CHAPTER 1. Introduction
1.4 Sample preparations on microfluidics for liquid biopsy
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Figure 1.10. Cell-free DNA purification on a lab-on-a-chip system. (a) Nanowire consisting of electroactive conducting polymer captures and releases the circulating cfDNA by the electrostatic interaction of negatively charged DNA and the conductive polymer,3-4 (b) a microfluidic solid-phase DNA extraction device using a surface-functionalized micropillar array,13 (c) a microfluidic chip, called vortex-GMACS, could achieve on-chip cfDNA purification using silica magnetic beads, which is controlled by hydrodynamics,25 (d) rapid fluorescence-based quantification of cfDNA from small volume (<10 µL) of plasma samples with septic patients,34 (e) DEP microarray device for the isolation and detection of cfDNA from chronic lymphocytic leukemia (CLL).39
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increase the binding of cfDNA to the silica surface of magnetic particles. Further optimizations of cfDNA purification processes, including the flow rate, the concentration of magnetic beads, and the magnetic field strength were carried out to achieve high yield of cfDNA.
Besides the field of liquid biopsy, cfDNA isolation has been done by use of a microfluidic device. Yang et al. demonstrated a simplified microfluidic device that enables to quantify the cfDNA in a small volume of plasma (<10 µL) in a short amount of time (5 min) for rapid assessment of septic patients.34 They implemented electrophoresis on the microfluidic device to isolate and concentrate cfDNA from plasma, and this concentrated cfDNA was quantified by fluorescence measurement.
Sonnenberg et al. reported the isolation and detection of cfDNA using a DEP microarray device.39 This method uses the fact that larger particles in blood, such as blood cells, can be isolated rather than nanoparticulate entities such as cfDNA by a low DEP field. This study achieved the isolation of cfDNA from 20 µL of whole blood from chronic lymphocytic leukemia (CLL) patients by the fluorescence detection of stained DNA in a high DEP field. Although these studies described some kinds of cfDNA isolation methods, they are not suitable to apply to the liquid biopsy field due to the limited sample volumes and yields.
1.4.2 EV isolation on microfluidics
The most widely used EV isolation method in biological applications is still the ultracentrifugation method (UC). However, it provides poor yields and purity of EVs, while also requiring a time-consuming process (> 6 hours), and bulk instruments for performing high g-force (150,000). Recently, therefore, various EV isolation techniques have been developed, and implemented in microfluidic devices based on the differences of immunoaffinity and size of extracellular vesicles.54 The most notable studies will be summarized according to the isolation principles in this section (Figure 1.11, Figure 1.12).
There are typical EV-specific markers such as CD9, CD63, and CD81, so their antibodies could be utilized to capture the EV with various methodologies. Shao et al. demonstrated a sensitive technique for profiling microvesicles (MVs) from the blood plasma of glioblastoma patients.14 In this system, the microvesicles were labeled with antibodies of their protein markers, then the antibodies were coupled with magnetic nano particles (MNPs) using the two-step bio-orthogonal approach (BOND-2). These microvesicle complexes were captured and concentrated on a filter by size-selective filtration, and quantified by NMR detection to profile the protein markers in the target microvesicles.
The same group reported a nano-plasmonic exosome (nPLEX) assay that enables a label-free and high- throughput quantitative analysis of exosomes.15-16 They adopted immunoaffinity-based EV isolation which directly performed on a sensing substrate (periodic gold nanoholes array), which was coated with
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Figure 1.11. Immunoaffinity-based EV isolation on a microfluidic device. (a) Enlarged size of magnetic beads-decorated microvesicle could be isolated by size-difference-based filtration prior to on-chip NMR detection,14 (b-c) immunoaffinity-based EV isolation directly on the SPR substrate.15-
16 (d) a microfluidic approach, total process of the isolation, enrichment of EVs, lysis of captured EVs, and detection was integrated on a single device.26, (e) ExoChip for on-chip isolation, quantification, and characterization of circulating exosomes.26-27
Figure 1.12 Size-difference-based EV isolation and other approaches on a microfluidic device.
(a) lab-on-a-disc system, called Exodisc, for EV isolation from urine and CCS,28 (b) sequential- filtration tool, called ExoTIC, can fractionation of EVs as a function of size,29 (c) acoustic EV separation on a chip,38 (d) DEP chip for EV isolation from blood plasma.42
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the target antibodies. Captured EVs were detected and quantified by a surface plasmon resonance (SPR)-based assay.
Other groups also demonstrated an immunoaffinity-based isolation of circulating EVs on a microfluidic device.26-27 He et al. described a microfluidic approach that could achieve the isolation and enrichment of EVs, the lysis of captured EVs, and detection on a single microfluidic chip.26 EVs were isolated from 30 µL of plasma using EV specific antibody-coated magnetic beads, and subsequently lysed by the method of chemical lysis. The EV lysate (intra-vesicular proteins) was captured on antibody-coated magnetic beads. Finally, these EV proteins were detected by a chemifluorescence- based sandwich immunoassay.
Although the immunoaffinity-based isolation provides highly pure EV samples, it relies highly on the kind of capture antibodies, so any EVs that have low expressions of target molecules cannot be isolated. Circulating cancer EVs are heterogeneous, coming from the heterogeneity of cancer, which means all EVs exhibit different expression levels of surface proteins. Therefore, lots of studies have focused on size-difference-based EV isolation in microfluidics (Figure 1.12).28-29, 38, 99 Woo et al.
developed fully automated EV isolation on a disc called Exodisc, integrated with two nano-sized filters (600 nm, 20 nm).28 This platform enables EV isolation starting from a few mL of raw biological samples such as urine, and cell-culture supernatant (CCS) in > 95% of recovery with high purity within 30 min.
It provides >100-fold higher mRNA concentrations compared with the UC method. Moreover, ELISA of urinary EV isolated from bladder cancer patients could be performed on the disc, and the results showed high levels of EVs marker expression (CD9, CD81). Liu et al. presented a sequential filtration tool called ExoTIC (exosome total isolation chip) that enabled ~4-1000-fold higher EV recovery than the UC method.29 Notably, ExoTIC could perform the size fractionation of EV from a heterogeneous EVs population as a function of their size by using a module of nano-sized membranes (200, 100, 80, 50, 30 nm). Differing from size-filtration method, Wu et al. reported the acoustic separation of EVs based on the differences of particle size.38 This platform provides label-free and a contact-free isolation directly from whole blood that obtains highly intact EVs for further downstream analysis.
Though the immunoaffinity-based and size-difference-based isolation of EVs have mainly been achieved in the microfluidic field, unique approaches have also been implemented by using the functional utility of microfluidics. For an example, Chen et al. proposed a dielectrophoretic (DEP) method to isolate EVs from blood plasma (Figure 1.12d).42 EVs were captured on the edges of the microelectrodes embedded in a PDMS chip while the DEP force was generated, then captured EVs were eluted when the DEP signal was turned off. The whole isolation process was easily performed, and it took only 30 min.
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Recent trends in isolation technology for circulating cell-free DNA and extracellular vesicles in microfluidics for liquid biopsy have been reviewed in this section. Together with the clinical significance of liquid biopsy is rapidly growing, the microfluidic approach is expected to have an increasingly important role in this field.