(291a) Multichannel Acoustic Separator for High-Throughput Multiplexed Biomolecule Detection on Soft Biofunctional Particles

Detection of biomarkers, including antibodies, antigens, and other physiologically relevant molecules, is essential for patient diagnosis and disease management. Multiplexed assays, which simultaneously detect multiple biomarkers in a single test, can significantly enhance diagnostic confidence. However, conventional detection assays, such as enzyme-linked immunosorbent assays (ELISA), are generally not suitable for rapid multiplexed detection, especially for biomarkers of disparate physical morphology (e.g., proteins, small molecules). Moreover, traditional biosensing techniques often require complex processing steps that introduce potential points of error and make detecting multiple biomarkers tedious. To address these challenges, we report a novel biospecific particle-based, acoustofluidics-enabled system for the simultaneous purification, preparation, and detection of multiple proteins and small molecules from complex biofluids. The central innovations of this technology are i) a class of fluorescently barcoded, functional negative acoustic contrast particles (fNACPs) that are modularly modified with antifouling polymers terminated in biorecognition motifs for the specific capture of biomarkers and ii) a 3D-printed, high-throughput multichannel acoustofluidic separator that interfaces with 12-channel pipettes for the purification of fNACPs from whole blood. Here, we show the rapid, sensitive, and simultaneous capture, purification, and detection of an antibody (Ab), antigen (Ag), and small molecule using the fNACP-based, acoustofluidic separator system.

When subjected to a half-wavelength standing wave within a microfluidic channel, positive acoustic contrast particles (PACPs) with diameters much smaller than the wavelength are forced to the pressure node, located along the middle of the channel, by primary radiation force. Conversely, NACPs are forced to the antinodes along the walls of the channel. Beyond this, particles and cells in the channel also experience a secondary radiation force, which is attractive between particles at close distances. Therefore, after acoustophoresis to the walls of the channel, NACPs subsequently aggregate. Finally, particles in an acoustofluidic channel experience a lateral acoustic radiation force that originates from gradients in the local acoustic potential along the channel and acts parallel to the channel, toward regions of local acoustic minima or maxima. At high acoustic radiation forces, this can result in acoustic trapping, which we define as the immobilization of NACPs along the walls of a straight channel in the presence of an applied flow, due to both the lateral component of the primary acoustic radiation force and the secondary acoustic radiation force exceeding the force on NACPs from Stokes’ drag. In this work, we synthesized NACPs via homogenization and polymerization of a Sylgard 184 and triethoxyvinylsilane polymer precursor. Following polymerization, we isolated a low-polydispersity fraction of the NACPs by sequential vortexed filtration through 40, 30, and 20 μm filters, collecting the NACPs retained by the 20 μm filter. The surfaces of NACPs were functionalized by conjugating the particles with biotin-polyethylene glycol (PEG)-silane linkers and streptavidin, in that order. Because of its four biotin-binding sites, addition of streptavidin to the fNACPs enables subsequent conjugation of a variety of biotinylated recognition elements to allow detection of disparate biomarker types. Here, to create fNCAPs specific to a model Ab, Ag, and small molecule, we functionalized fluorescently barcoded fNACPs with biotinylated ovalbumin Ag (OVA), anti-prostate specific antigen Ab (anti-PSA), and pyrabactin resistance 1 (PYR1) protein for the detection of anti-OVA Ab, PSA Ag, and the small molecule abscisic acid (ABA), respectively. The second enabling feature of this assay is the use of a 3D-printed, high-throughput acoustofluidic separator that interfaces with 12-channel multichannel pipettes to rapidly purify captured biomolecules on the fNACPs from biofluids. The separator was designed using Fusion CAD software and printed on a Prusa SL1S 3D printer. The device houses a 12-channel trapping array, fabricated by bonding 12 square glass capillaries to a piezoelectric transducer in parallel. The transducer is actuated by an applied AC signal at 750 kHz, which generates a pressure node along the center of each channel and antinodes along the walls of each channel. To assess trapping performance, 5x10⁴ NACPs were mixed with 1000 μL buffer, and the mixture was passed through each of the trapping channels with acoustics engaged, yielding waste and enriched samples. These samples were analyzed via flow cytometry to quantify NACP retention. To evaluate the fNACP assay performance and limit of detection (LOD), anti-OVA Ab, PSA Ag, and ABA were spiked into 100 μL buffer or porcine blood containing 10⁴ fNACPs of each type (i.e., Anti-OVA, PSA, and ABA-specific) and 10⁴ nonfunctionalized NACPs as an internal control. After target capture, the fNACPs and NACPs were purified using the multichannel acoustofluidic separator and incubated with fluorescently tagged secondary labels. After this incubation, the fNACPs were purified once more and analyzed via flow cytometry.

Using the described NACP synthesis and filtration approach, we isolated a low polydispersity fraction of NACPs with a mean size of ~25 μm. While the use of smaller particles in assays may offer the potential for faster particle-biomarker binding interactions due to increased diffusivity, we expected that larger particle sizes facilitate faster, more robust trapping of particles under flow, as the magnitude of acoustic radiation forces scales proportionally with the volume of particles (cubically with the radius) [2]. The discriminant forces acting on fNACPs and blood cells enabled their efficient (over 95% retention) separation in short timescales (<60 sec) using the 3D-printed, multichannel acoustofluidic separator. By functionalizing fluorescently barcoded fNACPs with a biotinylated OVA, we demonstrate the detection of anti-OVA antibodies at picomolar levels (60 pM), a sensitivity competitive with commercial ELISAs. We then show the sensitive detection of PSA by anti-PSA-functionalized fNACPs at physiological levels. Finally, we report the detection of small molecules by functionalizing fNACPs with a plant hormone receptor-inspired biorecognition motif. This third type of fNACP addresses an important limitation of traditional biosensing assays. While many immunoassays detect protein biomarkers, small molecules (i.e., molecules < 1000 Da) also serve as important indicators of disease, metabolic function, and toxin exposure. However, detecting small molecules is challenging due to their miniscule size and similarities in structure. We addressed this issue by functionalizing fNACPs with PYR1, which changes conformation when bound to the small molecule ABA, allowing complex formation with a secondary fluorescent protein, HAB1, and resultantly providing a means to quantify ABA concentration. Through barcoding the three types of fNACPs, we show simultaneous detection of all three biomarkers from whole blood using a multichannel separator that can be carried out with minimal user engagement in under 70 minutes.