Magnetic enrichment of immuno-specific extracellular vesicles for mass spectrometry using biofilm-derived iron oxide nanowires.

Immuno-specific enrichment of extracellular vesicles (EVs) can provide important information into cellular pathways underpinning various pathologies and for non-invasive diagnostics, including mass spectrometry-based analyses. Herein, we report an optimised protocol for immuno-magnetic enrichment of specific EV subtypes and their subsequent processing with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Specifically, we conjugated placental alkaline phosphatase (PLAP) antibodies to magnetic iron oxide nanowires (NWs) derived from bacterial biofilms and demonstrated the utility of this approach by enriching placenta-specific EVs (containing PLAP) from cell culture media. We demonstrate efficient PLAP+ve EV enrichment for both NW-PLAP and Dynabeads™-PLAP, with high PLAP protein recovery (83.7 ± 8.9% and 83.2 ± 5.9%, respectively), high particle-to-protein ratio (7.5 ± 0.7 × 109 and 7.1 ± 1.2 × 109, respectively), and low non-specific binding of non-target EVs (7 ± 3.2% and 5.4 ± 2.2%, respectively). Furthermore, our optimized EV enrichment and processing approach identified 2518 and 2545 protein groups with LC-MS/MS for NW-PLAP and Dynabead™-PLAP, respectively, with excellent reproducibility (Pearson correlation 0.986 and 0.988). These findings demonstrate that naturally occurring iron oxide NWs have comparable performance to current gold standard immune-magnetic beads. The optimized immuno-specific EV enrichment for LC-MS/MS method provides a low-cost and highly-scalable yet efficient, high-throughput approach for quality EV proteomic studies.

Fabrication of 3D continuous-flow reverse-transcription polymerase chain reaction microdevice integrated with on-chip fluorescence detection for semi-quantitative assessment of gene expression.

We fabricate a three-dimensional (3D) microdevice operated with minimal peripheral accessories, including a portable pump for semi-automated sample delivery and a single heater for temperature control, for performing reverse transcription polymerase chain reaction (RT-PCR) integrated with a downstream fluorescence detection module for semi-quantitative assessment of gene expression. The microdevice was fabricated by wrapping a polytetrafluoroethylene (PTFE) tube around a pre-designed polycarbonate mold to create a seamless microchannel for both the reverse transcription (RT) of RNA and the amplification of complementary DNA. In addition, a silicone tube, which underwent a two-step surface modification mediated by polyethyleneimine and glutaraldehyde coating, was connected at the outlet to capture amplicons downstream of the PTFE tube for on-site fluorescence detection. This fabrication method enabled continuous-flow RT-PCR (CF RT-PCR) using the 3D CF RT-PCR microdevice as a reactor, a single heater for the temperature control of both RT and PCR processes, and a disposable plastic syringe for semi-automated sample delivery. The microdevice was successfully implemented for the identification of the ß-actin gene, a constitutively expressed gene in all cells, and the sphingosine-1-phosphate lyase 1 gene, a potential pharmacological target gene in the diagnosis of cancer, diabetes, and atherosclerosis. This portable integrated microdevice offers a potential approach towards preliminary studies of gene expression and identification of RNA viruses.

Microdevice-based solid-phase polymerase chain reaction for rapid detection of pathogenic microorganisms.

We demonstrate the integration of DNA amplification and detection functionalities developed on a lab-on-a-chip microdevice utilizing solid-phase polymerase chain reaction (SP-PCR) for point-of-need (PON) DNA analyses. First, the polycarbonate microdevice was fabricated by thermal bonding  to contain microchambers as reservoirs for performing SP-PCR. Next, the microchambers were subsequently modified with polyethyleneimine and glutaraldehyde for immobilizing amine-modified forward primers. During SP-PCR, the immobilized forward primers and freely diffusing fluorescence-labeled reverse primers cooperated to generate target amplicons, which remained covalently attached to the microchambers for the fluorescence detection. The SP-PCR microdevice was used for the direct identifications of two widely detected foodborne pathogens, namely Salmonella spp. and Staphylococcus aureus, and an alga causing harmful algal blooms annually in South Korea, Cochlodinium polykrikoides. The SP-PCR microdevice would be versatilely applied in PON testing as a universal platform for the fast identification of foodborne pathogens and environmentally threatening biogenic targets.