With the growth in microfluidic technologies in the COVID-19 era, a substantial advance in the Lab-on-a-Chip (LOC) system has been presented, enabling miniaturization and integration of complex functions routinely performed by hand in the traditional analytical processes1–3. One of the most active applications in the LOC is observed in biomedical research with Point-of-care (POC) diagnostic devices. The POC diagnostic system can rapidly deliver diagnostic outcomes to patients providing the users with many benefits over the traditional diagnostic methods such as straightforward and easy-to-use interface, portability, less sample and reagent consumption, etc4.
In biomedical research, size-based particle separation plays a significant role in the sample preparation process and the detection of target species5,6. As the size of analytes in biological research range from angstrom to millimetre scale, the separation mechanism of choice depends on their dimensions. During the past decade, many size-based separation techniques have been established for biomedical applications5,7. Enormous progress has been made, and size-based separation technologies move towards the miniaturized platforms to meet the requirements for point of care testing8–10. Many detection techniques require isolating target species of interest from solutions during sample preparation to provide unbiased and accurate insights into the biological process or phenomena. Since the dimension of biological structures ranges from angstrom to millimetre scale, the separation method varies depending on the target size due to the growing need for robust and reproducible size-based particle separation techniques. Especially, significant efforts have been put into developing a miniaturized size-selective particle sorting method for point of care applications such as LOC particle sorters8–10. However, the LOC based separation techniques are limited to the sub-micron scale (such as cells, viruses, bacteria), which results in significant sample loss, diagnostics misinterpretation, and poor detection limit11–14. Developing robust and reliable size-based particle separation techniques is emerging as an essential prerequisite for nanoscale measurement to obtain insights into the various biological systems on a nanometer scale.
For nanoscale biomolecule sorting, biomedical research has carried out several size-dependent nanoparticle separation techniques such as technology-based on external fields application, physical sieving and size-selective precipitation. Unfortunately, commonly used current technologies for NP separation were still not sufficiently efficient in selectively sorting particles based on their size or molecular weight for proper POC use due to their complex hands-on processes and incompatibility with downstream analytical devices. A miniaturized, compact, cost-effective platform for size-based separation is more favourable than one in centralized laboratories to eliminate the need for bulky and sophisticated instruments and extensive input material and minimize the complexity of the device operation.
For nanoscale biomolecule separation, several size-dependent nanoparticle separation techniques have been employed. Based on the separation mechanism, these techniques fall into three categories: the use of external fields (centrifugal15–17, electrical18–20 and magnetic21,22), physical barriers for sieving23, and size-dependent precipitation24. One of the most widely used methods based on the application of external fields is field flow fractionation (FFF). FFF is a particle separation technique taking advantage of the field exerted to a solution flowing through a channel. The field is perpendicular to the fluid flow, which results in the separation of particles suspended in the carrier solution due to the difference in travelling speed depending on their size and molecular weight. Despite its variations and versatility, it has not been widely used as a size based nanoparticle separation method due to its low throughput and extensive input material required. Alternatively, a size-dependent precipitation method has been devised. Size-dependent precipitation occurs when the stability, physical or chemical properties change depending on the surface chemistry of the nanoparticles. Sieving is another alternative for the size-selective fractionation of nanoparticles. Chromatography and membrane filtration employs physical barriers such as a column or physical hole to control elution and retention depending on the size of particles. Unfortunately, these traditional size based nanoparticle separation techniques result in huge sample loss, therefore, large input material is required. In addition, bulky instrumentation is involved in generating external field during the separation process. Due to the complexity of these technologies, there has been increasing demand for the development of miniaturized and compact platform for size-based separation, which can be used as a cost-effective preparative method in point of care application.
There have been many microfluidic approaches to bring the size-selective nanoparticle separation into miniaturized platforms. One of the most common separation mechanisms utilized in the microfluidic platform is membrane-based filtration because of its high resolution and straightforward procedure25–28. Gaborski et al.23 demonstrated nanoparticle fractionation using porous nanocrystalline silicon membranes with resolution as high as 5 nm, potentially applicable to Lab-on-a-Chip system for dead-end filtration. However, membrane filtration typically suffers from low efficiency associated with clogging and formation of cake layer near membrane filter29, leading to low flow rate, sample loss, and high fluid resistance30. Also, the fabrication of nanopores involves a complicated and time-consuming micro-fabrication process due to nanoscale particle separation. To circumvent this limitation, "filter-free" microfluidic separation techniques have been developed. Microfluidic approaches based on electrophoresis, field-flow fractionation (FFF)31,32 and centrifugation15,17 have been devised and implemented for size-based nanoparticle separation and fractionation. However, these techniques still rely on external fields which usually need the bulky instrument to manipulate particle motion.
Furthermore, most passive separation techniques require expensive fabrication to build microstructures. For a point-of-care application, lab on a chip system that can be realized with a simple fabrication process, providing a user-friendly interface enabling easy sample handling, yet with high resolution with minor sample loss is necessary. For these reasons, the lack of reliable size-selective nanoparticle separation is a bottleneck in analyses of nanoscale biomolecules such as ribosome profiling, which requires size-selective separation of monosome from polysome molecules33.
As membrane filtration can provide the most straightforward processing and high resolution, it has many advantages over filter-free alternatives. Hence it can be an excellent size-selective nanoparticle separation tool as long as the problems associated with membrane fouling during the process is addressed.As a part of its effort, many publications and research works have improved membrane filtration by utilizing ultrasonic treatment of membrane filters. However, most results focused on bulk processing for industrial use, such as wastewater processing, food processing, and ultrapure water production34,35. Typically, they employed ultrasonic bath, which requires high energy consumption and extensive input material, and it may not be suitable for biological applications where available input material is low.
To realize simple and rapid size-selective nanoparticle separation for POC application, we have developed a LOC system based on dead-end filtration by incorporating commercially available track-etched membrane filters with different pore sizes cut-off. Sample containing polydisperse nanoparticles has been processed in the microfluidic platform with embedded membrane filters. The dead-end filtration may cause clogging and membrane fouling due to the formation of a cake layer near the membrane surface, leading to low flow rate and membrane and sample loss damage. We have introduced ultrasonic treatment by integrating ultrasonic transmitters with a membrane filtration system online to overcome this issue. Ultrasonic assisted membrane filtration was modified to bring it into a miniaturized microfluidic platform. The functionality of this novel LOC based technology was confirmed by demonstrating size based gold and polymer nanoparticle separation.