End-Functionalized Poly(vinylpyrrolidone) for Ligand Display in Lateral Flow Device Test Lines

Lateral flow devices are rapid (and often low cost) point-of-care diagnostics—the classic example being the home pregnancy test. A test line (the stationary phase) is typically prepared by the physisorption of an antibody, which binds to analytes/antigens such as viruses, toxins, or hormones. However, there is no intrinsic requirement for the detection unit to be an antibody, and incorporating other ligand classes may bring new functionalities or detection capabilities. To enable other (nonprotein) ligands to be deployed in lateral flow devices, they must be physiosorbed to the stationary phase as a conjugate, which currently would be a high-molecular-weight carrier protein, which requires (challenging) chemoselective modifications and purification. Here, we demonstrate that poly(vinylpyrrolidone), PVP, is a candidate for a polymeric, protein-free test line, owing to its unique balance of water solubility (for printing) and adhesion to the nitrocellulose stationary phase. End-functionalized PVPs were prepared by RAFT polymerization, and the model capture ligands of biotin and galactosamine were installed on PVP and subsequently immobilized on nitrocellulose. This polymeric test line was validated in both flow-through and full lateral flow formats using streptavidin and soybean agglutinin and is the first demonstration of an “all-polymer” approach for installation of capture units. This work illustrates the potential of polymeric scaffolds as anchoring agents for small-molecule capture agents in the next generation of robust and modular lateral flow devices and that macromolecular engineering may provide real benefit.


Mass Spectrometry
Low resolution mass spectra (LRMS) were recorded on a Bruker Esquire 2000 spectrometer using electrospray ionisation (ESI). M/z values are reported in Daltons.

FT-IR Spectroscopy
Fourier Transform-Infrared (FT-IR) spectroscopy measurements were carried out using an Agilent Cary 630 FT-IR spectrometer, in the range of 650 to 4000 cm -1 .

Size Exclusion Chromatography
Size exclusion chromatography (SEC) analysis was performed on an Agilent Infinity II MDS instrument equipped with differential refractive index (DRI), viscometry (VS), dual angle light scatter (LS) and variable wavelength UV detectors. The system was equipped with 2 x PLgel Mixed D columns (300 x 7.5 mm) and a PLgel 5 µm guard column. The mobile phase used was DMF (HPLC grade) containing 5 mM NH4BF4 at 50 ℃ at flow rate of 1.0 mL.min -1 .

X-ray Photoelectron Spectroscopy (XPS)
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 analyzed 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.

Dynamic Light Scattering
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 analyzed using Malvern DTS 7.03 software. All determinations S7 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.

UV-vis Spectroscopy
Absorbance measurements were recorded on an Agilent Cary 60 UV-Vis Spectrophotometer and on a BioTek Epoch microplate reader.

Transmission Electron Microscopy
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.

Image Collection of Lateral Flow Dipsticks and Devices
All devices were scanned using a Kyocera TASKalfa 5550ci printer to a pdf file that was converted to a jpeg. The jpeg was analyzed in ImageJ 1.51. 1 None of the images in this ESI have been image adjusted i.e. no changes/enhancements have been made from the original scan images. The main paper images may have been enhanced cropped to improve clarity.

Solvent drying
4Å molecular sieves were activated either by heat or using microwave energy (600W). A 20% w/v. of sieves:solvent was used, the solvent was degassed with nitrogen for 30 minutes with the sieves present and then left overnight before the solvent was used.

Synthesis of 2-(dodecylthiocarbanothionylthio)-2-methyl propionic acid (DMP)
This was synthesized, according to a previously published procedure. 2 2.00 g (9.88 mmol) 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.

Synthesis of pentafluorophenyl-2-dodecylthiocarbonothioylthio)-2-methylpropanoate (PFP-DMP)
This was synthesized, 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 0.429 g (1.46 mmol) of sodium citrate tribasic dihydrate was added. The reaction was allowed to reflux for 30 minutes before cooling to room temperature over 3 hours. The solution was centrifuged at 13 krpm for 30 minutes and the pellet resuspended in 40 mL of water to give an absorbance at 520 nm of ~1Abs.

Gold Nanoparticle Polymer Coating Functionalization -16 nm
10 mg of polymer was agitated overnight with 10 mL of 16 nm AuNPs ~1Abs at UVmax. The solution was centrifuged at 13 krpm for 30 minutes and the pellet resuspended in 10mL of water, the solution was centrifuged again at 13 krpm for 30 minutes and the pellet resuspended in 1 mL aliquots and centrifuged at 14.5 krpm for 10 minutes. The pellets were combined into a 1 mL solution with an absorbance at 520 nm of ~10 Abs.

Gold Nanoparticle Polymer Coating Functionalization -40 nm
10 mg of polymer was agitated overnight with 10 mL of 40 nm AuNPs ~1Abs at UVmax. The solution was centrifuged at 6 krpm for 10 minutes and the pellet resuspended in 10mL of water, the solution was centrifuged again at 6 krpm for 10 minutes and the pellet resuspended in 1 mL aliquots and centrifuged at 6 krpm for 10 minutes. The pellets were combined into a 1 mL solution with an absorbance at UVmax of ~10 Abs.

Lateral Flow Strip Production, Running and Analysis Protocols
The procedure to produce flow-through and lateral flow devices was identical, apart from the deposition of the analyte directly onto the nitrocellulose (flow-through), versus application of tests lines onto the nitrocellulose (lateral flow). This is a truncated protocol from Baker et al., provided for clarity. 6

Protocol for Manufacturing Lateral Flow Strips
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.

Protocol for Test Line Addition to the Lateral Flow Strips
1 µL of the test line solution was added to the test strip using a micropipette fitted with 10 µL tip, the test line was spotted ~1 cm from the non-wick end of the strip. The strips were dried at 37 ℃ in an oven for 30 minutes. The tests strips were allowed to cool to room temperature before testing.

Protocol for Running Lateral Flow Test Without Target Analyte in 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 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.

Protocol for Running Lateral Flow Test with Target Analyte in 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.

S25
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.

Standard Protocol for Lateral Flow Strip Analysis
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 analyzed 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. of CaCl2, 0.8 g (0.8% w/v., 123 mmol.dm -3 ) of NaN3, 0.5 g (0.5% w/v., 4.07 mmol.dm -3 ) of Tween-20 and 10 g (10% w/v.) of poly(vinyl pyrrolidone)400 (PVP400, Average Mw ~40,000) were dissolved in 100 mL of water. The buffer was not pH adjusted.

Intensity Calculations
The average background was determined by calculating the mean grey value of points between 0 -60 relative distance units, subtracted from 255 (the grey value of clean nitrocellulose); excluding points from aggregation at the solvent front, points contributing to the signal peak and points in the wick. This average background value was subtracted from the lowest grey value of the signal peak (subtracted from 255) to give intensity.

Signal-to-Noise Calculations
The signal (intensity of test) was then divided by the noise (intensity of control) value to give a signal-to-noise value. Figure S4. Normalized size exclusion chromatography RI molecular weight distributions of telechelic PHEA obtained in DMF versus PMMA standards. S28 Figure S5. Normalized size exclusion chromatography RI molecular weight distributions of PVP obtained in DMF versus PMMA standards.