of AU565. This might be due to the stickier cell surface leading to varying degree of non-specific binding of the circuit components. Therefore, it was necessary to include a negative control, where no antibody was conjugated to the DNA circuit, for each cell lines tested to interpret the assay results meaningfully. However, such non-specific binding was less of an issue in our SPC than in typical ELISA assay since two different initiator strands need to be non-specifically bound in close proximity to each other and with proper orientation to trigger HCR. Hence, intermediate washing step was not critical to the success of our assay though one could choose to perform a single washing step at the end of reaction to reduce variations in the results (data not shown).

Figure 2. Detection of (a) HER2:HER2 homodimer membrane fraction and (b) cytosol fraction, and (c) HER2:HER3 heterodimer membrane fraction and (d) cytosol fraction extracted from AU565, MDA-MB-231 and MCF10A cell lines. The protein content in each fraction was adjusted to 100 µg/mL.The data are shown as mean ± standard deviation (n = 3).

Dimer Detection in Cell ELISA Format

Direct Visualization of Receptor Clusters

Our SPC was next applied for the in situ detection of receptor clusters on fixed cells. The performance of SPC on cell surface, which was now a heterogeneous phase reaction, was evaluated by characterizing the efficiency of signal transduction (Figure S3) and HCR (Figure S4). The cells were seeded, fixed and blocked in 96-well plate, as per standard cell ELISA protocol. This was followed by the one-pot addition of SPC to the fixed cells and the FRET signal was monitored over time (Figure S5). The circuit operation remained robust at the cell level where significant positive signals developed over time only in presence of all circuit components, i.e. anti-HER2-I1, anti-HER2-I2 or anti-HER3-I2, HP1, HP2 and positive AU565 cell, for both HER2:HER2 and HER2:HER3 dimers. Though equilibrium was not attained even after 4 h, the FRET ratio for the test on AU565 cells was already statistically more significant (p < 0.005) than all other controls by the first time point of 30 min (Figure 3a). The negative control was taken as the SPC without antibody recognition moiety, which accounted for the inherent circuit leakage. The lack of appreciable signal upon the omission of either anti-HER2-I1 and anti-HER2-I2 or anti-

The long, localized DNA fluorescent chain generated from HCR FRET readout was used to visualize the interaction sites directly. We first performed time-lapse confocal imaging on AU565 cells upon the one-pot addition of our SPC (Video SV1). Visible signals started to form within 10 min of reaction for both HER2:HER2 and HER2:HER3 dimers, and bright FRET signals was observed within 30 min (Figure 4). No distinct, localized FRET signal was seen for the negative control (no antibody conjugated). The time-lapse study further confirmed that 30 min was sufficient for generating sufficient signal over background noise as previously concluded from the cell ELISA results. All the analysis from this point onwards was fixed at 30 min reaction time. This is faster than the conventional immunohistochemistry method used for clinical diagnostics. However, it is not suitable for probing the dynamics of receptor clustering in real time if they occurred at a time scale faster than the circuit operation. Instead, it should be used to profile strong interactions with long binding time, e.g. 30 – 60 min of binding between T-cell and antigen-presenting cells,35

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Analytical Chemistry to detect the surface receptor profiles on different cell lines under confocal imaging. Again, strong FRET signal developed only for HER2:HER2 and HER2:HER3 dimers in AU565 cells (Figure 5). The other two cell lines with no HER2 and HER3 expression did not develop appreciable FRET signal. Similarly, the negative controls returned no FRET signal.

or interactions captured at a particular time point within fixed samples. The localization of the FRET signal on the AU5656 cell surface was confirmed by Z-stack analysis (Video SV2). The specificity of the circuit operation was confirmed by omitting different circuit components in a series of controls (Figure S6). There was no signal present in the negative control where no antibody recognition moiety was conjugated. Neither was there any visible signal when only one of the initiators was used which again confirmed that two recognition events were needed as inputs to the circuit. Lastly, no signal was observed when only the HCR hairpins were added, which suggests that any non-specifically bound hairpins were too diffuse on the cell membrane to be viewed visibly under our confocal settings.

Figure 5. Direct visualization of HER2:HER2 homodimer and HER2:HER3 heterodimer under confocal microscopy after 30 min of incubation in three cell lines: AU565 (high expression), MCF10A (normal, basal-to-no expression) and MDA-MB-231 (triple negative breast cancer cell, no expression). Scale bar shown corresponds to 50 µm.

One of the striking advantage of the SPC is its simple operation for the in situ detection and visualization of receptor clustering events. While the detailed working mechanism of our circuit involved multiple reaction steps, the final form of the assay operates autonomously based on the inputs detected. Its one-pot format reduces processing time, laborious work and more importantly, cuts down the chance of human errors. Moreover, it is compatible with existing bio-analytical methods, i.e. antibody recognition, ELISA and confocal imaging. These are practical features for eventual translational studies.

Figure 4. Confocal images at various time points (0, 10, 20 and 30 min) after the one-pot SPC was added to AU565 cells to probe for HER2:HER2 homodimers (first column) and HER2:HER3 heterodimers (second column). Negative control where no antibody was conjugated is shown in the third column. An enlarged image of the cells is shown in the inset of the confocal image at 30 min. Scale bar shown corresponds to 50 µm.

Next, our circuit is “plug-and-play” where different recognition moieties, e.g. antibody, aptamer or small molecules, can be attached to the initiator strands to detect specific target(s) of interest. Similar concept of such versatile, one-pot assay was demonstrated by other groups though their strategies are largely applicable only for detecting single protein target(s).36,37 However, proteins seldom function as single entities and are often part of a larger protein complex or interactome network.38-40 Additional information can be harnessed from the plethora of biomolecular interaction events, such as post-translational modifications and cell surface

Detecting Expression Profiles of Different Receptor Clusters Having established the speed and specificity of circuit operation for direct visualization, we proceeded to test its ability

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markers as major diagnostics biomarkers and drug targets.41-

Video of z-stacking for HER2:HER3 heterodimer on AU565 cell (AVI)

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Nonetheless one should note a few caveats in interpreting the results obtained using this method. First, the circuit was designed to recognize biomolecular events in close proximity with the inherent assumption that they are indicative of interaction events. However, it is possible that the circuit can be falsely triggered in presence of neighbouring pairs of HER2 or HER3 but not necessarily existing as true dimers. This is a common problem for existing molecular toolbox, e.g. fluorescence co-localization.44 One way to minimize the detection of false clustering events is to reduce the spacer length of the initiators to constrain the effective radius of detection. Next, the same antibody conjugated to either I1 or I2 was used to detect for HER2 homodimer which inevitably will compete for the same binding epitope. This means that some homodimers recognized by a pair of HER2-I1 or HER2-I2 will not generate HCR signal leading to some false negatives in the results. It is possible to use different HER2 antibody to eliminate the competitive effect;12 however, the false positive rate will increase correspondingly, i.e. the antibody pair can bind to a single protein target (assuming their binding epitope is different) to give HCR signal even though the homodimer is not present. In its current form, the split proximity circuit is suitable for diagnostics applications with its advantage in speed and simplicity. We look forward to refining the circuit sequence design and reaction conditions so that it can be applied for live cell analysis. This will facilitate the spatiotemporal mapping of receptor clustering events in response to specific ligand stimulation or changes in the environment and expand the application of this toolbox for studying upstream biological events.

AUTHOR INFORMATION Corresponding Author To whom correspondence should be addressed. Tel: +65 6516 1699; Fax: +65 6779 1936; Email: [email protected]

ACKNOWLEDGMENT This work was supported by research funding from the Singapore Millennium Foundation and the Singapore Ministry of Education Academic Research Fund Tier 1. Funding for open access charge: Singapore Ministry of Education Academic Research Fund Tier 1. Y. S. A. would like to thank the National University of Singapore and Ministry of Education for the President Graduate Fellowship.

REFERENCES (1) Cochran, J. R.; Aivazian, D.; Cameron, T. O.; Stern, L. J. Receptor clustering and transmembrane signaling in T cells. Trends Biochem. Sci 2001, 26, 304-310. (2) Gedye, C. A.; Hussain, A.; Paterson, J.; Smrke, A.; Saini, H.; Sirskyj, D.; Pereira, K.; Lobo, N.; Stewart, J.; Go, C.; Ho, J.; Medrano, M.; Hyatt, E.; Yuan, J.; Lauriault, S.; Kondratyev, M.; van den Beucken, T.; Jewett, M.; Dirks, P.; Guidos, C. J.; Danska, J.; Wang, J.; Wouters, B.; Neel, B.; Rottapel, R.; Ailles, L. E. Cell Surface Profiling Using High-Throughput Flow Cytometry: A Platform for Biomarker Discovery and Analysis of Cellular Heterogeneity. PLoS ONE 2014, 9, e105602. (3) Joensson, H. N.; Samuels, M. L.; Brouzes, E. R.; Medkova, M.; Uhlén, M.; Link, D. R.; Andersson-Svahn, H. Detection and Analysis of Low-Abundance Cell-Surface Biomarkers Using Enzymatic Amplification in Microfluidic Droplets. Angew. Chem. Int. Ed. 2009, 48, 2518-2521. (4) Christopoulos, A. Allosteric binding sites on cell-surface receptors: novel targets for drug discovery. Nat. Rev. Drug Discov. 2002, 1, 198-210. (5) Bethani, I.; Skånland, S. S.; Dikic, I.; Acker-Palmer, A. Spatial organization of transmembrane receptor signalling. EMBO J. 2010, 29, 2677. (6) Heldin, C.-H. Dimerization of cell surface receptors in signal transduction. Cell 1995, 80, 213-223. (7) Rubin, I.; Yarden, Y. The Basic Biology of HER2. Ann. Oncol. 2001, 12, S3-S8. (8) Yarden, Y. Biology of HER2 and Its Importance in Breast Cancer. Oncology 2001, 61(suppl 2), 1-13. (9) Weigel, M. T.; Dowsett, M. Current and emerging biomarkers in breast cancer: prognosis and prediction. Endocr.-Relat. Cancer 2010, 17, R245-R262. (10) Vogel, C. L.; Cobleigh, M. A.; Tripathy, D.; Gutheil, J. C.; Harris, L. N.; Fehrenbacher, L.; Slamon, D. J.; Murphy, M.; Novotny, W. F.; Burchmore, M.; Shak, S.; Stewart, S. J.; Press, M. Efficacy and Safety of Trastuzumab as a Single Agent in FirstLine Treatment of HER2-Overexpressing Metastatic Breast Cancer. J. Clin. Oncol. 2002, 20, 719-726. (11) Sergina, N. V.; Rausch, M.; Wang, D.; Blair, J.; Hann, B.; Shokat, K. M.; Moasser, M. M. Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3. Nature 2007, 445, 437-441.

Conclusions Taken in its entirety, the split proximity circuit was successfully used to detect receptor clusters in both cell lysate (homogeneous phase) and on fixed cells (heterogeneous phase). Under both conditions, the circuit successfully operated with good specificity and minimal circuit leakage to generate significant signal over background noise within 30 min and in absence of intermediate washing steps. Circuit computation on the cell surface successfully differentiated the receptor expression profiles on three cell types. The formation of a localized, amplified HCR FRET signal facilitated the direct visualization of receptor clustering in situ for additional spatial information which is useful for detection works.45

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxx DNA sequences, additional western blot, ELISA and confocal imaging results (PDF) Video of time-lapse confocal imaging (AVI)

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Analytical Chemistry


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Localized Visualization and Autonomous Detection of Cell Surface

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