Microfluidic system using a homobifunctional imidoester for simultaneous biomolecules isolation

This study reports the use of a homobifunctional imidoester (HI)-based microfluidic system for simultaneous DNA and protein isolation from a single solid or liquid biopsy sample. The microfluidic analysis of biomolecules has significantly enhanced the speed, sensitivity, and efficiency of diagnostic evaluation [1-2]. However, most microfluidic-based systems to date have focused on nucleic acid isolation, and the simultaneous isolation of various biomolecules has rarely been investigated with this technology. Conventional methods for the isolation of biomolecules include the use of filters/membranes, organic chemical reagents, and the large instruments such as vacuums and centrifuges. However, our methods differs from that HI based microfluidic system only took 40 min for the isolation of both biomolecules in a single process without the use of hazardous chemical reagents or need for large instruments. The principle of the HI-based microfluidic system is illustrated in Figure 1. The inner-channel of the microfluidic platform is activated with APTES, leading to the formation of an amine-reactive group of the organic molecules (Step 1). A mixture of sample is loaded into the microfluidic platform (Step 2). The HI then captures DNA onto the amine-modified channel surface through both electrostatic interactions and covalent bonding (Step 3) [2]. The DNA is next extracted with elution buffer (Step 4). Optimization of the microfluidic system is illustrated in Figure 2. To enhance the efficiency of the biomolecule isolation, the most efficient (100% capture rate) and inexpensive microfluidic-channel design was selected from among several candidates using simulation and experimental results. Fundamental characterization of the HI-based microfluidic system is illustrated in Figure 3. To validate the effectiveness of our new HI-based microfluidic system, we used it to test the ability of different HIs such as DMP, DTBP, and DMA to directly bind DNA. Validation of the HI-based microfluidic system is illustrated in Figure 4. The DNA extracted using the HI-based microfluidic system was analyzed by PCR and real-time PCR. The proteins isolated by the HI-based microfluidic system were assessed by western blot and silver staining of HI-system and RIPA method. Compared to the RIPA method which requires more than 30 min for protein isolation, we were able to rapidly isolate proteins using the HI-based microfluidic system. Clinical utility of the HI-based microfluidic system in human tissue specimens is illustrated in Figure 5. Both DNA and protein from 23 patient-derived samples were isolated using the HI-based microfluidic platform and were identified by PCR, silver staining, and western blotting. These tests revealed the successful isolation of both DNA and protein from solid and liquid samples using the HI-based microfluidic platform. After presentation of results, this HI-based microfluidic system shows good rapidity, affordability, and portability in the isolation of biomolecules from limited samples for subsequent clinical analysis.