We demonstrate proof of concept for a point-of-care diagnostic that is used for the detection of chloride channel accessory 1 (CLCA1), a key regulator of mucus production. The prototypical device utilizes ultrasound-confined polystyrene (PS) microspheres held in a longitudinal standing bulk acoustic wave (LSBAW) as reaction substrates. Pressure field amplification between two included pillar arrays enables immobilization of these antigen-coated beads in a predetermined low-pressure region perpendicular to the direction of flow. Bronchoalveolar lavage (BAL) samples from IL-13 stimulated pigs were incubated overnight in a solution of untreated PS beads prior to channel introduction. A PZT- 8 piezoceramic transducer was used to actuate the channel (f1,E = 575 kHz) to focus and confine the beads in a linear zero-pressure node. A solution of two proprietary anti-CLCA1 monoclonal antibodies (mAbs) modified with sulfocyanine3 (Cy3) NHS ester were flowed (7 μL/min, 15 min) into the channel and incubated for 1.5 hours. A solution of phosphate-buffered saline was then used to remove excess antibodies prior to channel/bead cluster imaging. The bead solution was collected, under no acoustic actuation, at the outlet for analysis by flow cytometry. Increased fluorescence over control samples (p<0.0001) demonstrated that the LSBAW platform can serve as a functional immunoassay to allow for serial, contactless reagent washes and fluorescent probe introduction.
We report a synthesis platform for manipulation, modification, and potential production of nanoscale reagents using microscale carrier particles confined to a longitudinal acoustic trap. Pairs of perforated “pseudo-walls” enhance the local acoustic pressure field to immobilize reaction substrates (polymeric or glass beads) from a heterogeneous suspension. Nodal regions (pressure minima) of the acoustic field are perpendicular to the inflow direction to enable continuous, contactless, serial chemical reactions on the microparticle surface for, e.g., antibody (Ab) attachment, alteration, and recovery. Here, we discuss the potential of longitudinal standing bulk acoustic wave (LSBAW) architectures for creation of antibody conjugates (ACs) at research and production scales. Results prove the feasibility of combining acoustic manipulation of microcarriers with steps constituting the synthesis and purification of ACs including mixing, washing, buffer exchange, conjugation reaction, and release/recovery. 10-μm diameter hollow glass spheres (HGS) were modified to create a Protein G-terminated self-assembled monolayer (SAM). Modified HGS were introduced and focused to the midline of the acoustic trap. Focused beads were then incubated with a solution of green fluorescent secondary antibodies (rabbit anti-goat IgG Alexa Fluor 488) for 45 minutes before flushing the channel with phosphate buffered saline (1xPBS). N- hydroxysuccinimide (NHS) chemistry was used to attach a sulfo-cyanine3 (Cy3) NHS ester dye to the immobilized antibodies to demonstrate a common conjugation technique. Finally, a low-pH release buffer was used for recovery. Flow cytometry and gel electrophoresis confirm successful AC assembly without damage due to ultrasound exposure. Potential scale-up strategies are also discussed.