(592d) Sensing and Sound: Biospecific, Acoustic-Responsive Particles for Multiplexed Biomarker Detection

Detection of biomarkers, including antibodies, antigens, and other physiologically relevant molecules, is essential for patient diagnosis and disease management. The simultaneous detection of multiple biomarkers in multiplexed assays can enhance confidence in testing results and diagnoses. However, conventional biomolecule detection assays, such as enzyme-linked immunosorbent assays (ELISA), are not suitable for simultaneous detection, and multiple individual assays are often required. Moreover, traditional biosensing techniques often also require complex processing steps that introduce potential points of error and make detection of multiple biomarkers a tedious task that involves significant user engagement. In addition to proteins, small molecules (e.g., molecules under 1000 Da), can serve as important additional indicators of disease progression, metabolic function, and toxin exposure, among other physiological states. However, compared to detection of proteins, which is often aided by capture of target biomarkers by antibody recognition elements, detection of small molecules is more challenging; due to the miniscule size and similarities in structure of small molecules, antibodies typically lack the selectivity necessary for rigorous quantitation of small molecules in biofluids. This limitation often prevents detection of small molecules through traditional assays. Thus, there is a need for simplified and rapid multiplexed biosensing assays that can evaluate the presence and concentration of disparate biomarker types.

To address these challenges, we show a novel biospecific particle-based system for the simultaneous purification and detection of multiple proteins and small molecules from biofluids. One enabling innovation of this technology is the use of a new class of fluorescently barcoded, functional negative acoustic contrast particles (fNACPs) that are modified with antifouling polymers terminated in biorecognition motifs for the specific capture of biomarkers. Due to the poly(ethylene glycol) linkers between the particle surfaces and capture antigen, nonspecific adsorption is extremely low at physiological conditions. Moreover, because of the modular functionalization approach we employ, the fNACPs can be tuned to detect a range of target biomarkers. The second enabling innovation is the use of an acoustofluidic separation device to rapidly purify fNACPs and captured biomolecules from biofluids. This system makes use of acoustofluidic trapping channels, which produce a pressure node along the center of the channels and antinodes along the walls of the channels. fNACPs are forced to the antinodes of the standing wave due to their negative contrast, where they are trapped by secondary acoustic radiation forces. We show that the discriminant forces acting on particles and blood cells enables their efficient (over 99%) separation from whole blood in <60 seconds. To capture antibodies, we show the functionalization of fNACPs with antigen recognition elements; after capture, the antibodies are labeled by secondary fluorescent antibodies and analyzed by flow cytometry. Using this system, we demonstrate the detection of anti-OVA antibodies at picomolar levels (35-60 pM) from whole blood, a sensitivity competitive with commercial ELISAs. Moreover, while ELISA can require 3-5 hours and considerable user engagement to yield results, the fNACP-enabled assay requires only ~70 minutes to detect biomarkers, with only ~10 minutes of this time involving user engagement. To capture and detect small molecules, we functionalize fNACPs with a plant hormone receptor-inspired biorecognition element and reporter system. Upon binding small molecules, the protein recognition element forms a recognition element-small molecule-fluorescent protein reporter complex, enabling detection of small molecules. By fluorescently barcoding discrete populations of fNACPs based on biomarker specificity, we show multiplexed detection of three individual biomarkers in a single assay by flow cytometry. Overall, this fNACP-based, acoustofluidics-enabled assay offers a simple and versatile approach for the rapid, sensitive, specific, and simultaneous detection of a range of target biomolecules, including proteins and small molecules. The methodology for fNACP production and functionalization is currently patent-pending.