key: cord-0865432-hljr9q0e authors: Baker, Alexander N.; Muguruza, Asier R.; Richards, Sarah-Jane; Georgiou, Panagiotis G.; Goetz, Stephen; Walker, Marc; Dedola, Simone; Field, Robert A.; Gibson, Matthew I. title: Lateral Flow Glyco-Assays for the Rapid and Low-Cost Detection of Lectins–Polymeric Linkers and Particle Engineering Are Essential for Selectivity and Performance date: 2022-02-01 journal: Adv Healthc Mater DOI: 10.1002/adhm.202101784 sha: 66fed7ea27c8985fb96c3e1a8cea5ce6468d4862 doc_id: 865432 cord_uid: hljr9q0e Lateral flow immuno-assays, such as the home pregnancy test, are rapid point-of-care diagnostics that use antibody-coated nanoparticles to bind antigens/analytes (e.g., viruses, toxins or hormones). Ease of use, no need for centralized infrastructure and low-cost, makes these devices appealing for rapid disease identification, especially in low-resource environments. Here glycosylated polymer-coated nanoparticles are demonstrated for the sensitive, label-free detection of lectins in lateral flow and flow-through. The systems introduced here use glycans, not antibodies, to provide recognition: a “lateral flow glyco-assay,” providing unique biosensing opportunities. Glycans are installed onto polymer termini and immobilized onto gold nanoparticles, providing colloidal stability but crucially also introducing assay tunability and selectivity. Using soybean agglutinin and Ricinus communis agglutinin I (RCA(120)) as model analytes, the impact of polymer chain length and nanoparticle core size are evaluated, with chain length found to have a significant effect on signal generation—highlighting the need to control the macromolecular architecture to tune response. With optimized systems, lectins are detectable at subnanomolar concentrations, comparable to antibody-based systems. Complete lateral flow devices are also assembled to show how these devices can be deployed in the “real world.” This work shows that glycan-binding can be a valuable tool in rapid diagnostics. The samples were attached to electrically-conductive carbon tape, mounted on to a sample bar and loaded into a Kratos Axis Ultra DLD spectrometer which possesses a base pressure below 1 x 10 -10 mbar. XPS measurements were performed in the main analysis chamber, with the sample being illuminated using a monochromated Al Kα x-ray source. The measurements were conducted at room temperature and at a take-off angle of 90° with respect to the surface parallel. The core level spectra were recorded using a pass energy of 20 eV (resolution approx. 0.4 eV), from an analysis area of 300 μm x 700 μm. The spectrometer work function and binding energy scale of the spectrometer were calibrated using the Fermi edge and 3d5/2 peak recorded from a polycrystalline Ag sample prior to the commencement of the experiments. In order to prevent surface charging the surface was flooded with a beam of low energy electrons throughout the experiment and this necessitated recalibration of the binding energy scale. To achieve this, the C-C/C-H component of the C 1s spectrum was referenced to 285.0 eV. The data was analysed in the CasaXPS package, using Shirley backgrounds and mixed Gaussian-Lorentzian (Voigt) lineshapes. For compositional analysis, the analyser transmission function has been determined using clean metallic foils to determine the detection efficiency across the full binding energy range. Hydrodynamic diameters (Dh) and size distributions of particles were determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS with a 4 mW He-Ne 633 nm laser module operating at 25 ℃. Measurements were carried out at an angle of 173° (back scattering), and results were analysed using Malvern DTS 7.03 software. All determinations were repeated 5 times with at least 10 measurements recorded for each run. Dh values were calculated using the Stokes-Einstein equation where particles are assumed to be spherical. Absorbance measurements were recorded on an Agilent Cary 60 UV-Vis Spectrophotometer and on a BioTek Epoch microplate reader. Dry-state stained TEM imaging was performed on a JEOL JEM-2100Plus microscope operating at an acceleration voltage of 200 kV. All dry-state samples were diluted with deionized water and then deposited onto formvar-coated copper grids. All devices were scanned using a Kyocera TASKalfa 5550ci printer to a pdf file that was converted to a jpeg. The jpeg was analysed in ImageJ 1.51. 1 This was synthesised, according to a previously published procedure. 2 2.00 g (9.88 mmmol) of 1-dodecane thiol was added dropwise to stirring 2.10 g (9.89 mmol) of K3PO4 in 30mL of acetone at RTP, the mixture was left to stir for 25 minutes to form a white suspension. This was synthesised, according to a previously published procedure. To 500 mL of water was added 0.163 g (0.414 mmol) of gold(III) chloride trihydrate, the mixture was heated to reflux and 14.6 mL of water containing 0. This solution was then used as a seed solution, and three further portions of 1.6 mL of 25 mM HAuCl4 were added with 20 min between each addition. Following completion of this step 1 mL was taken for DLS and UV/Vis analysis. The sample was diluted by adding 135 mL of MilliQ water and 4.9 mL of 60 mM sodium citrate. This solution was then used as a seed solution, and the process was repeated with three further additions of 2.5 mL of 25 mM HAuCl4, this solution was analysed by DLS and UV/Vis and target size of 40 nm was reached, so the solution was allowed to cool. The procedure to produce flow-through and lateral flow devices was identical, apart from the deposition of the analyte directly to the nitrocellulose (flow-through), versus application of tests lines to the nitrocellulose (lateral flow). Backing cards were cut to size by removal of 20 mm using a guillotine. Nitrocellulose was added to the backing card by attaching the plastic backing of the nitrocellulose to the selfadhesive on the card. The wick material was then added to the backing card so it overlaps with the nitrocellulose by ~5 mm. The lateral flow strips were cut to size of width 2-3 mm. The tests strips were allowed to cool to room temperature before testing. The running buffer of total volume 50 µL was made as follows; 5 µL AuNPs (OD10), 5 µL lateral flow assay buffer -10 × HEPES buffer, 40 µL water. The running solution was then agitated on a roller for 5 minutes. 45 µL of this solution was added to a 0.2 mL PCR tube, standing vertically. A small "v" (~3 mm) was cut into the test strips at the non-wick end and the strips added to the PCR tubes, so they protrude from the top and the immobile phase (1 cm from non-wick end) is not below the solvent line. There was one test per tube. All tests were run in triplicate. The tests were run for 20 minutes before removal from the tubes. The test strips were allowed to dry at room temperature for ~5 minutes. The test strips were mounted test-face down onto a clear and colourless piece of acetate sheeting. The Protocol for Running Lateral Flow Test Without Target Analyte in Buffer was used for the flow-through assays as the target analyte is deposited on the nitrocellulose as a "test line" i.e. the analyte is not in the running buffer. The running buffer of total volume 50 µL was made as follows; 5 µL AuNPs (OD10), 5 µL lateral flow assay buffer -10 × HEPES buffer, 40 µL of water -x µL, where x is the volume of target analyte added to make the required concentration of the lectin. The running solution was then agitated on a roller for 5 minutes. 45 µL of this solution was added to a 0.2 mL PCR tube, standing vertically. A small "v" (~3 mm) was cut into the test strips at the non-wick end and the strips added to the PCR tubes, so they protrude from the top and the immobile phase (1 cm from non-wick end) is not below the solvent line. There was one test per tube. All tests were run in triplicate. The tests were run for 20 minutes before removal from the tubes. The test strips were allowed to dry at room temperature for ~5 minutes. The test strips were mounted test-face down onto a clear and colourless piece of acetate sheeting. The acetate sheets were scanned using a Kyocera TASKalfa 5550ci printer to a pdf file that was converted to a jpeg, scans were taken within 1 hour of strip drying. The jpeg was analysed in ImageJ 1.51 1 using the plot profile function to create a data set exported to Microsoft Excel for Mac. The data was exported to Origin 2019 64Bit and trimmed to remove pixel data not from the strip surface. The data was aligned and averaged (mean). The data was then reduced by number of groups to 100 data points (nitrocellulose and wick) and plotted as Grey value (scale) vs Relative distance along the 100 data points. were dissolved in 100 mL of water. The buffer was not pH adjusted. Nitrocellulose was added to the backing card by attaching the plastic backing of the nitrocellulose to the self-adhesive on the card. The wick material was then added to the backing card so it overlaps with the nitrocellulose by ~5 mm. The strips were then cut to size of width ~3 mm so they sit in the cassettes without the need for excess force to fit. Tests lines were then 10% w/v. of poly(vinyl pyrrolidone)400 (Average Mw ~40,000 g.mol -1 ), 50% w/v. trehalose, 10% w/v. sucrose and 0.1% w/v. Tween-20 were added to distilled water and allowed to dissolve. 8 µL 10× HEPES buffer (20% PVP400) was added to 72 µL distilled water. 80 µL was added to the sample pad and allowed to absorb. The test was run for 10 minutes before scanning the cassettes using a Kyocera TASKalfa 5550ci printer, the images were exported to a pdf file that was converted to a jpeg. Within ~1 hour the strips were removed from the cassettes and added to acetate sheets. These were scanned using a Kyocera TASKalfa 5550ci printer to a pdf file that was converted to a jpeg, acetate scans were taken within 1 hour of strip drying. The jpegs were analysed in Image J 1.51 using the plot profile function to create a data set exported to Microsoft Excel for Mac. The data was exported to Origin 2019 64Bit and trimmed to remove pixel data not from the strip surface. The data was aligned and averaged (mean). The data was then reduced by number of groups to 100 data points (just the nitrocellulose surface) and plotted as Grey value (scale) vs Relative distance along the 100 data points. 8 µL 10× HEPES buffer (20% PVP400) was added to 72 µL of water -x µL, where x is the volume of target analyte added to make the required concentration of the lectin. 80 µL was added to the sample pad and allowed to absorb. The test was run for 10 minutes before scanning the cassettes using a Kyocera TASKalfa 5550ci printer, the images were exported to a pdf file that was converted to a jpeg. Within ~1 hour the strips were removed from the cassettes and added to acetate sheets. These were scanned using a Kyocera TASKalfa 5550ci printer to a pdf file that was converted to a jpeg, acetate scans were taken within 1 hour of strip drying. The jpegs were analysed in Image J 1.51 using the plot profile function to create a data set exported to Microsoft Excel for Mac. The data was exported to Origin 2019 64Bit and trimmed to remove pixel data not from the strip surface. The data was aligned and averaged (mean). The data was then reduced by number of groups to 100 data points (just the nitrocellulose surface) and plotted as Grey value (scale) vs Relative distance along the 100 data points. Relative distance pixel 1 to 10 and 51 to 60 (area around the test line), excluding pixels that contributed to the signal peak were averaged (mean). This average was subtracted from the lowest grey value between 11 to 50 (test line region). AuNP System Signal:Noise NIH Image to ImageJ: 25 Years of Image Analysis Optimization of the Polymer Coating for Glycosylated Gold Nanoparticle Biosensors to Ensure Stability and Rapid Optical Readouts Synthesis of Glycosylamines: Indentification and Quantification of Side Products Improved Synthesis of Glycosylamines and a Straightforward Preparation of N-Acylglycosylamines as Carbohydrate-Based Detergents The Critical Importance of Size on Thermoresponsive Nanoparticle Transition Temperatures: Gold and Micelle-Based Polymer Nanoparticles Kinetically Controlled Seeded Growth Synthesis of Citrate-Stabilized Gold Nanoparticles of up to 200 Nm: Size Focusing versus Ostwald Ripening