Hydrodynamic Flow Confinement Research Articles

Localized hydrodynamic flow confinement assisted nanowire sensor for ultrasensitive protein detection

Affordable methods for ultra-sensitive biomarkers detection may improve the standard of living in resource-constrained countries. Nanowire biosensor is preponderant in ultra-sensitive protein detection. However, current strategies for nanowire sensor (NWS) fabrication often require sophisticated instruments, being inaccessible in less-resourced laboratories. Herein, we circumvent this challenge by developing a simple methodology, localized hydrodynamic flow confinement assisted nanowire sensor fabrication, enable the detection with limits of detection (LOD) for IgA and IgG measurement were 0.089 fg/mL and 0.93 fg/mL, respectively, demonstrating a 10-fold increase in detection sensitivity compared with the published NWS. Noteworthy, an X–Y positioner combined with a homemade microchemical pen (MCP) for tunable chemical deposition were sufficient to complete the fabrication of the nanowire biosensor without other expensive and demanding equipment. Overall, a particularly accessible, competitive, and low-cost approach of nanowire sensor fabrication for ultra-sensitive protein detection was developed, which could widely facilitate the application of nanowire biosensors. Besides, the nanowire sensor can also be employed to detect other analytes of interest by the use of different stimuli-responsive biosystems.

Localized Hydrodynamic Flow Confinement Assisted Nanowire Sensor for Ultrasensitive Protein Detection

Localized Hydrodynamic Flow Confinement Assisted Nanowire Sensor for Ultrasensitive Protein Detection

Spatially Resolved Genetic Analysis of Tissue Sections Enabled by Microscale Flow Confinement Retrieval and Isotachophoretic Purification.

We have developed a method for spatially resolved genetic analysis of formalin-fixed paraffin-embedded (FFPE) cell block and tissue sections. This method involves local sampling using hydrodynamic flow confinement of a lysis buffer, followed by electrokinetic purification of nucleic acids from the sampled lysate. We characterized the method by locally sampling an array of points with a circa 200 μm diameter footprint, enabling the detection of single KRAS and BRAF point mutations in small populations of RKO and MCF-7 FFPE cell blocks. To illustrate the utility of this approach for genetic analysis, we demonstrate spatially resolved genotyping of FFPE sections of human breast invasive ductal carcinoma.

Spatially Resolved Genetic Analysis of Tissue Sections Enabled by Microscale Flow Confinement Retrieval and Isotachophoretic Purification

AbstractWe have developed a method for spatially resolved genetic analysis of formalin‐fixed paraffin‐embedded (FFPE) cell block and tissue sections. This method involves local sampling using hydrodynamic flow confinement of a lysis buffer, followed by electrokinetic purification of nucleic acids from the sampled lysate. We characterized the method by locally sampling an array of points with a circa 200 μm diameter footprint, enabling the detection of single KRAS and BRAF point mutations in small populations of RKO and MCF‐7 FFPE cell blocks. To illustrate the utility of this approach for genetic analysis, we demonstrate spatially resolved genotyping of FFPE sections of human breast invasive ductal carcinoma.

Deep-Reaching Hydrodynamic Flow Confinement: Micrometer-Scale Liquid Localization for Open Substrates With Topographical Variations.

We present a method for nonintrusive localization and reagent delivery on immersed biological samples with topographical variation on the order of hundreds of micrometers. Our technique, which we refer to as the deep-reaching hydrodynamic flow confinement (DR-HFC), is simple and passive: it relies on a deep-reaching hydrodynamic confinement delivered through a simple microfluidic probe design to perform localized microscale alterations on substrates as deep as 600 μm. Designed to scan centimeter-scale areas of biological substrates, our method passively prevents sample intrusion by maintaining a large gap between the probe and the substrate. The gap prevents collision of the probe and the substrate and reduces the shear stress experienced by the sample. We present two probe designs: linear and annular DR-HFC. Both designs comprise a reagent-injection aperture and aspiration apertures that serve to confine the reagent. We identify the design parameters affecting reagent localization and depth by DR-HFC and study their individual influence on the operation of DR-HFC numerically. Using DR-HFC, we demonstrate localized binding of antihuman immunoglobulin G (IgG) onto an activated substrate at various depths from 50 to 600 μm. DR-HFC provides a readily implementable approach for noninvasive processing of biological samples applicable to the next generation of diagnostic and bioanalytical devices.

Microfluidic Probes and Quadrupoles: A new era of open microfluidics

The microfluidic probe (MFP) is a noncontact technology that applies the concept of hydrodynamic flow confinement (HFC) within a small gap to eliminate the need for closed microfluidic conduits and, therefore, overcome the conventional closed-system microfluidic limitation. Since its invention, the concept has experienced continuing advancement with several applications, ranging from manipulating mammalian cells and printing protein arrays to performing microfabrication.

Centimeter-Scale Surface Interactions Using Hydrodynamic Flow Confinements.

We present a device and method for selective chemical interactions with immersed substrates at the centimeter-scale. Our implementations enable both, sequential and simultaneous delivery of multiple reagents to a substrate, as well as the creation of gradients of reagents on surfaces. The method is based on localizing submicroliter volumes of liquids on an immersed surface with a microfluidic probe (MFP) using a principle termed hydrodynamic flow confinement (HFC). We here show spatially defined, multiplexed surface interactions while benefiting from the probe capabilities such as non-contact scanning operation and convection-enhanced reaction kinetics. Three-layer glass-Si-glass probes were developed to implement slit-aperture and aperture-array designs. Analytical and numerical analysis helped to establish probe designs and operating parameters. Using these probes, we performed immunohistochemical analysis on individual cores of a human breast-cancer tissue microarray. We applied α-p53 antibodies on a 2 mm diameter core within 2.5 min using a slit-aperture probe (HFC dimension: 0.3 mm × 1.2 mm). Further, multiplexed treatment of a tissue core with α-p53 and α–β-actin antibodies was performed using four adjacent HFCs created with an aperture-array probe (HFC dimension: 4 × 0.3 mm × 0.25 mm). The ability of these devices and methods to perform multiplexed assays, present sequentially different liquids on surfaces, and interact with surfaces at the centimeter-scale will likely spur new and efficient surface assays.

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe.

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

Selective local lysis and sampling of live cells for nucleic acid analysis using a microfluidic probe.

Heterogeneity is inherent to biology, thus it is imperative to realize methods capable of obtaining spatially-resolved genomic and transcriptomic profiles of heterogeneous biological samples. Here, we present a new method for local lysis of live adherent cells for nucleic acid analyses. This method addresses bottlenecks in current approaches, such as dilution of analytes, one-sample-one-test, and incompatibility to adherent cells. We make use of a scanning probe technology - a microfluidic probe - and implement hierarchical hydrodynamic flow confinement (hHFC) to localize multiple biochemicals on a biological substrate in a non-contact, non-destructive manner. hHFC enables rapid recovery of nucleic acids by coupling cell lysis and lysate collection. We locally lysed ~300 cells with chemical systems adapted for DNA or RNA and obtained lysates of ~70 cells/μL for DNA analysis and ~15 cells/μL for mRNA analysis. The lysates were introduced into PCR-based workflows for genomic and transcriptomic analysis. This strategy further enabled selective local lysis of subpopulations in a co-culture of MCF7 and MDA-MB-231 cells, validated by characteristic E-cadherin gene expression in individually extracted cell types. The developed strategy can be applied to study cell-cell, cell-matrix interactions locally, with implications in understanding growth, progression and drug response of a tumor.

Two-Aperture Microfluidic Probes as Flow Dipole: Theory and Applications.

A microfluidic probe (MFP) is a mobile channel-less microfluidic system under which a fluid is injected from an aperture into an open space, hydrodynamically confined by a surrounding fluid, and entirely re-aspirated into a second aperture. Various MFPs have been developed, and have been used for applications ranging from surface patterning of photoresists to local perfusion of organotypic tissue slices. However, the hydrodynamic and mass transfer properties of the flow under the MFP have not been analyzed, and the flow parameters are adjusted empirically. Here, we present an analytical model describing the key transport properties in MFP operation, including the dimensions of the hydrodynamic flow confinement (HFC) area, diffusion broadening, and shear stress as a function of: (i) probe geometry (ii) aspiration-to-injection flow rate ratio (iii) gap between MFP and substrate and (iv) reagent diffusivity. Analytical results and scaling laws were validated against numerical simulations and experimental results from published data. These results will be useful to guide future MFP design and operation, notably to control the MFP “brush stroke” while preserving shear-sensitive cells and tissues.

Systematic analysis of microfluidic probe design and operation.

Microfluidic probes are an emerging tool used in a wide range of applications including surface biopatterning, immunohistology, and cell migration studies. They control flow above a surface by simultaneously injecting and aspirating fluids from a pen-like structure positioned a few tens of microns above a surface. Rather than confining flows inside microchannels they rely on recirculating flow patterns between the probe tip and the substrate to create a hydrodynamic flow confinement (HFC) zone in which reagents can be locally delivered to the surface. In this paper, we provide a theoretical model, supported by numerical simulations and experimental data, describing the extent of the HFC as a function of the two most important probe operation parameters, the ratio of aspiration to injection flow rate, and the distance between probe apertures. Two types of probes are studied: two-aperture microfluidic probes (MFPs) and microfluidic quadrupoles (MQs). In both cases, the model yields very accurate results and suggests a simple underlying theory based on 2D potential flows to understand probe operation. We further highlight how the model can be used to precisely control the probe's "brush stroke" while in surface patterning mode. The understanding of probe operation made possible through the provided analytical model should lay the bases for computer-controlled probe calibration and operation.

Hierarchical Hydrodynamic Flow Confinement: Efficient Use and Retrieval of Chemicals for Microscale Chemistry on Surfaces

We devised, implemented, and tested a new concept for efficient local surface chemistry that we call hierarchical hydrodynamic flow confinement (hierarchical HFC). This concept leverages the hydrodynamic shaping of multiple layers of liquid to address challenges inherent to microscale surface chemistry, such as minimal dilution, economical consumption of reagent, and fast liquid switching. We illustrate two modes of hierarchical HFC, nested and pinched, by locally denaturing and recovering a 26 bp DNA with as little as 2% dilution and by efficiently patterning an antibody on a surface, with a 5 μm resolution and a 100-fold decrease of reagent consumption compared to microcontact printing. In addition, valveless switching between nanoliter volumes of liquids was achieved within 20 ms. We believe hierarchical HFC will have broad utility for chemistry on surfaces at the microscale.

ChemInform Abstract: Microfluidics in the “Open Space” for Performing Localized Chemistry on Biological Interfaces

AbstractReview: 74 refs.

Microfluidic probes for use in life sciences and medicine

Microfluidic probes (MFPs) combine the concepts of microfluidics and of scanning probes and constitute a contact-free and channel-free microfluidic system. Whereas classically the sample is introduced into the microfluidic device, with a MFP, the microfluidic stream is applied to the sample. MFPs use hydrodynamic flow confinement instead of walls to constrain a microfluidic stream between the MFP tip and a substrate. Because MFPs are free to move, they can be used to process large areas and samples in a selective manner. The development of MFP technology is recent and has numerous potential applications in several fields, most notably in the life sciences. In this review, we discuss the concept of MFPs and highlight their application in surface biopatterning, controlling the cellular microenvironments, local processing of tissue slices, and generating concentration gradients of biochemicals. We hope that this manuscript will serve as an interdisciplinary guide for both engineers as they further develop novel MFPs and applications and for life scientists who may identify novel uses of the MFP for their research.

Multilayered microfluidic probe heads

Microfluidics have emerged as powerful processing and analytical tools. However, as they are usually closed systems, they are difficult to use with large samples and surfaces that cannot pass inside their conduits. Here, we extend our previously reported microfluidic probe (MFP) concept, in which a focused stream of liquid is scanned over a surface, for which heads were fabricated using one Si wafer and a molded poly(dimethylsiloxane) (PDMS) block. The new heads comprise a microfabricated Si lid, a chip for hydrodynamic flow confinement (HFC) and standard ports and fittings. The HFC chip carries the smallest structures needed for focusing the liquid on the surface; the Si lid is bonded to the HFC chip using a polyimide adhesive and has macroscopic vias for bonding standard ports and fittings. These heads are easy to fabricate at high yield, have high-resolution potential and are compatible with a large number of chemicals and solvents. We exemplify this by patterning proteins on a surface, directly developing a resist using a high-pH developer, and producing tapered structures in a resist. This work should foster the adoption of MFPs for processing, patterning and analyzing surfaces under microfluidic conditions.