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Objective Human papillomavirus (HPV)–associated oropharyngeal cancer (OPC) is a lethal disease with increasing incidence; however, technologies for early detection are limited. Nanomotors are synthetic nanostructures that can be powered by different mechanisms and functionalized for specific applications, such as biosensing. The objective of this investigation was to demonstrate an in vitro proof of concept for a novel nanomotor-based cancer detection approach toward in vivo detection of HPV-OPC. Study Design In vitro cell line incubated with ultrasound-propelled nanomotors. Setting Basic science and engineering laboratories. Subjects and Methods Ultrasound-powered gold nanowire nanomotors were functionalized with graphene oxide and dye-labeled single-stranded DNA for the specific intracellular detection of HPV16 E6 mRNA transcripts. Nanomotors were incubated with HPV-positive or HPV-negative human OPC cells under static conditions or with an applied ultrasound field for 15 minutes. The resulting intracellular fluorescence was assessed with fluorescence microscopy and analysis software. Results Nanomotors incubated with RNA extracted from HPV-positive OPC cells resulted in 60.7% of maximal fluorescence recovery, while incubation with RNA extracted from HPV-negative cells produced negligible fluorescence. Nanomotor incubation with intact HPV-negative cells produced minimal fluorescence (0.01 au), while incubation with HPV-positive cells produced a detectable signal (0.43 au) under static conditions and had 2.3-times greater intensity when powered with ultrasound. Conclusion Acoustically powered nanomotors can successfully identify HPV16 E6 mRNA transcripts extracellularly and within intact cells. This work represents the first step toward a novel, practical approach to address the challenge of visually detecting HPV-OPC in real time.

The microfluidic silicon mold was fabricated according to previous work [22]. Shortly, a 50 nm layer of Cr was deposited (Temescal BJD 1800) on a 4" silicon wafer to act as an etch mask. Next, photolithography was used to pattern the microfluidic channels and the unmasked portions were etched by Deep Reactive Ion Etching (Plasmalab Oxford P100), yielding 100 μm tall patterns.Depth measurements were performed using a Dektak 150 surface profiler (Veeco, Plainview, NY).Then, a 70 nm layer of poly(methyl methacrylate) (PMMA 950 A2, MicroChem, USA) was spincasted onto the Si master, followed by soft baking at 180 °C. A silicone layer of 500 μm (Dow Corning, Sylgard 184) was then spin-casted onto the Si master to yield the final microfluidic

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