Exosomes carry microRNA biomarkers, occur in higher abundance in cancerous patients than in healthy ones, and because they are present in most biofluids, including blood and urine, these can be ...obtained noninvasively. Standard laboratory techniques to isolate exosomes are expensive, time consuming, provide poor purity, and recover on the order of 25% of the available exosomes. We present a new microfluidic technique to simultaneously isolate exosomes and preconcentrate them by electrophoresis using a high transverse local electric field generated by ion‐depleting ion‐selective membrane. We use pressure‐driven flow to deliver an exosome sample to a microfluidic chip such that the transverse electric field forces them out of the cross flow and into an agarose gel which filters out unwanted cellular debris while the ion‐selective membrane concentrates the exosomes through an enrichment effect. We efficiently isolated exosomes from 1× PBS buffer, cell culture media, and blood serum. Using flow rates from 150 to 200 μL/h and field strengths of 100 V/cm, we consistently captured between 60 and 80% of exosomes from buffer, cell culture media, and blood serum as confirmed by both fluorescence spectroscopy and nanoparticle tracking analysis. Our microfluidic chip maintained this recovery rate for more than 20 min with a concentration factor of 15 for 10 min of isolation.
Pathological degeneration of axons disrupts neural circuits and represents one of the hallmarks of neurodegeneration
. Sterile alpha and Toll/interleukin-1 receptor motif-containing protein 1 (SARM1) ...is a central regulator of this neurodegenerative process
, and its Toll/interleukin-1 receptor (TIR) domain exerts its pro-neurodegenerative action through NADase activity
. However, the mechanisms by which the activation of SARM1 is stringently controlled are unclear. Here we report the cryo-electron microscopy structures of full-length SARM1 proteins. We show that NAD
is an unexpected ligand of the armadillo/heat repeat motifs (ARM) domain of SARM1. This binding of NAD
to the ARM domain facilitated the inhibition of the TIR-domain NADase through the domain interface. Disruption of the NAD
-binding site or the ARM-TIR interaction caused constitutive activation of SARM1 and thereby led to axonal degeneration. These findings suggest that NAD
mediates self-inhibition of this central pro-neurodegenerative protein.
Liquid biopsy, screening cancer non‐invasively and frequently by detecting and quantifying molecular markers in physiological fluids, would significantly improve cancer survival rate but it remains a ...distant goal. The key obstacles presented by the highly heterogeneous samples are rapid/high‐yield purification and precise/selective marker capture by their antibody and oligo probes. As irregular expressions of these molecular biomarkers are the key signals, quantifying only those from the cancer cells would greatly enhance the performance of the screening tests. The recent discovery that the biomarkers are carried by nanocarriers, such as exosomes, with cell‐specific membrane proteins suggests that such selection may be possible, although a new suite of fractionation and quantification technologies would need to be developed. Although under‐appreciated, membrane microfluidics has made considerable contributions to resolving these issues. We review the progress made so far, based on ion‐selective, track‐etched, and gel membranes and advanced electrophoretic and nano‐filtration designs, in this perspective and suggest future directions.
Microfluidic Devices for Bioapplications Yeo, Leslie Y.; Chang, Hsueh-Chia; Chan, Peggy P. Y. ...
Small (Weinheim an der Bergstrasse, Germany),
January 3, 2011, Letnik:
7, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Harnessing the ability to precisely and reproducibly actuate fluids and manipulate bioparticles such as DNA, cells, and molecules at the microscale, microfluidics is a powerful tool that is currently ...revolutionizing chemical and biological analysis by replicating laboratory bench‐top technology on a miniature chip‐scale device, thus allowing assays to be carried out at a fraction of the time and cost while affording portability and field‐use capability. Emerging from a decade of research and development in microfluidic technology are a wide range of promising laboratory and consumer biotechnological applications from microscale genetic and proteomic analysis kits, cell culture and manipulation platforms, biosensors, and pathogen detection systems to point‐of‐care diagnostic devices, high‐throughput combinatorial drug screening platforms, schemes for targeted drug delivery and advanced therapeutics, and novel biomaterials synthesis for tissue engineering. The developments associated with these technological advances along with their respective applications to date are reviewed from a broad perspective and possible future directions that could arise from the current state of the art are discussed.
Microfluidics, which entails the manipulation of fluids and bioparticles at micron and submicron dimensions, is a promising technology that could potentially revolutionize an entire spectrum of chemical and biological processes. This review provides an overview of current technological advances and developments to date across wide and varied themes in medicine and biotechnology from genomic, proteomic, and cellomic analysis to drug delivery, drug screening, and point‐of‐care diagnostics.
Solid-state nanopores allow high-throughput single-molecule detection but identifying and even registering all translocating small molecules remain key challenges due to their high translocation ...speeds. We show here the same electric field that drives the molecules into the pore can be redirected to selectively pin and delay their transport. A thin high-permittivity dielectric coating on bullet-shaped polymer nanopores permits electric field leakage at the pore tip to produce a voltage-dependent surface field on the entry side that can reversibly edge-pin molecules. This mechanism renders molecular entry an activated process with sensitive exponential dependence on the bias voltage and molecular rigidity. This sensitivity allows us to selectively prolong the translocation time of short single-stranded DNA molecules by up to 5 orders of magnitude, to as long as minutes, allowing discrimination against their double-stranded duplexes with 97% confidence.
Current biomarkers for myocardial infarction (MI) diagnosis are typically late markers released upon cell death, incapable of distinguishing between ischemic and reperfusion injury and can be ...symptoms of other pathologies. Circulating microRNAs (miRNAs) have recently been proposed as alternative biomarkers for MI diagnosis; however, detecting the changes in the human cardiac miRNA profile during MI is extremely difficult. Here, to study the changes in miRNA levels during acute MI, a heart‐on‐chip model with a cardiac channel, containing human induced pluripotent stem cell (hiPSC)‐derived cardiomyocytes in human heart decellularized matrix and collagen, and a vascular channel, containing hiPSC‐derived endothelial cells, is developed. This model is exposed to anoxia followed by normoxia to mimic ischemia and reperfusion, respectively. Using a highly sensitive miRNA biosensor that the authors developed, the exact same increase in miR‐1, miR‐208b, and miR‐499 levels in the MI‐on‐chip and the time‐matched human blood plasma samples collected before and after ischemia and reperfusion, is shown. That the surface marker profile of exosomes in the engineered model changes in response to ischemic and reperfusion injury, which can be used as biomarkers to detect MI, is also shown. Hence, the MI‐on‐chip model developed here can be used in biomarker discovery.
In this study, a tissue‐engineered human heart anoxia and reperfusion tissue (HEART) model is developed and combined with a near real‐time miRNA concentration sensor to test the viability of miRNAs as novel heart attack biomarkers. As a proof of concept, it is also observed that the HEART has comparable miRNA concentrations and exosome profiles to time matched clinical samples.
The ion flux dynamics across a straight nanoslot is imaged to understand the nonequilibrium phenomenon of overlimiting current density across a nanoporous membrane. With a slow ac field, an ...ion-depletion front is generated intermittently from one end of the nanochannel, and a vortex instability first predicted by Rubinstein, Staude, and Kedem Desalination 69, 101 (1988).10.1016/0011-9164(88)80013-4 is found to arrest the self-similar diffusive front growth. This electrokinetic instability evolves into a stationary interfacial vortex array that specifies the overlimiting current, independent of external stirring or convective flow.
We report a bifurcated continuous field-flow fractionation (BCFFF) chip for high-yield and high-throughput (20 min) extraction of nucleic acids from physiological samples. The design uses a membrane ...ionic transistor to sustain low-ionic strength in a localized region at a junction, such that the resulting high field can selectively isolate high-charge density nucleic acids from the main flow channel and insert them into a standardized buffer in a side channel that bifurcates from the junction. The high local electric field and the bifurcated field-flow design facilitate concentration reduction of both divalent cation (Ca
2+
) and molecular PCR inhibitors by more than two orders of magnitude, even with high-throughput continuous loading. The unique design with a large (>20 mM mm
−1
) on-chip ionic-strength gradient allows miniaturization into a high-throughput field-flow fractionation chip that can be integrated with upstream lysing and downstream PCR/sensor modules for various nucleic acid detection/quantification applications. A concentration-independent 85% yield for extraction and an overall post-PCR yield exceeding 60% are demonstrated for a 111 bp dsDNA in 10 μL of human plasma, compared to no amplification with the raw sample. A net yield four times larger than a commercial extraction kit is demonstrated for miR-39 in human plasma.
We report a new Bifurcated Continuous Field-Flow Fractionation (BCFFF) microfluidic chip for isolation and purification of nucleic acids from blood plasma with high and concentration-independent yield. The platform is ideal for isolation and quantification of small miRNAs.
There has been increasing evidence that micro and messenger RNA derived from exosomes play important roles in pancreatic and other cancers. In this work, a microfluidics-based approach to the ...analysis of exosomal RNA is presented based on surface acoustic wave (SAW) exosome lysis and ion-exchange nanomembrane RNA sensing performed in conjunction on two separate chips. Using microRNA hsa-miR-550 as a model target and raw cell media from pancreatic cancer cell lines as a biological sample, SAW-based exosome lysis is shown to have a lysis rate of 38%, and an ion-exchange nanomembrane sensor is shown to have a limit of detection of 2 pM, with two decades of linear dynamic range. A universal calibration curve was derived for the membrane sensor and used to detect the target at a concentration of 13 pM in a SAW-lysed sample, which translates to 14 target miRNA per exosome from the raw cell media. At a total analysis time of ~1.5 h, this approach is a significant improvement over existing methods that require two overnight steps and 13 h of processing time. The platform also requires much smaller sample volumes than existing technology (~100 μL as opposed to ~mL) and operates with minimal sample loss, a distinct advantage for studies involving mouse models or other situations where the working fluid is scarce.
In this study, novel nanocomposite membranes were fabricated from single-walled carbon nanotubes (SWNTs) and a triptycene-containing polyimide. The desirable interfacial morphology and homogenous ...dispersion of SWNTs were achieved by strong π-π stacking and supramolecular shape-fitting interactions between the nanotube and the paddlewheel-shaped triptycene moieties in polymer matrix. Mixed-matrix membranes (MMMs) containing 2–15 wt% of as-purchased SWNTs (AP-SWNTs) were prepared and tested for their gas transport properties. While all MMMs showed greatly enhanced permeabilities with well-maintained selectivities, a non-linear dependence of permeability on filler content was observed. Another series of MMMs with purified SWNTs (P-SWNTs) and acid-treated SWNT (A-SWNTs) were also prepared. The functionalized A-SWNT MMM exhibited the best separation performance at 2 wt% filler content among all MMMs, which can be attributed to the improved interfacial affinity with carboxylic acid functionalized surface of A-SWNTs.
•Single-walled carbon nanotube (SWNTs) were incorporated into triptycene-based polyimide to fabricate mixed-matrix membranes.•Strong interfacial interaction achieved between SWNTs and triptycene moieties.•The effect of content, surface functionality, and aspect ratio of SWNTs were studied.•MMMs showed superior gas separation performance at low filler loading (2 wt%).
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