Platinum Nanocatalyst Amplification: Redefining the Gold Standard for Lateral Flow Immunoassays with Ultrabroad Dynamic Range

Paper-based lateral flow immunoassays (LFIAs) are one of the most widely used point-of-care (PoC) devices; however, their application in early disease diagnostics is often limited due to insufficient sensitivity for the requisite sample sizes and the short time frames of PoC testing. To address this, we developed a serum-stable, nanoparticle catalyst-labeled LFIA with a sensitivity surpassing that of both current commercial and published sensitivities for paper-based detection of p24, one of the earliest and most conserved biomarkers of HIV. We report the synthesis and characterization of porous platinum core–shell nanocatalysts (PtNCs), which show high catalytic activity when exposed to complex human blood serum samples. We explored the application of antibody-functionalized PtNCs with strategically and orthogonally modified nanobodies with high affinity and specificity toward p24 and established the key larger nanoparticle size regimes needed for efficient amplification and performance in LFIA. Harnessing the catalytic amplification of PtNCs enabled naked-eye detection of p24 spiked into sera in the low femtomolar range (ca. 0.8 pg·mL–1) and the detection of acute-phase HIV in clinical human plasma samples in under 20 min. This provides a versatile absorbance-based and rapid LFIA with sensitivity capable of significantly reducing the HIV acute phase detection window. This diagnostic may be readily adapted for detection of other biomolecules as an ultrasensitive screening tool for infectious and noncommunicable diseases and can be capitalized upon in PoC settings for early disease detection.

showing the pH dependency of antibody conjugation, with pH 6 (pH adjusted using 10 mM HEPES buffer) being optimal. Physisorption is maximally achieved at a pH close to the isoelectric point of the protein to be conjugated. (b) Plot of test line intensity and "test line to background ratio" against varying antibody coverage on PtNCs. Antibody coverage densities were calculated using the cross-sectional area of the antibody assuming a ca. 10 nm diameter protein, and surface area of PtNC sphere (using Z-average diameter from DLS).
"Max" corresponds to the theoretical maximum number of antibodies that can pack onto the PtNC surface in a monolayer. The highest test line signal intensity was achieved when particles were conjugated in the presence of 2 times the "max" number of antibodies to theoretically saturate the surface. The signal was also quantified using the ratio of the intensity of the test line (for 50 pg·mL -1 spiked FBS) to the intensity of the test line for the blank sample (background). Test line signal intensity from the blank control sample indicates nonspecific binding. Test line to background ratio was highest at incubation with 2*max antibody coverage. Particles conjugated in less than an excess of antibodies resulted in PtNC aggregation during the conjugation, which led to elevated nonspecific binding in the blank sample and decreased test line to background ratio. (c) The number of antibodies conjugated to PtNCs (ca. 120 nm diameter) after incubation of a fixed particle concentration in optimal antibody coverage solution (2*max) for 3 h. When PtNCs are incubated in a 2-fold excess of antibodies with respect to surface area, a monolayer is formed. Red dotted line indicates the theoretical number of antibodies to fully coat PtNCs. After antibody modification, the particles were centrifuged and the concentration of antibody in the supernatant (unbound fraction) was measured using a Thermo Scientific Micro-BCA (bicinchoninic acid) Protein Assay Kit. (d) DLS measurements showing normalized particle size distribution (intensity) for bare PtNCs (black) and antibody modified PtNCs (red). (e) Percent of activity retained for PtNCs which have been modified with antibodies and subsequently blocked in 2 wt% beta-casein blocking solution. Antibody modified particles retain ca. 35% activity compared to as-synthesized PtNCs. All data are averaged from ≥ 3 independent measurements where error bars represent the standard deviation from the mean. image of the test strips was acquired using an iPhone 6 camera. Images were taken with test strips laid on the same area of a standard lab bench. One image from each independent experiment (n ≥ 3) was analyzed (a). Next, the image was imported into ImageJ software, and (b) converted to a 16-bit grayscale image. Next, using the Gel Analyzer function, rectangular regions surrounding the test line were selected (c). A rectangular region with a defined aspect ratio of 4:3 was used, with the width of the region spanning the test line on an individual strip, and the height being 1.5 times the width of the test line. The selected rectangular regions were analyzed using the Gel Analyzer function in ImageJ. In brief, the profile plots of the test lines were generated (d), peaks of interest were defined, and then the peak areas were measured using the Wand Tool. Peak areas corresponding to "test line signal intensity" were compared between test strips within a single image. For six week accelerated aging experiment, test line intensities were normalized to a standard line (e) (highlighted by the star), which was present in all images, to account for variation in lighting between time points.

Synthesis general experimental
All reagents and starting materials were obtained from chemical suppliers, unless specifically stated otherwise, and were used as received. Reactions were monitored by thin layer chromatography using pre-coated SIL G/UV 254 plates purchased from VWR. Flash chromatography was carried out manually using Kiesegel 60 M 0.04/0.063 mm silica gel or automatically using a BioTage Isolera with KP-Snap or KP-Sil columns. NMR spectra were recorded using a Bruker AC300, AC500 or AC600 spectrometer (300 MHz, 500 MHz and 600 MHz respectively). Chemical shifts (δ) are given in ppm units relative to the solvent reference and coupling constants (J) are measured in Hertz. Proton ( 1 H) NMR multiplicities are shown as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (double doublet), dt (double triplet), etc. HMBC, HSQC and DEPT were employed to aid with accurate assignments. Infrared spectra were recorded on a Perkin Elmer Spectrum 100 FTIR spectrometer (ATR mode). High and low resolution mass spectrometry of organic molecules was provided by the EPSRC Mass Spectrometry facility at Swansea using an LTQ Orbitrap XL.

Chemical Biology General Experimental
All buffers were passed through a microfilter before use to remove particulates and the pH adjusted using 1 M HCl or 1 M NaOH. pH was measured using a Hanna Instruments pH 210 electronic pH meter. For desalting Zeba™ Spin Desalting columns, 7 KDa MWCO, were employed. Protein concentrations were determined photometrically using a Varian Cary 100 Bio UV-Visible spectrophotometer operating at 21 °C. For small scale centrifugation Eppendorf 5415 R and VWR Galaxy 14D microcentrifuges were employed. An Eppendorf Thermomixer Comfort heating block was used for temperature and agitation controlled experiments.
Non-reducing 16% acrylamide gels were made using standard procedures. A 4% stacking gel was utilised. Samples (70 µM) were mixed 5:1 with a 5× R-250 Dye SDS-loading buffer, heated for 5 minutes at 75 °C and loaded onto the gel with a total volume of 4 µL. Samples were run at constant current (30 mA) for 40 minutes in 1 × SDS running buffer and stained with Coomasie.

LCMS was performed on a Waters Acquity uPLC connected to Waters Acquity Single Quad
Detector and a photodiode array. Flow rate was set at 0.600 ml/min. A Hypersil Gold C4 (50 × 2.1 mm) column at 50 °C was used for separation. Solvent A is H2O (0.1% formic acid), solvent B is MeCN (0.1% formic acid). Mobile phase: 95:5 A:B; gradient over 4 min to 5:95 A:B. MS mode ES+; scan range: m/z ¼ 250-2,000; scan time: 0.25 s. A capillary voltage of 3.5 kV and a cone voltage of 50 V were employed. Injection volumes of 10 µl at 10 µM were used. All deconvoluted mass spectra were produced using the software provided by the manufacturer.