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Article Cite This: ACS Omega 2019, 4, 11515−11521
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Glass Chemistry to Analyze Human Cells under Adverse Conditions Angela N. Marquard,† Jonathan C. T. Carlson,†,‡,* and Ralph Weissleder*,†,§ †
Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge Street, CPZN 5206, Boston, Massachusetts 02114, United States ‡ MGH Cancer Center, Massachusetts General Hospital, Boston, Massachusetts 02114, United States § Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, United States
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S Supporting Information *
ABSTRACT: Emerging point-of-care diagnostic tests capable of analyzing whole mammalian cells often rely on the attachment of harvested cells to glass surfaces for subsequent molecular characterization. We set out to develop and optimize a kit for the diagnosis of lymphoma in low- and middle-income countries where access to advanced healthcare testing is often absent or prone to error. Here, we optimized a process for the lyophilization of neutravidin-coated glass and cocktails of antibodies relevant to lymphoma diagnosis to establish long-term stability of reagents required for point-ofcare cell capture technology. Lyophilized glass slides showed no decline in their performance compared to freshly prepared neutravidin glass and preserved capture efficiency for 5 weeks under easily attainable storage conditions. We demonstrate the successful performance of the low-cost, lyophilized kit in a cell capture assay to enable true point-of-care analyses under adverse conditions. We anticipate that the strategy can be expanded to other cancer cell types or cell-derived vesicle analysis.
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INTRODUCTION Global healthcare is slowly becoming a reality given significant advances in bioengineering, miniaturization, affordable manufacturing costs, and the rising socioeconomic status of many low- and middle-income countries (LMIC). However, significant hurdles persist, particularly in the development of cell-based assays. Such assays are of particular importance in global oncology, where they would allow healthcare providers to establish the diagnosis and subtype of cancer1−4 as well as enhancing the diagnosis of communicable diseases, particularly those where tissue or biofluid sampling is often required (i.e., Legionella,5 mycobacteria, or Pneumocystis species6). For many of these applications, it is often necessary to capture cells onto glass slides for subsequent interrogation by plasmonic, fluorescence, holographic, or other means of imaging. Yet, adverse environmental conditions in primary care settings, paired with a lack of reagents and a scarcity of highly trained specialists, have made such evaluations difficult and prone to error. We have previously developed a holographic method to diagnose lymphoma in LMIC; the device uses contrastenhanced microholography and a deep-learning algorithm to directly analyze percutaneously obtained fine-needle aspirates.1 Lymphomas constitute a heterogeneous collection of diseases with diverse natural histories and treatment responses. Clinical prognostication and selection of appropriate therapeutic options for each subtype require accurate classification, based, in developed countries, on review of cellular markers, histology, and molecular analysis by specialized hematopathologists. In our prior work, we demonstrated the feasibility and high accuracy of the holographic device in characterizing cells; © 2019 American Chemical Society
we also developed a concise algorithm to classify benign vs malignant and low-grade vs aggressive cellular populations from clinical biopsies, based on a tailored panel of cell markers.1 The diagnostic accuracy of this approach was prospectively validated in 40 patients referred for imageguided aspiration of nodal mass lesions suspicious for lymphoma. The studies, however, were conducted with freshly prepared reagents in fluid-based form and required multistep protocols for the preparation of the analytical toolkit at the time of analysis, which is exceptionally challenging in subSaharan countries. In the current study, we thus extended these proof-of-principle experiments to develop glass capture technology and experimental conditions that are suitable for long-term storage, single-step point-of-care reconstitution, and more resistant to heat and humidity. Specifically, we optimized a capture technology for cells onto lyophilized neutravidincoated glass slides and developed quality control procedures to assure uniformity. We show that these improved methods are superior compared to commercially available reagents and enable true point-of-care analyses under adverse conditions in highly reproducible form. Finally, the methods may be extendable to vesicles7 and other biomarkers.
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RESULTS Figure 1 provides an overview of the three experimental aspects: (i) glass activation, for deposition of a high-density neutravidin monolayer; (ii) kit preparation, incorporating both Received: April 11, 2019 Accepted: June 19, 2019 Published: July 3, 2019 11515
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Figure 1. Glass chemistry and schematic overview. (a) Glass coverslips modified with a high-density layer of neutravidin are lyophilized for longterm storage stabilized by trehalose. (b) Cells are immunostained with a lyophilized antibody cocktail that contains both capture and staining antibodies. (c) Cells are captured via biotinylated antibodies in a flow-cell cartridge containing rehydrated neutravidin glass for analysis.
biotinylated antibodies for cellular capture and fluorescently labeled imaging probes; (iii) cellular analysis. Because the stringent storage and handling conditions specified by the manufacturers of commercially available streptavidin-coated glass slidesshipped on ice or dry ice, stored in sealed packaging at −20/−80 °Cwere poorly suited for assay implementation in the LMIC context we sought, we optimized our own approach for the preparation and long-term storage of capture slides (Figure 1a). Antibody cocktails containing both biotinylated antibodies for cell capture and labeled antibodies as additional markers were also lyophilized in a single kit with trehalose and other excipients (see Materials and Methods) to prevent protein denaturation (Figure 1b). Finally, cells stained with the antibody kit are captured onto the reconstituted neutravidin glass for imaging and analysis (Figure 1c). Freshly cleaned glass coverslips in a multislide holder (50 coverslips per batch) are silanized with 3-(aminopropyl)triethoxysilane (APTES) followed by treatment with glutaraldehyde.1,8 Serial preparations demonstrated that rigorous stirring of the reaction mixture at each stage was necessary to produce a homogeneous surface. In the final step, reductive amination chemistry conjugates lysine residues on neutravidin to the aldehyde-functionalized surface, yielding high-density neutravidin-coated glass (Figure 1a). To explore the preservation of the neutravidin activity after long-term storage, the glass slides were lyophilized in a range of buffer (succinate, histidine, phosphate), pH (5−7), and lyoprotectant (sucrose, trehalose) conditions (data not shown). We found that the presence of trehalose as a lyoprotectant (Figure 1a) in Milli-Q water provided optimum reconstitution, as buffer salts deposited on the glass surface during the lyophilization process made the recovery of an optically clear substrate challenging within the planned LMICcompatible assay techniques. Subsequent experiments (vide infra) were conducted to define the relationship between the trehalose concentration and slide durability in storage.
Methods to measure the surface density of coated glass or silica surfaces have frequently relied on highly specialized techniques such as atomic force microscopy and X-ray photoelectron spectroscopy.9,10 We sought to benchmark the neutravidin functionalization of the glass surface by more readily available fluorescence imaging. To that end, we first assessed the ability of a direct biotin-fluorophore conjugate (AF594-biotin) to optically interrogate neutravidin/streptavidin density. The signal/background of the resulting images was poor, even at 3 s exposure times (Figure S1). We thus implemented a secondary antibody-based amplification step to indirectly visualize the neutravidin surface layer. Functionalized and blocked protein-coated surfaces were incubated with the biotinylated mouse anti-CD20 capture antibody used in our cellular assays, followed by a suitable fluorescent secondary antibody (AlexaFluor647 goat anti-mouse). The resulting signal amplification enabled us to readily quantify the surface coating density and visualize defects in the surface, whether mechanical (e.g., pipette tip scratches) or chemical (from inhomogeneous labeling). The surface of neutravidin coverslips showed approximately 10 times higher fluorescence intensity over background than a commercial streptavidin glass slide (Figure 2a). Control images with no primary antibody were equivalent to the no-coating control, verifying the specificity of staining. Comparative fluorescence images demonstrate the lower surface modification density of the commercial glass and comparably low spatial heterogeneity (Figure 2b). Both commercial streptavidin glass and our neutravidin coverslips revealed a susceptibility to mechanical damage to the protein layer that was readily visualized with our imaging method, highlighting a need for gentle handling. Reconstitution of the lyophilized neutravidin glass was tested by comparing B-cell capture efficiency to that of freshly prepared neutravidin glass. We used B-cell lymphoma cell lines (Daudi and DB) and the Jurkat T-cell leukemia cell line to create a standardized panel that encompasses the key positive/ 11516
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which demonstrated only 5% mean capture efficiency (Figure 2c). Given these encouraging results, we next attempted to define the importance of lyoprotectants in the slide preparation and to quantify effects of temperature and storage conditions on the cell capture (Figure 3). Stabilization of the neutravidin
Figure 2. Neutravidin surface imaging and cell capture efficiency. Surface modification of prepared neutravidin glass and a commercial streptavidin slide was analyzed by fluorescence microscopy after staining with biotinylated antibody and secondary antibody fluorescence amplification. (a) Fluorescence intensity plot of a 2 mm cross-section of immunostained glass. (b) Fluorescence image comparison of lyophilized neutravidin glass and a commercial streptavidin slide. The prepared neutravidin glass showed a greater modification density than commercial streptavidin glass. Scale bar = 200 μm. (c) Capture efficiency of lyophilized neutravidin glass by biotinylated antibodies was compared to freshly prepared neutravidin glass and commercial streptavidin glass with a B-cell lymphoma cell line (DB) and a nonspecific T-cell leukemia cell line (Jurkat). p = 0.98 between fresh and lyophilized glass capture, p < 0.0001 between both fresh/lyophilized and commercial glass capture.
negative markers for clinical biopsy samples.1 CD19/20+ DB cells were incubated with biotinylated anti-CD19 and antiCD20 antibodies (Table S1) and then rigorously washed to remove any excess/unbound antibody, which can markedly inhibit capture. Cells in suspension were pipetted into a 1 mm adhesive silicone flow cell applied to the neutravidin coverslips, incubated for 10 min with rotatory rocking, imaged to quantify baseline density, and then washed with a syringe pump (see Materials and Methods for further details). Capture efficiencies were defined by the number of cells retained in the postwash image per field of view and found to be 85−90% for both fresh and lyophilized glass, indicating no loss in neutravidin functionality post-lyophilization (Figure 2c). DB cells stained with matching concentrations of a biotinylated isotype control antibody exhibited capture efficiencies of less than 12% for both fresh/lyophilized glass. Lyophilized neutravidin glass did not show an increase in nonspecific capture of CD19/20 negative Jurkat cells compared to fresh neutravidin (18% for lyophilized neutravidin, 23% for fresh neutravidin). The performance of the high-density prepared neutravidin glass was far superior compared to a commercial streptavidin slide,
Figure 3. Optimization and stability of lyophilized neutravidin glass. (a) Capture efficiency of lyophilized neutravidin coverglass stabilized with different concentrations of trehalose (p = 0.99 between fresh and 1% trehalose). (b) Capture efficiency was stable after 24 h at 4−37 °C (p ≥ 0.6 for all comparisons). After 1 week of storage, a decline in capture efficiency was observed for cells stored at 30 °C (p = 0.49) and 37 °C (p < 0.01). (c) Slides stored at 4 °C for 5 weeks performed equivalently to the freshly prepared glass (p = 0.86).
surface by trehalose for long-term storage was investigated and optimized by soaking the neutravidin glass in various concentrations of trehalose prior to lyophilization at room temperature overnight. The surface chemistry stabilization of the lyophilized slides was examined with the B-cell capture protocol (as above) upon reconstitution with PBS (Figure 3a). When neutravidin was stabilized with 1% trehalose solution, DB cell capture efficiency was equivalent to that of the fresh neutravidin glass. In the absence of trehalose, capture efficiency was markedly reduced (38%) compared to the fresh glass. At higher trehalose concentration, capture efficiency decreased to 11517
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Figure 4. Biological characterization of the lyophilized antibody panel. Flow cytometry validation of the activity of lyophilized antibodies (gray) compared to fresh antibodies (red).
Figure 5. Multichannel imaging of captured cells. Fluorescent images of lymphoma cells stained with kappa light chain, lambda light chain, and Ki67 after capture by biotinylated CD19 and CD20 antibodies (DAPI stained for visualization) onto the lyophilized neutravidin glass. Daudi and DB B cells (CD19/20+) show high capture, whereas Jurkat cells (CD19/20−) show low capture, as expected. All cells are positive for Ki67. Scale bar = 50 μm.
at 37 °C. Lyophilized neutravidin slides stored at room temperature still maintained 85% capture efficiency after 4 weeks, consistent with a decay t1/2 of ≥10 weeks, more than sufficient for brief interruptions in refrigeration during shipping or transit to remote sites (e.g., satellite hospitals or remote clinics). Appealingly, the lyophilized neutravidin glass stored at 4 °C demonstrated capture efficiency equal to that of freshly prepared neutravidin glass even after 5 weeks (Figure 3c). To extend a lyophilized kit to the full ensemble of reagents for molecular cell labeling, we also lyophilized biotinylated anti-CD19 and anti-CD20, anti-kappa light chain, anti-lambda
82%. Based on these results, 1% trehalose was used for further neutravidin glass stability kinetics. Batches of stabilized, lyophilized neutravidin glass were stored under desiccated conditions at a range of environmentally relevant temperatures and serially tested for B-cell capture. Lyophilized glass could withstand temperatures up to 37 °C for 24 h with no decrease in cell capture efficiency (Figure 3b). Only minor declines in cell capture were observed after 1 week in storage at room temperature (96%). However, after 1 week at higher temperatures, capture efficiency decreased to 81% with storage at 30 °C and 40% with storage 11518
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of the cells to the durable neutravidin surface via CD19 and CD20 enriches the bulk fine-needle aspirate for the relevant Bcell subpopulation, enabling the three-channel analysis of clonality (kappa, lambda) and proliferation rate (Ki67) for diagnostic classification. Immunophenotypic isolation thus reduces the number of independent channels required for imaging, a key technical challenge for portable, low-cost fluorescence microscopes and/or smartphone-based imaging systems.12 Extensive optimization of the stabilization of individual monoclonal antibodies by sugars and other excipients during lyophilization has been achieved in the pharmaceutical industry for antibodies licensed as lyophilized powders.13−15 Nevertheless, only a few commercial methods are available for lyophilizing multiple-antibody cocktails for diagnostic assays, all of which rely on proprietary buffer formulations and techniques.16,17 Here, we demonstrated a simple, low-cost strategy for preparing lyophilized, ready-to-use antibody cocktails containing up to five different antibody markers, stabilized with only common laboratory reagents. Several previous studies have used glass activation for biosensors18 and cell capture applications.19 Although these laboratory-specific approaches for the preparation of monolayer functionalized glass (e.g., aminosilanes, aldehydes) have an extended track record,20−22 the methods have been tailored for on-demand preparation, precisely the scenario we set out to avoid. In keeping with the objective of procedural simplicity at the point of analysis, the lyophilized neutravidin glass and antibody cocktail require only rehydration (≤5 min) to be ready for use. Moreover, our approach is unique in that it tackles the clinical problem of lymphoma diagnostics in LMIC. The most common lymphoma subtypes in sub-Saharan Africa are diffuse large B-cell lymphoma, Burkitt’s lymphoma, classical Hodgkin’s lymphoma, and chronic lymphocytic leukemia.23,24 Optimal standard therapies differ for the various subtypes of lymphoma, as does the expected clinical course across a broad spectrum of outcomes (i.e., from indolent and non-lifethreatening to rapidly progressive and potentially fatal); the current lack of new technology to facilitate correct diagnosis/ prognosis is thus a major problem.25,26 Immunohistochemical studies and other key elements of molecular pathology continue to have very limited availability in the LMIC of Africa.27 We hope that the approach here is a first step toward triaging patients with suspected lymphoma to the most appropriate treatment and/or clinical observation strategy. Beyond the tested antibody cocktails, we anticipate that the strategy can be expanded to other cancer cell types (e.g., breast, melanoma, or lung cancer) or cell-derived vesicle analysis.7 Initial optimization experiments for the lyophilization of breast cancer antibody cocktails are currently underway for clinical applications.
light chain, and anti-Ki67 antibodies in a custom formulation containing trehalose, polysorbate 20, and succinic acid as stabilizers (see Materials and Methods), which we arrived at after iteratively screening a range of buffer/excipient compositions for each antibody individually. This antibody cocktail contains the most relevant molecular markers to distinguish between aggressive and indolent lymphoma subtypes in clinical lymphoma diagnosis.11 Optimization of the lyophilization conditions revealed that pH and buffer composition had a large impact on the activity of the lyophilized antibodies as certain antibodies had markedly poorer staining performance in particular buffers (Figure S2). Successful lyophilization of the antibodies was validated by flow cytometry quantification of the corresponding targets in Daudi (CD19/20+, kappa+), DB (CD19/20+, lambda+), or Jurkat cells (CD19/20−, Ki67+) before and after lyophilization. The fluorescence signal of the lyophilized antibodies showed little to no change compared to the fresh antibody staining (Figure 4). To demonstrate the performance of the capture assay with the full array of lyophilized materials, lymphoma cells stained with lyophilized/reconstituted fluorescently labeled kappa light chain, lambda light chain, and Ki67 were imaged after capturing by lyophilized/reconstituted biotinylated antiCD19 and anti-CD20 antibodies onto lyophilized/reconstituted neutravidin glass (Figure 5). Daudi and DB cells showed efficient capture, as expected, relative to low levels of nonspecific binding for Jurkat cells. All cells were DAPI stained for visualization. As expected, kappa and Ki67 expressions were high in Daudi cells, whereas lambda and Ki67 expressions were high in DB cells; Jurkat cells were positive only for Ki67, completing the panel and verifying capture and immunostaining of cancer cells with the lyophilized kit and functionalized glass.
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DISCUSSION The analysis of human cells immobilized on glass slides represents the mainstay of modern, nondestructive cytoanalysis. Typically such studies are performed in well-equipped laboratories and clinical facilities specialized in immunolabeling. However, bringing these technologies to point-of-care sites has been far more challenging, especially in LMIC where additional hurdles and adverse environmental conditions exist. Here, we show that glass slides can be activated, lyophilized, and used for immunocapture and subsequent analysis, all in kit format. As a part of NCI’s Center for Global Health and their Affordable Cancer Technologies program (https://www. cancer.gov/about-nci/organization/cgh), we are preparing to use the developed technology to perform clinical trials in Africa with previously developed analysis techniques for cell capture such as artificial intelligence diffraction analysis4 and a low-cost contrast-enhanced micrography device that has been validated in a clinical trial for the molecular diagnosis of lymphoma.1 The trials will be conducted in Botswana in both hospital and rural health center settings. Refrigeration (4 °C) is available for longer-term storage at a central facility but not readily maintained during periods of shipping or transport to rural locations for on-site use. The major advantages of this approach are manifold including: (i) more efficient use of expensive specialty reagents; (ii) expanding the shelf-life of “kits” from hours to weeks or months; (iii) simplification of multistep procedures so that they can be performed by a less skilled workforce, and (iv) reduction in assay cost. The capture
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MATERIALS AND METHODS Neutravidin Glass Preparation/Lyophilization. For reagents and supplies, see Table S1. Glass coverslips (22 mm × 22 mm, Gold Seal) were loaded into coverslip racks (Electron Microscopy Sciences) and activated by immersing the glass in 10 M NaOH for 20 min followed by 5 min sonication in Milli-Q water 2 times. Coverslips were treated by immersion in a large beaker with 2% (3-aminopropyl)triethoxysilane (APTES) in 95:5 EtOH/H2O for 30 min with stirring, then rinsed 3 times with fresh washes of 95% ethanol. The ethanol-washed slides were then submerged in 11519
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conjugated anti-rabbit secondary antibody (for kappa, lambda, and Ki67) and Alexa Fluor 488-conjugated anti-mouse secondary antibody (for CD19 and CD20) were used. Fluorescent signals were measured with a CytoFLEX flow cytometer (Beckman Coulter) and compared to fresh antibody cocktails and cells with no primary antibody staining as a negative control (Figure S5). Fluorescent images of captured cells were acquired on an Olympus BX-63 upright automated epifluorescence microscope. All cells were stained with DAPI and visualized postcapture with a DAPI filter cube. Alexa Fluor 647-conjugated anti-kappa light chain, Alexa Fluor 532-conjugated anti-lambda light chain, and Alexa Fluor 488-conjugated anti-Ki67 markers were excited with traditional Cy5, Cy3, and FITC filter cubes, respectively. Cell Culture. Daudi (Burkitt’s lymphoma), DB (germinal center B-cell-like DLBCL), and Jurkat (T-cell leukemia) cell lines were purchased from the American Type Culture Collection (ATCC). All cell lines were maintained in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 IU penicillin, and 100 μg/mL of streptomycin at 37 °C under a humidified atmosphere of 5% CO2. Cell lines were routinely tested for mycoplasma contamination with the MycoAlert Mycoplasma Detection Kit (Lonza). Statistics. Statistical analyses and data plotting were performed in GraphPad Prism 7. Error bars represent the standard deviation. A one-way analysis of variance (Tukey correction for multiple comparisons) was used to determine statistical significance.
2.5% glutaraldehyde in PBS for 2.5 h and then rinsed with a 5 min sonication in Milli-Q water 2 times. Neutravidin (ThermoFisher) was reconstituted from the lyophilized powder to prepare a stock solution of 0.1 mg/mL in PBS and was deposited onto the glutaraldehyde glass via reductive amination with 4.5 mM NaBH3CN by soaking the coverslips in the reaction mixture in a shallow Petri dish for 2.5 h, followed by three washes with PBS. For long-term storage, neutravidincoated slides were soaked in 1% trehalose in Milli-Q water for 30 min. Excess water was removed, and slides were lyophilized overnight at room temperature on a VirTis FreezeMobile 25L freeze dryer (SP Scientific). Neutravidin Surface Fluorescence Imaging. Commercial and prepared neutravidin glass was first blocked with 1% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Glass slides were incubated with biotinylated anti-CD20 capture antibody (15 μg/mL) for 30 min, washed, then incubated with Alexa Fluor 647-conjugated secondary antibody (4 μg/mL) for 30 min followed by washing with PBS. Fluorescent images of the stained surfaces were acquired on an Olympus BX-63 upright automated epifluorescence microscope excited with a Cy5 filter cube at 1 s exposure time. Antibody Lyophilization. Solutions containing 1 mg/mL antibody, 60 mM trehalose, 0.01% polysorbate 20 in 5 mM succinate buffer at pH 5 were frozen by immersing in liquid nitrogen, then lyophilized at 4 °C for 5 h followed by lyophilization at room temperature overnight on a VirTis FreezeMobile 25L freeze dryer (SP Scientific). Assay Procedure. B-cell capture assay was performed as before.1 Briefly, lyophilized antibody cocktails containing biotinylated anti-CD19 and anti-CD20 and either kappa light chain, lambda light chain, or anti-Ki67 antibodies were reconstituted (10 μg/mL) and incubated with fixed, permeabilized (0.005% saponin) cells for 30 min at room temperature. Cells were washed 4 times with the assay buffer to remove any unbound antibody. Lyophilized neutravidin coverglass was reconstituted for 5 min in PBS, then incorporated into a flow-cell cartridge consisting of a stickertype hybridization chamber (9 mm diameter and 1 mm depth, Grace Bio-labs), and blocked with 1% BSA for 1 h at room temperature. Antibody-labeled cells (10 000 based on a hemocytometer count of cell culture suspensions) were introduced into the flow cell and incubated for 10 min on a variable speed nutating mixer (Fisher Scientific, 30 rpm) to prevent nonspecific settling onto the glass. CD19/20+ cells were captured by the neutravidin surface, and unbound cells were removed by washing with 1 mL of total buffer volume, delivered at a rate of 40 mL/h with a syringe pump and flexible 1 mm tubing. Pilot experiments demonstrated an important correlation between chamber depth and appropriate flow rate for optimum differential capture, with lower flow rates required for shallower chambers. The assay buffer consisted of PBS supplemented with 2% BSA and 2% fetal bovine serum (FBS). Images were taken before and after washing (Figures S3 and S4) and quantified by first applying a global threshold followed by automatic particle counting in ImageJ to determine capture efficiency. Flow Cytometry and Cell Imaging. To validate the activity of the lyophilized antibodies, flow cytometry was performed in lymphoma cell lines. Cells (500 000) were stained with lyophilized antibody cocktails containing antiCD19, anti-CD20, and either anti-kappa light chain, antilambda light chain, or anti-Ki67 (10 μg/mL). Alexa Fluor 405-
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b01036.
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List of antibodies and reagents; neutravidin surface staining with biotin-fluorophore; antibody lyophilization optimization; representative prewash and postwash images in the cell capture assay; flow cytometry data with additional negative controls (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (J.C.T.C.). *E-mail:
[email protected] (R.W.). ORCID
Ralph Weissleder: 0000-0003-0828-4143 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by NIH grants CA202637, CA202064, and CA206890. We would like to thank Dr Christopher Rodell for helpful discussion in regard to glass functionalization and Dr Katy Yang for assistance in gathering flow cytometry data. We also thank Phil McFarland for his help with the capture assay. Finally, we would like to thank the entire Harvard/Botswana UH3 team for many valuable discussions and their support. 11520
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DOI: 10.1021/acsomega.9b01036 ACS Omega 2019, 4, 11515−11521
