Periodic Reporting for period 1 - BiFlowFilter (Bidirectional flow filter: microfluidic device for separation of biomolecules)
Período documentado: 2020-03-01 hasta 2021-08-31
Diagnostic analyses of sub-microliter volumes offer significant advances in biological research and medicine. Advanced diagnostic technologies (i.e. next generation sequencing) already require small volume samples and offer detection limits as low as a few molecules per specimen. Yet, sample preparation for such assays is sensitive, including steps of separating unwanted components from the crude biological sample and exchanging the chemical composition. Current separation techniques (i.e. chromatography, dialysis, diafiltration) are designed for large volume samples (> 10-100 µL) and are inadequate for sub-microliter volumes. The bottleneck for realizing the existing diagnostic techniques on actual small samples (i.e. rare biopsies, single cell analyses) lies lack of an appropriate technology for treatment of sub-microliter volumes.
We suggested to design a novel device for rapid and high-purity extraction of molecules and particles from sub-microliter volumes using our recently developed bidirectional flow filter (BFF). The technology leverages microscale flow patterning using field-effect electroosmosis to generate adjacent flow streams in opposite directions inside a microfluidic channel. The parallel but opposite streams allow separation of analytes based on their diffusivities. The system could potentially process tens of nanoliters per minute and separate components regardless of their charge, thus allowing the sample preparation needed for sub-microliter volumes. Furthermore, since our system is based on the diffusivities of the substances and not on their net charge or structure, separation of substances that cannot be distinguished using electrophoresis methods can be acheived.
Our first task in the development was adaptation of the BFF to biological samples. We analyzed a series of buffers in a range of pH and ionic strengths. We included chemical additives both to enhance fluid flow and to reduce protein adhesion to the device walls, which we found detrimental to the flow control. We tested various coatings to reduce protein adhesion, including PAcrAm-g-(PMOXA,Si,NH2) which was designed by expert coating company (SuSoS) to reduce protein adhesion to surfaces. We fabricated BFF devices with different dielectric layers to increase flow velocities at non-acidic pH and improve device stability, and designed BFF channels of various geometries (length and width of stream lines, number of stream lines) to increase the throughput. Further studies are needed to improve BFF performance for diagnostics.
To allow extraction of analytes, we introduced several geometric changes to the original BFF device, which was not designed for extraction. The novel BFF configuration allowed extraction of fluorescent beads from small molecules with efficiency of >80% and an extraction purity >99.5%. This novel BFF design can already be used for diffusion-based separation and extraction of biological substances that are compatible with low ionic strength and acidic conditions (PH 3.8). We successfully separated genomic DNA (~48 Kbp) from short DNA fragments (~200 bp), and were able to elute purified genomic DNA from the outlet reservoir. Yet, more research is needed for separation of samples that contain proteins or samples at pH > 5.5 and > 40 mM total salts.
We suggested to design a novel device for rapid and high-purity extraction of molecules and particles from sub-microliter volumes using our recently developed bidirectional flow filter (BFF). The technology leverages microscale flow patterning using field-effect electroosmosis to generate adjacent flow streams in opposite directions inside a microfluidic channel. The parallel but opposite streams allow separation of analytes based on their diffusivities. The system could potentially process tens of nanoliters per minute and separate components regardless of their charge, thus allowing the sample preparation needed for sub-microliter volumes. Furthermore, since our system is based on the diffusivities of the substances and not on their net charge or structure, separation of substances that cannot be distinguished using electrophoresis methods can be acheived.
Our first task in the development was adaptation of the BFF to biological samples. We analyzed a series of buffers in a range of pH and ionic strengths. We included chemical additives both to enhance fluid flow and to reduce protein adhesion to the device walls, which we found detrimental to the flow control. We tested various coatings to reduce protein adhesion, including PAcrAm-g-(PMOXA,Si,NH2) which was designed by expert coating company (SuSoS) to reduce protein adhesion to surfaces. We fabricated BFF devices with different dielectric layers to increase flow velocities at non-acidic pH and improve device stability, and designed BFF channels of various geometries (length and width of stream lines, number of stream lines) to increase the throughput. Further studies are needed to improve BFF performance for diagnostics.
To allow extraction of analytes, we introduced several geometric changes to the original BFF device, which was not designed for extraction. The novel BFF configuration allowed extraction of fluorescent beads from small molecules with efficiency of >80% and an extraction purity >99.5%. This novel BFF design can already be used for diffusion-based separation and extraction of biological substances that are compatible with low ionic strength and acidic conditions (PH 3.8). We successfully separated genomic DNA (~48 Kbp) from short DNA fragments (~200 bp), and were able to elute purified genomic DNA from the outlet reservoir. Yet, more research is needed for separation of samples that contain proteins or samples at pH > 5.5 and > 40 mM total salts.