AuPt Nanoparticles – “Black Gold” Multiplexed Immunoassays Novel Plasmonic Signalers for Lateral Flow Immunoassays Kristin B. Cederquist*, Megan R. Grima, Andrew A. Mills, Kori M. Fetters, Jessica R. Gregg, Nusayba M. Tabbah, Yuki Ichikawa, and Bing Liu Nanobio Department  IMRA America, Inc.  1044 Woodridge Avenue, Ann Arbor, MI 48105  *kcederqu@imra.com AuPt Nanoparticles – “Black Gold” Blue Gold Introduction Gold nanoparticles have long been valued for their characteristic and distinctive red color – a direct result of their surface plasmon resonance, which occurs at ~520 nm. Citrate reduction is the most common synthesis method, but this gives particles with a natively adsorbed citrate coating, which can hinder downstream bioconjugation efficiency. To prevent this, IMRA America has developed i-colloid: nanoparticles for lateral flow made by pulsed laser ablation of bulk metal targets. These particles exhibit no surface chemistry because of their fabrication method, and their optical properties can be tuned by changing composition or morphology. Exchanging a pure gold target for one that is 75% Au and 25% Pt results in a brown/black colloidal suspension. These nanoparticles offer the same easy conjugation and high antibody loading capacity of pure Au with a significantly altered color profile. Head-to-head comparison in an hCG lateral flow immunoassay revealed that AuPt could perform equally to Au when particles were conjugated with anti-hCG antibodies using identical procedures. Fluorescence Using careful procedures, IMRA scientists have devised a means to controllably aggregate gold nanoparticles into new and stable architectures. This results in nanostructures that absorb ~2x more light in the visible spectrum. Because of the emergence of a secondary peak around 620 nm in the particles’ extinction profile, they appear a deep indigo in color. Because blue gold is fabricated from typical gold nanoparticles, the antibody conjugation procedure is almost identical. Even in the presence of the aggregator molecule, antibodies exhibit high coverage on the surface of the particles. Blue gold showed identical performance to regular 40 nm gold nanoparticles in a lateral flow assay for hCG. Though the number of blue gold particles is lower than the number of red gold particles per unit volume because of controlled aggregation, their increased visual absorption leads to a higher amount of color per binding event. Au Competitors AuPt Au Nanoparticles 1100 mIU/mL hCG 0 mIU/mL hCG 40 nm i-colloid gold nanoparticles have been incorporated into lateral flow immunoassay tests, both in-house and at customer facilities. For in-house testing, particles were modified with anti-β-hCG monoclonal antibodies. Goat anti-hCG and goat anti-mouse IgG were printed onto Millipore HF135 membranes with a BioDot ZX1010 printer as the test and control lines, respectively. Membranes were mounted on DCN backing cards with wick pads and cut into 0.5 cm-wide strips using a BioDot CM5000 cutter. i-colloid nanoparticles were found to exhibit enhanced sensitivity in dose-response curves over competitor particles produced by citrate reduction. This could be attributed to higher antibody loading on i-colloid. Multiplexed Immunoassays To examine the multiplexing capability of these particles, we printed anti-hCG and anti-cardiac troponin I (cTnI) onto cellulose membrane and assembled them into half-strips. We then immobilized anti-cardiac troponin I (cTnI) to Au nanoparticles and anti-hCG to AuPt nanoparticles and introduced both particles into different permutations of protein targets (both, one or the other, or neither) just before introducing them to immunoassay test strips. The color of both particles was retained on-strip. Furthermore, signals in the presence of one target were close to those attained when either both or no targets were added, suggesting that the particles were sufficiently blocked. 100 nm 100 ng/mL hCG 0 ng/mL hCG References Cederquist, K. B.; Liu, B.; Grima, M. R.; Dalack, P. J.; Mahorn, J. T. “Laser-fabricated gold nanoparticles for lateral flow immunoassays.” Coll. Surf. B 2017, 149, 351-357. Zhang, D.; Gökce, B.; Barcikowski, S. “Laser synthesis and processing of colloids: fundamentals and applications.” Chem. Rev. 2017, 117, 3990-4103.