Measuring the DNA Cargo of Viruses Using Nanofluidics*

Adeno-associated viruses (AAVs) are engineered to deliver therapeutic DNA for gene therapy. However, AAV manufacturing is far from perfect, producing only a small portion of full viruses with the therapeutic gene. Real-time quality control in continuous AAV manufacturing requires characterizing the ratio of full to empty viruses. Here, we developed a nanofluidic approach for distinguishing full from empty viruses by measuring mass. Our approach uses nanochannel resonators, which measure nanoparticle mass from a proportional change in the device's resonant frequency. Single AAVs weigh only a few attograms, producing too low of a signal-to-noise ratio; we thus measure the average mass of AAV populations. We theoretically derived the relationship between the average AAV mass of a tested solution and the variance in resonant frequency. Using this relationship, we experimentally measured AAV mass, producing results consistent with standard, yet slower, biochemical methods. Using Monte-Carlo simulations of AAVs advecting and diffusing within the nanochannel, we gained additional insight into our measurements. With our approach, we aim to offer a real-time, high resolution characterization of AAV mass to enable quality control in continuous AAV manufacturing.