(432e) Highthroughput Nanofluidics: A Disposable Virus Enrichment and Purification Ultrafiltration Device for Sensitive and Rapid Screening for Infectious Respiratory Diseases

Isolation and purification pretreatment steps are necessary for any sensor technology. The current SARS-CoV-2 RT-PCR tests cannot meet unprecedented global demand for rapid and reliable diagnosis, particularly when new variants threaten to initiate another pandemic, precisely because the PCR pretreatment technologies are inadequate in speed and yield. Column-based RNA-extraction sample pretreatment method, for example, is widely used but it is limited in sample capacity, which eventually leads to compromised sensitivity, and is time-consuming (0.5-1h). Ultracentrifugation is often used in the lab for virus isolation, but it is only for research due to the extended operation time and low accessibility. Nanofiltration with ultrafilration membrane can also be used. However, virus isolation yield of conventional ultrafiltration membranes is extremely low because of adsorption and filter cake formation.These deficiencies lead to false negatives, particularly for early stage infections with low viral load, and prevent pooled testing with large sample volumes. Rapid screening of early stage patients is the key to the effectiveness of any pandemic control program. There is hence an urgent need for large-volume RNA-extraction-free protocols that are directly compatible with established PCR-based testing workflow. This technology would transform COVID-19 testing.

We have developed an asymmetric nanoporous membrane (ANM) filtration technology, based on ion-track nanoporous membranes, to preconcentrate viruses from virus transfer medium with high yield and yet high throughput. At its core, the ANM contains thin and low-tortuosity (straight) nanopores with a highly asymmetric (conical) geometry but uniform pore tip size (<3% CV). As a result, retention is accomplished exclusively by the nanopore orifice with no penetration of the virus into the membrane, thus significantly minimizing virus loss in the membrane. The high recovery of the isolated viruses can be simply achieved by filtering the virus sample transport medium (~ 50µl concentrated retentate from 1ml of input solution). With asymmetric etching, we can improve the throughput 20 fold with a conic pore geometry whose hydrodynamic resistance is much smaller than that of a straight cylinder. This significantly enhanced hydrodynamic permeability allows processing of realistic sample with a very low negative operating pressure (-0.8 atm), such as that from a blood vacutainer tube. Surface modification also minimizes streaming potential and the resulting Induced-Charge Electro-Osmotic vortices that can corrupt the ultrafiltration efficiency. These outstanding features of ANM make it ideal for Point-of-Care ultrafiltraion of saliva samples for virus enrichment and isolation.

We demonstrated that our device could concentrate SARS-CoV-2 or lentiviruses from at least 10mL of virus transfer medium at a flow rate of more than 200uL/min. The yield of our device is high (~70%) and consistent over a large range of loading concentrations. The viruses can be collected from the device or directly lysed by adding surfactants or thermal lysis. The lysed samples can then be directly detected by qRT-PCR without further purification. A PCR Ct value improvement of more than 4 (16 fold) is achieved compared to the common virus isolation workflow, which gives our device better sensitivity and lower false-positive rates. It essentially improves the sensitivity of the PCR tests from several hundred viral particles per sample to less than 10 viral particles. This translates into a 30% improvement in sensitivity or reduction in false negatives in a testing population with less than 1% positivity rates. We also demonstrate that the high throughput enables pooled screening of multiple saliva samples with large load volume.