Reducing false negatives of point-of-care diagnostic tests: A bifurcated continuous field-flow fractionation (BCFFF) chip for high-yield and high-throughput nucleic acid extraction and purification

Generally, DNA quantification and RNA quantification, normally referred to as nucleic acid quantification, is commonly performed to determine the average concentration of DNA or RNA in a sample prior to proceeding with downstream experiments. In practice, detection and quantification of nucleic acids play essential roles in various applications, including: clinical diagnostics, food safety, forensic analysis, and environmental monitoring.  For example, it is used to determine COVID-19 load in screening tests during the current pandemics. In addition, the discovery of host nucleic acid biomarkers for different diseases has opened up numerous new and future applications. At present, the nucleic acid analysis technologies available, such as: PCR, microarray and sequencing, can provide sensitive and selective quantification. However, they all demand a high-yield pre-treatment step to extract and purify the nucleic acid targets and insert them into a standardized buffer, for sensitive and accurate quantification, independent of the actual detection platform. As of now, these extraction methods are based on liquid extraction into immiscible liquids and solid extraction by high-affinity binding columns. Overall, the multiple and noncontinuous operation steps of the current microfluidic-based extraction modules also render their integration with downstream PCR or other detection modules extremely difficult and hence the entire assay must be done in a centralized laboratory.

Therefore, one can hypothesize that a high-throughput and high-yield extraction chip module that can be integrated with downstream steps in an integrated continuous-flow platform would significantly elevate the detection sensitivity, quantification accuracy and usability of nucleic acid analysis technologies, particularly for Point-of-Care screening tests that do not need to be sent to a centralized lab. In this line of thoughts, a team of researchers from the University of Notre Dame: Mr. Chenguang Zhang, Professor Satyajyoti Senapati and Professor Hsueh-Chia Chang, in collaboration with Dr. Gongchen Sun at the Georgia Institute of Technology developed a novel on-chip microfluidic extraction technology that could isolate nucleic acids from an inhibitor-rich plasma sample and insert them into a standardized PCR buffer at high yield and throughput. Their work is currently published in the research journal, Lab on a Chip.

The research team focused on developing a field-flow fractionation design which could extract charged nucleic acids from a continuous flow by electrophoresis. To achieve this, the researchers adopted a design that used a membrane ionic transistor to sustain low-ionic strength in a localized region at a junction, such that the resulting high field could selectively isolate high-charge density nucleic acids from the main flow channel and insert them into a standardized buffer in a side channel that bifurcated from the junction.

The authors reported that the high local electric field and the bifurcated field-flow design facilitated concentration reduction of both divalent cation and molecular PCR inhibitors by more than two orders of magnitude, even with high-throughput continuous loading.  Moreover, their exceptional design comprising of a large on-chip ionic-strength gradient allowed miniaturization into a high-throughput field-flow fractionation chip that could be integrated with upstream lysing and downstream PCR/sensor modules for various nucleic acid detection/quantification applications.

In summary, the study presented the design and validation of a bifurcated continuous field-flow fractionation (BCFFF) chip for high-yield and high-throughput extraction of nucleic acids from physiological samples. Remarkably, the team demonstrated that the developed BCFFF platform was versatile and could be applied for isolation of both long dsDNAs and short miRNAs, without changing the device configuration or the operation. Even better, their technique recorded a net yield four times larger than a commercial extraction kit for miR-39 in human plasma. In a statement to Medicine Innovates, Professor Hsueh-Chia Chang further emphasized that with their integrated chip, an extraction yield from plasma higher than 80% for different nucleic acids with different lengths, ranging from long dsDNA fragments to short miRNAs could be obtained.