Immiscible Filtration Assisted By Surface Tension Research Articles

Magnetic Nanoparticle-Based Microfluidic Platform for Automated Enrichment of High-Purity Extracellular Vesicles.

Extracellular vesicles (EVs) are available in various biological fluids and have highly heterogeneous sizes, origins, contents, and functions. Rapid enrichment of high-purity EVs remains crucial for enhancing research on EVs in tumors. In this work, we present a magnetic nanoparticle-based microfluidic platform (ExoCPR) for on-chip isolation, purification, and mild recovery of EVs from cell culture supernatant and plasma within 29 min. The ExoCPR chip integrates bubble-driven micromixers and immiscible filtration assisted by surface tension (IFAST) technology. The bubble-driven micromixer enhances the mixing between immunomagnetic beads and EVs, eliminating the need for manual pipetting or off-chip oscillatory incubation. The high-purity EVs were obtained after passing through the immiscible phase interface where hydrophilic or hydrophobic impurities nonspecifically bound to SIMI were removed. The ExoCPR chip had a capture efficiency of 75.8% and a release efficiency of 62.7% for model EVs. We also demonstrated the powerful performance of the ExoCPR in isolating EVs from biological samples (>90% purity). This chip was further employed in clinical plasma samples and showed that the number of GPC3-positive EVs isolated from hepatocellular carcinoma patients was significantly higher than that of healthy individuals. This ExoCPR chip may provide a promising tool for EV-based liquid biopsy and other fundamental research.

Quantitative analysis of respiratory viruses based on lab-on-a-chip platform.

The quantitative analysis of respiratory viruses is of great importance for rapid diagnosis, precision medicine, and prognosis. Several current quantitative analysis systems have been proposed and commercialized. Although they have been proven in trials, quantitative analyzes based on real samples are still complex, time-consuming, and expensive. Therefore, they are not able to directly quantify real samples. In this work, we presented a lab-on-a-chip platform combined with an automated control system to achieve quantitative analysis from samples to results. We developed a multilayer integrated chip to rapidly extract and quantify RNA of coronavirus disease 2019 (COVID-19) pseudovirus from large-volume nasal swab samples. The dependence of the magnetic bead size and the interfacial effect was studied for the first time, and the conditions of immiscible filtration assisted by surface tension (IFAST) method for nucleic acid extraction were optimized to increase the nucleic acid recovery rate up to 85%. Inside the chip, a pneumatic valve was developed for automatic opening and closing of the liquid channel. The integrated chip platform and automatic control system presented here are advantageous for use in resource-limited settings (RLS). In addition, our method can be extended to other respiratory viruses and other sample types.

A lab-on-a-chip platform for integrated extraction and detection of SARS-CoV-2 RNA in resource-limited settings

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the unprecedented global pandemic of coronavirus disease-2019 (COVID-19). Efforts are needed to develop rapid and accurate diagnostic tools for extensive testing, allowing for effective containment of the infection via timely identification and isolation of SARS-CoV-2 carriers. Current gold standard nucleic acid tests require many separate steps that need trained personnel to operate specialist instrumentation in laboratory environments, hampering turnaround time and test accessibility, especially in low-resource settings. We devised an integrated on-chip platform coupling RNA extraction based on immiscible filtration assisted by surface tension (IFAST), with RNA amplification and detection via colorimetric reverse-transcription loop mediated isothermal amplification (RT-LAMP), using two sets of primers targeting open reading frame 1a (ORF1a) and nucleoprotein (N) genes of SARS-CoV-2. Results were identified visually, with a colour change from pink to yellow indicating positive amplification, and further confirmed by DNA gel electrophoresis. The specificity of the assay was tested against HCoV-OC43 and H1N1 RNAs. The assay based on use of gene N primers was 100% specific to SARS-CoV-2 with no cross-reactivity to HCoV-OC43 nor H1N1. Proof-of-concept studies on water and artificial sputum containing genomic SARS-CoV-2 RNA showed our IFAST RT-LAMP device to be capable of extracting and detecting 470 SARS-CoV-2 copies mL−1 within 1 h (from sample-in to answer-out). IFAST RT-LAMP is a simple-to-use, integrated, rapid and accurate COVID-19 diagnostic platform, which could provide an attractive means for extensive screening of SARS-CoV-2 infections at point-of-care, especially in resource-constrained settings.

A Microfluidic Device for Rapid Screening of E. coli O157:H7 Based on IFAST and ATP Bioluminescence Assay for Water Analysis.

We present a simple microfluidic system for rapid screening of Escherichia coli (E. coli) O157:H7 employing the specificity of immunomagnetic separation (IMS) via immiscible filtration assisted by surface tension (IFAST), and the sensitivity of the subsequent adenosine triphosphate (ATP) assay by the bioluminescence luciferin/luciferase reaction. The developed device was capable of detecting E. coli O157:H7 from just 6 colony forming units (CFU) in 1 mL spiked buffer within 20 min. When tested with wastewater discharged effluent samples, without pre-concentration, the device demonstrated the ability to detect 104 CFU per mL seeded; suggesting great potential for point-of-need microbiological water quality monitoring.

HIV Viral RNA Extraction in Wax Immiscible Filtration Assisted by Surface Tension (IFAST) Devices

The monitoring of viral load is critical for proper management of antiretroviral therapy for HIV-positive patients. Unfortunately, in the developing world, significant economic and geographical barriers exist, limiting access to this test. The complexity of current viral load assays makes them expensive and their access limited to advanced facilities. We attempted to address these limitations by replacing conventional RNA extraction, one of the essential processes in viral load quantitation, with a simplified technique known as immiscible filtration assisted by surface tension (IFAST). Furthermore, these devices were produced via the embossing of wax, enabling local populations to produce and dispose of their own devices with minimal training or infrastructure, potentially reducing the total assay cost. In addition, IFAST can be used to reduce cold chain dependence during transportation. Viral RNA extracted from raw samples stored at 37°C for 1 week exhibited nearly complete degradation. However, IFAST-purified RNA could be stored at 37°C for 1 week without significant loss. These data suggest that RNA isolated at the point of care (eg, in a rural clinic) via IFAST could be shipped to a central laboratory for quantitative RT-PCR without a cold chain. Using this technology, we have demonstrated accurate and repeatable measurements of viral load on samples with as low as 50 copies per milliliter of sample.

Weak protein–protein interactions revealed by immiscible filtration assisted by surface tension

Biological mechanisms are often mediated by transient interactions between multiple proteins. The isolation of intact protein complexes is essential to understanding biochemical processes and an important prerequisite for identifying new drug targets and biomarkers. However, low-affinity interactions are often difficult to detect. Here, we use a newly described method called immiscible filtration assisted by surface tension (IFAST) to isolate proteins under defined binding conditions. This method, which gives a near-instantaneous isolation, enables significantly higher recovery of transient complexes compared to current wash-based protocols, which require reequilibration at each of several wash steps, resulting in protein loss. The method moves proteins, or protein complexes, captured on a solid phase through one or more immiscible-phase barriers that efficiently exclude the passage of nonspecific material in a single operation. We use a previously described polyol-responsive monoclonal antibody to investigate the potential of this new method to study protein binding. In addition, difficult-to-isolate complexes involving the biologically and clinically important Wnt signaling pathway were isolated. We anticipate that this simple, rapid method to isolate intact, transient complexes will enable the discoveries of new signaling pathways, biomarkers, and drug targets.

Exclusion-Based Capture and Enumeration of CD4+ T Cells from Whole Blood for Low-Resource Settings

In developing countries, demand exists for a cost-effective method to evaluate human immunodeficiency virus patients' CD4(+) T-helper cell count. The TH (CD4) cell count is the current marker used to identify when an HIV patient has progressed to acquired immunodeficiency syndrome, which results when the immune system can no longer prevent certain opportunistic infections. A system to perform TH count that obviates the use of costly flow cytometry will enable physicians to more closely follow patients' disease progression and response to therapy in areas where such advanced equipment is unavailable. Our system of two serially-operated immiscible phase exclusion-based cell isolations coupled with a rapid fluorescent readout enables exclusion-based isolation and accurate counting of T-helper cells at lower cost and from a smaller volume of blood than previous methods. TH cell isolation via immiscible filtration assisted by surface tension (IFAST) compares well against the established Dynal T4 Quant Kit and is sensitive at CD4 counts representative of immunocompromised patients (less than 200 TH cells per microliter of blood). Our technique retains use of open, simple-to-operate devices that enable IFAST as a high-throughput, automatable sample preparation method, improving throughput over previous low-resource methods.

Characterization of Molecules Binding to the 70K N-Terminal Region of Fibronectin by IFAST Purification Coupled with Mass Spectrometry

Fibronectin (Fn) is a large glycoprotein present in plasma and extracellular matrix and is important for many processes. Within Fn the 70 kDa N-terminal region (70k-Fn) is involved in cell-mediated Fn assembly, a process that contributes to embryogenesis, development, and platelet thrombus formation. In addition, major human pathogens including Staphlycoccus aureus and Streptococcus pyogenes bind the 70k-Fn region by a novel form of protein-protein interaction called β-zipper formation, facilitating bacterial spread and colonization. Knowledge of blood plasma and platelet proteins that interact with 70k-Fn by β-zipper formation is incomplete. In the current study, we aimed to characterize these proteins through affinity purification. For this affinity purification, we used a novel purification technique termed immiscible filtration assisted by surface tension (IFAST). The foundation of this technology is immiscible phase filtration, using a magnet to draw paramagnetic particle (PMP)-bound analyte through an immiscible barrier (oil or organic solvent) that separates an aqueous sample from an aqueous eluting buffer. The immiscible barrier functions to remove unbound proteins via exclusion rather than dilutive washing used in traditional isolation methods. We identified 31 interactors from plasma, of which only seven were previously known to interact with Fn. Furthermore, five proteins were identified to interact with 70k-Fn from platelet lysate, of which one was previously known. These results demonstrate that IFAST offers advantages for proteomic studies of interacting molecules in that the technique requires small sample volumes, can be done with high enough throughput to sample multiple interaction conditions, and is amenable to exploratory mass spectrometric and confirmatory immuno-blotting read-outs.

The VerIFAST: an integrated method for cell isolation and extracellular/intracellular staining

Isolation and characterization of a specific subset of cells from a large heterogeneous population is necessary for studying rare subpopulations of cells. Existing methods require transfer or wash steps that risk causing loss of the rare cell population of interest. Integrated methods reduce loss, making these methods especially useful for reliable isolation of rare cell populations. In this report, we demonstrate the VerIFAST, a device that builds upon the simplified workflow of the Immiscible Filtration Assisted by Surface Tension (IFAST) to integrate a method for cellular isolation with methods for extra- and intracellular staining. First, a front-end purification step allows cells and unwanted particulates to passively settle out of the operational path of the paramagnetic particles, resulting in good efficiency of capture (>80%) and purity (>70%) with a single virtual wall traverse. Second, a Sieve Chamber is used downstream of the isolation chamber that removes excess unbound paramagnetic particles (PMPs) and performs complex multi-step washing procedures without centrifugation or transfer steps. Further, cellular staining can be performed in the device and is demonstrated for extracellular epithelial cell adhesion molecule (EpCAM), intracellular pan-cytokeratins, and Ki-67.

Automated Operation of Immiscible Filtration Assisted by Surface Tension (IFAST) Arrays for Streamlined Analyte Isolation

The purification of analytes is an important prerequisite for many analytical processes. Although automated infrastructure has dramatically increased throughput for many of these processes, the upstream analyte purification throughput has lagged behind, partially due to the complexity of conventional isolation processes. Here, we demonstrate automated operation of arrays of a new sample preparation technology--immiscible filtration assisted by surface tension (IFAST). IFAST uses surface tension to position an immiscible liquid barrier between a biological sample and downstream buffer. Paramagnetic particles are used to capture analytes of interest and draw them across the immiscible barrier, thus resulting in purification in a single step. Furthermore, the planarity of the IFAST design enables facile and simultaneous operation of multiple IFAST devices. To demonstrate the application of automation to IFAST, we successfully perform an array of 48 IFAST-based assays to detect the presence of a specific antibody. This assay array uses only a commercial automated liquid handler to load the devices and a custom-built magnet actuator to operate the assays. Automated operation of the IFAST devices resulted in more repeatable results relative to manual operation.

Streamlining Immunoassays with Immiscible Filtrations Assisted by Surface Tension

Immunoassays are utilized for a wide variety of clinical and biomedical research applications. In typical immunoassays, analytes are captured, labeled, and quantified on a single surface (e.g., the bottom of a well plate). In order to minimize the background, this type of assay must be washed multiple times between each of these steps to ensure residual reagents (e.g., unbound labeling antibody) are removed from the system. In this manuscript, the immunoassay is fundamentally reconfigured, such that each reagent is confined to its own well and no wash steps are required. Using immiscible filtration assisted by surface tension (IFAST), a technique developed for nucleic acid and whole cell purifications, immunoassays can be drastically simplified such that all reagent manipulation is performed at the start of the assay (i.e., no pipetting steps are necessary during the assay). Analytes are bound to paramagnetic particles via antibodies and drawn through oil barriers between four isolated compartments: (1) sample well, (2) primary antibody labeling well, (3) secondary antibody labeling well, and (4) readout buffer well. Using this technique, we have demonstrated repeatable detection of as little as 188 fg of protein. IFAST immunoassay functionality is demonstrated by detecting a well accepted prostate cancer biomarker, prostate specific antigen (PSA). Assay performance was assessed by measuring known concentrations of recombinant PSA protein. The assay was then used to measure PSA concentrations in conditioned media and human plasma samples.

Both LRP5 and LRP6 Receptors Are Required to Respond to Physiological Wnt Ligands in Mammary Epithelial Cells and Fibroblasts

A canonical Wnt signal maintains adult mammary ductal stem cell activity, and this signal requires the Wnt signaling reception, LRP5. However, previous data from our laboratory have shown that LRP5 and LRP6 are co-expressed in mammary basal cells and that LRP6 is active, leading us to question why LRP6 is insufficient to mediate canonical signaling in the absence of LRP5. Here, we show that at endogenous levels of LRP5 and LRP6 both receptors are required to signal in response to some Wnt ligands both in vitro (in mouse embryonic fibroblasts and mammary epithelial cells) and in vivo (in mammary outgrowths). This subgroup of canonical ligands includes Wnt1, Wnt9b, and Wnt10b; the latter two are expressed in mammary gland. In contrast, the ligand commonly used experimentally, Wnt3a, prefers LRP6 and requires just one receptor regardless of cellular context. When either LRP5 or LRP6 is overexpressed, signaling remains ligand-dependent, but the requirement for both receptors is abrogated (regardless of ligand type). We have documented an LRP5-6 heteromer using immiscible filtration assisted by surface tension (IFAST) immunoprecipitation. Together, our data imply that under physiological conditions some Wnt ligands require both receptors to be present to generate a canonical signal. We have designed a model to explain our results based on the resistance of LRP5-6 heteromers to a selective inhibitor of E1/2-binding Wnt-LRP6 interaction. These data have implications for stem cell biology and for the analysis of the oncogenicity of LRP receptors that are often overexpressed in breast tumors.

Abstract 4272: An integrated platform for quantifying gene expression in co-cultured cells

Abstract The quantification of mRNA is a ubiquitous and critical tool for understanding cellular mechanisms in cancer. While RT-PCR is often the endpoint, the success of the analysis depends not only on the PCR reaction, but also on an entire process flow linking living cells to the PCR endpoint. For cultured cells, this process flow includes the culture itself, cell lysis, mRNA extraction and purification, and RT-PCR. While much research has been targeted to streamlining and increasing throughput of the PCR process, the remainder of the process flow has largely been neglected. In this study, we link a new mechanism for purifying mRNA, Immiscible Filtration Assisted by Surface Tension (IFAST), with cell culture on a single platform. IFAST uses an immiscible liquid barrier (e.g., oil) to separate the cell culture / lysis region from an elution buffer. Using paramagnetic particles (PMPs) that selectively bind mRNA, we extracted mRNA from the lysate by using a magnet to draw the PMP-captured mRNA through the immiscible phase. This process, which takes only seconds, replaces multiple washing steps required by current mRNA isolation protocols, which typically take 15-60 minutes to complete. The simplicity of IFAST enables facile integration of culture and mRNA extraction on an easy-to-use chip that requires only a micropipette and magnet to operate. Once proof-of-concept was demonstrated with a single cell type, the platform was expanded to incorporate co-culture of two cell types, with breast cancer cells and stromal cells in separate compartments connected via diffusion ports to allow cytokine exchange. Each compartment has an IFAST device, such that mRNA can be collected independently from each side. RT-PCR expression levels from the integrated culture / IFAST platform had less variance than similar experiments with independent culture and mRNA purification components, possibly due to the elimination of error during transfer. mRNA purified with the integrated device had a yield and purity similar to “gold standard” kits. Breast cancer cells co-cultured with bone marrow stromal cells showed increased proliferation and morphological changes relative to breast cancer cells cultured alone. Additionally, mRNA extracted from these cells using the integrated device showed transcriptional changes consistent with estrogen response, even in hormone-independent conditions. We have developed a new platform linking compartmentalized cell co-culture with NA isolation. This technology simplifies and accelerates two processes ubiquitous in cancer biology, enabling the collection of additional endpoints with finite resources while reducing error associated with manipulation. Furthermore, co-cultures of breast cancer with stroma from a metastatic site induced proliferation and transcriptional signaling associated with pro-growth conditions, illustrating the utility of this platform for studying the metastatic microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4272. doi:1538-7445.AM2012-4272

Abstract 2373: An integrated approach for isolating CTCs to enable the interrogation of cellular heterogeneity using IFAST

Abstract Background: The isolation of circulating tumor cells (CTCs) from whole blood is enabling for the study of malignant cells from patients with metastatic disease through a minimally invasive procedure. The analytical methods following the isolation of CTCs can be used to gain information about the disease, but require a highly purified population of cells that are minimally disturbed from the isolation procedures. While enumeration of CTCs during therapy has been shown to predict overall survival in men with metastatic prostate cancer, current methodology is limited in its ability to obtain information about these cells beyond count. Recent findings also suggest that CTC populations are heterogeneous (e.g. EMT, stem cell markers), and enumeration of CTCs based solely on EpCAM surface expression does not permit evaluation of CTC subpopulations. Therefore, methods that can isolate these cells based on their heterogeneity and further evaluate surface markers in an integrated platform will be beneficial in progressing the clinical relevance of CTC collection. Method: We present a method to sequentially isolate cells from a single input sample by cellular surface markers. Using immiscible filtration assisted by surface tension (IFAST) technology, we have demonstrated the ability to purify cells from a mixed cell population while maintaining the viability of these cells. The principle of the technology is that a high interfacial energy between the cell sample and oil creates a barrier that is normally impassable due to surface tension. Magnetic beads are added that have antibodies specific to a cellular surface marker and bind to the cells. A magnetic force is then applied to move the cells to and across the interface between the fluid and the oil, leaving anything unbound to the beads behind, thus isolating the cells of interest. CTC isolation is challenging due to potential cell loss in pipette transfer and wash steps. Using the IFAST device, we are able to add whole buffycoat into the input of the device and sequentially isolate, stain, and image CTCs without any pipette transfer or wash steps. Results: We have demonstrated the ability to have a single source solution in which two unique cell populations are present representing a basal and luminal cell type (DU145s, LNCaPs) and demonstrate the ability to individually isolate each of the two populations using a cellular surface antibody unique to one of the cell types (DU145 - CD44, LNCaPs - EpCAM) from a buffycoat containing high background PBMCs with greater than 80% of the resulting cells being the cell of interest in each situation, and demonstrate immunostaining of the isolated cell population (vimentin, HLA-A2). Conclusions: IFAST technology can be used to isolate multiple fractions from a single buffycoat solution using sequential bead additions and pull-throughs, demonstrating an advantage of this technology in the assessment of CTC heterogeneity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2373. doi:1538-7445.AM2012-2373

Facile and rapid DNA extraction and purification from food matrices using IFAST (immiscible filtration assisted by surface tension)

Extraction and purification of DNA is a prerequisite to detection and analytical techniques. While DNA sample preparation methods have improved over the last few decades, current methods are still time consuming and labor intensive. Here we demonstrate a technology termed IFAST (Immiscible Filtration Assisted by Surface Tension), that relies on immiscible phase filtration to reduce the time and effort required to purify DNA. IFAST replaces the multiple wash and centrifugation steps required by traditional DNA sample preparation methods with a single step. To operate, DNA from lysed cells is bound to paramagnetic particles (PMPs) and drawn through an immiscible fluid phase barrier (i.e. oil) by an external handheld magnet. Purified DNA is then eluted from the PMPs. Here, detection of Clostridium botulinum type A (BoNT/A) in food matrices (milk, orange juice), a bioterrorism concern, was used as a model system to establish IFAST's utility in detection assays. Data validated that the DNA purified by IFAST was functional as a qPCR template to amplify the bont/A gene. The sensitivity limit of IFAST was comparable to the commercially available Invitrogen ChargeSwitch® method. Notably, pathogen detection via IFAST required only 8.5 μL of sample and was accomplished in five-fold less time. The simplicity, rapidity and portability of IFAST offer significant advantages when compared to existing DNA sample preparation methods.

Purification of cell subpopulations via immiscible filtration assisted by surface tension (IFAST)

The selective isolation of a sub-population of cells from a larger, mixed population is a critical preparatory process to many biomedical assays. Here, we present a new cell isolation platform with a unique set of advantages over existing devices. Our technology, termed Immiscible Filtration Assisted by Surface Tension, exploits physical phenomena associated with the microscale to establish fluidic barriers composed of immiscible liquids. By attaching magnetically-responsive particles to a target cell population via immunocapture, we can selectively transport this population across the immiscible barrier and into a separate aqueous solution. The high interfacial energy associated with the immiscible phase / aqueous phase boundaries prevents unwanted cells or other contaminants from inadvertently crossing the immiscible phase. We have demonstrated, using fluorescent particles, stromal cells, and whole blood as "background", that we can successfully isolate ~70% of a target breast cancer cell population with an average purity of >80%. Increased purity was obtained by coupling two immiscible barriers in series, a modification that only slightly increases operational complexity. Furthermore, several samples can be processed in parallel batches in a near-instantaneous manner without the requirement of any washing, which can cause dilution (negative selection) or significant uncontrolled loss (positive selection) of target cells. Finally, cells were observed to remain viable and proliferative following traverse through the immiscible phase, indicating that this process is suitable for a variety of downstream assays, including those requiring intact living cells.

One-step purification of nucleic acid for gene expression analysis via Immiscible Filtration Assisted by Surface Tension (IFAST)

The extraction and purification of nucleic acids from complex samples (e.g. blood, biopsied tissue, cultured cells, food) is an essential prerequisite for many applications in biology including genotyping, transcriptional analysis, systems biology, epigenetic analysis, and virus/bacterial detection. In this report, we describe a new process of nucleic acid extraction that utilizes "pinned" aqueous/organic liquid interfaces in microchannels to streamline the extraction mechanism, replacing all washing steps with a single traverse of an immiscible fluid barrier, termed Immiscible Filtration Assisted by Surface Tension (IFAST). Nucleic acids in biological samples are bound to paramagnetic particles and then drawn across the IFAST device (or array of IFAST devices) using a magnet. While the strength of the IFAST barrier is suitable for separation of nucleic acids from lysate in its current embodiment, its permeability can be selectively adapted by adjusting the surface tensions/energies associated with the cell lysate, the immiscible phase, and the device surface, enabling future expansion to other non-nucleic acid applications. Importantly, processing time is reduced from 15-45 minutes to less than 5 minutes while maintaining purity, yield, and scalability equal to or better than prevailing methods. Operation is extremely simple and no additional lab infrastructure is required. The IFAST technology thus significantly enhances researchers' abilities to isolate and analyze nucleic acids, a process which is critical and ubiquitous in an extensive array of scientific fields.