Display omitted
•Vertical flow assays (VFAs) with multistep reactions were performed in a 3D paper-based microfluidic device.•Programmed loading of all reagents into the analysis region was achieved ...by utilizing a delayed flow in a pressed paper.•Upper limit and sensitivity of the assays were increased without Hook effect compared to lateral flow assays (LFAs).•C-Reactive protein was detected for the prediction of cardiovascular disease in a clinical range (0.005–5μg/mL).
Although vertical flow assays (VFAs) have a number of advantages compared to lateral flow assays (LFAs) such as a short analysis time, no line interference, and no Hook effect, VFAs are not preferred as LFAs because of their complicated operation principle. In this study, we demonstrated VFAs with multistep reactions for the detection of C-reactive protein (CRP) based on the programmed reagent loading in a pressed region integrated 3D paper-based microfluidic device. The flow order of all reagents in a 3D paper-based microfluidic device was programmed based on the delayed flow caused by the pressed region as well as the geometry modification of the paper channel. After simultaneous loading of all the reagents required for assays, they are sequentially loaded into the analysis region with a programmed sequence. As a proof of concept, a high-sensitivity CRP (hs-CRP) detection with signal amplification was performed to predict the riskiness of cardiovascular disease within 15min. The detection limit was improved from 0.01 to 0.005μg/mL via a maximum 3.47-fold signal amplification. Additionally, the upper limit of hs-CRP detection increased to 5μg/mL without Hook effect. Finally, we successfully detected hs-CRP in a clinically relevant range (0.005–5μg/mL), while LFAs cannot cover due to the Hook effect.
This paper presents a rotary device designed for facile delivery of multiple reagents to a paper strip for multistep assays. Its purpose is to allow users to easily perform multistep assays and ...achieve sensitive detection. While the test strip remains stationary, rotating the top piece of the device aligns the reagent and absorbent pads to each end of the paper strip and initiates fluid flow. Further incremental rotation makes an adjacent pair of pads to align simultaneously, causing fluid flow of subsequent reagent that was preloaded in the reagent pad. In this work, various porous substrates were tested to observe their effect on overall flow rate of the system and multistep assays were performed to demonstrate its simple use. As a proof of concept, enzyme-linked immunosorbent assay was carried out to detect Escherichia coli O157:H7.
The development of a cost-effective and efficient bacterial detection assay is essential for diagnostic fields, particularly in resource-poor settings. Although antibodies have been widely used for ...bacterial capture, the production of soluble antibodies is still expensive and time-consuming. Here, we developed a nitrocellulose-based lateral flow assay using cell wall binding domains (CBDs) from phage as a recognition element and colloidal gold nanoparticles as a colorimetric signal for the detection of a model pathogenic bacterium, Bacillus cereus (B. cereus). To improve conjugation efficiency and detection sensitivity, cysteine-glutathione-S-transferase-tagged CBDs and maltose-binding protein-tagged CBDs were produced in Escherichia coli (E. coli) and incorporated in our assays. The sensitivity of the strip to detect B. cereus was 1×104 CFU/mL and the overall assay time was 20min. The assay showed superior results compared to the antibody-based approach, and did not show any significant cross-reactivity. This proof of concept study indicates that the lateral flow assay using engineered CBDs hold considerable promise as simple, rapid, and cost-effective biosensors for whole cell detection.
•The cell wall binding domain (CBD) of endolysin was tested for whole cell detection.•A lateral flow assay using CBD as a recognition element was developed.•The assay showed promising results for whole cell detection.
This paper presents a pressed paper-based dipstick that enables detection of foodborne pathogens with multistep reactions by exploiting the delayed fluid flow and channel partition formation on ...nitrocellulose (NC) membrane. Fluid behaviors are easily modified by controlling the amount of pressure and the position of pressed region on the NC membrane. Detection region of the dipstick is optimized by controlling flow rate and delayed time based on Darcy's law. All the reagents required for assay are dried on the NC membrane and they are sequentially rehydrated at the prepartitioned regions when the device is dipped into sample solution. In this manner, multistep reactions can be facilitated by one-step dipping of the dipstick into the sample solution. As a proof of concept, we performed detection of two fatal foodborne pathogens (e.g., Escherichia coli O157:H7 and Salmonella typhimurium) with signal enhancement. In addition, we expanded the utilization of channel partitions by developing a pressed paper-based dipstick into dual detection format.
Although the hanging drop methods have a number of advantages for spheroid culture, they suffer from reagent exchange procedures that depend on tedious and accurate liquid handling by manual ...pipetting or robotic arms. To simplify these procedures, we developed a method for liquid handling in a hanging drop array (HDA) chip for spheroid culture and analysis by integrating microfluidic channels operated by pushbuttons. Six finger-actuated microfluidic pumping units connected to a 3 × 3 HDA can draw or replenish reagents in an HDA chip without any external equipment. The initial cell seeding, medium exchange, and staining for further analysis can be simply done by pushing the buttons in the programmed order. After the assessment of the reagent exchange ratio of the device, BT474 spheroids of various sizes were cultured in the device for 7 days by exchanging the medium once a day and stained on the same device by exchanging the medium with staining reagents for the LIVE/DEAD assay. Furthermore, the cultured spheroids were embedded into collagen by exchanging the medium with a collagen solution to mimic a cancer metastasis environment.
Although immunomagnetic separation is a useful sample pretreatment method that can be used to separate target pathogens from a raw sample, it is challenging to remove unbound free magnetic ...nanoparticles (MNPs) for colorimetric detection of target pathogens. Here, size-based filtration was exploited for the rapid on-site detection of pathogens separated by immunomagnetic separation in order to remove unbound free MNPs using a finger-powered microfluidic device. A membrane filter and an absorbent pad were integrated into the device and a mixture of unbound free MNPs and MNP-bound
(
) O157:H7 was dispensed over the membrane filter by pressing and releasing the pressure chamber. A colorimetric signal was generated by MNP-bound
O157:H7 while unbound free MNPs were washed out by the absorbent. Furthermore, the colorimetric signals can be amplified using a gold enhancer solution when gold-coated MNPs were used instead of MNPs. As a result, 10
CFU/mL
O157:H7 could be detected by the enhanced colorimetric signal on a proposed device.
We report a contraction-expansion array (CEA) microchannel device that performs label-free high-throughput separation of cancer cells from whole blood at low Reynolds number (Re). The CEA ...microfluidic device utilizes hydrodynamic field effect for cancer cell separation, two kinds of inertial effects: (1) inertial lift force and (2) Dean flow, which results in label-free size-based separation with high throughput. To avoid cell damages potentially caused by high shear stress in conventional inertial separation techniques, the CEA microfluidic device isolates the cells with low operational Re, maintaining high-throughput separation, using nondiluted whole blood samples (hematocrit ~45%). We characterized inertial particle migration and investigated the migration of blood cells and various cancer cells (MCF-7, SK-BR-3, and HCC70) in the CEA microchannel. The separation of cancer cells from whole blood was demonstrated with a cancer cell recovery rate of 99.1%, a blood cell rejection ratio of 88.9%, and a throughput of 1.1 × 10(8) cells/min. In addition, the blood cell rejection ratio was further improved to 97.3% by a two-step filtration process with two devices connected in series.
We report a contraction–expansion array (CEA) microchannel that allows inertial size separation by a force balance between inertial lift and Dean drag forces in fluid regimes in which inertial fluid ...effects become significant. An abrupt change of the cross-sectional area of the channel curves fluid streams and produces a similar effect compared to Dean flows in a curved microchannel of constant cross-section, thereby inducing Dean drag forces acting on particles. In addition, the particles are influenced by inertial lift forces throughout the contraction regions. These two forces act in opposite directions each other throughout the CEA microchannel, and their force balancing determines whether the particles cross the channel, following Dean flows. Here we describe the physics and design of the CEA microfluidic device, and demonstrate complete separation of microparticles (polystyrene beads of 4 and 10
μm in diameter) and efficient exchange of the carrier medium while retaining 10
μm beads.
Current organ‐on‐a‐chip technologies confront limitations in effectively recapitulating the intricate in vivo microenvironments and accommodating diverse experimental conditions on a single device. ...Here, a novel approach for constructing a multi‐composition tumor array on a single microfluidic device, mimicking complex transport phenomena within tumor microenvironments (TMEs) and allowing for simultaneous evaluation of drug efficacy across 12 distinct conditions is presented. The TME array formed by bioprinting on a microfluidic substrate consists of 36 individual TME models, each characterized by one of three different compositions and tested under four varying drug concentrations. Notably, the TME model exhibits precise compartmentalization, fostering the development of self‐organized vascular endothelial barriers surrounding breast cancer spheroids affecting substance transport. Multivariable screening and analysis of diverse conditions, including model complexity, replicates, and drug concentrations, within a single microfluidic platform, highlight the synergistic potential of integrating bioprinting with microfluidics to evaluate drug responses across diverse TME conditions comprehensively.
A novel approach is reported by integrating bioprinting and microfluidic technologies to create a multi‐composition tumor array that mimics the complex transport in the tumor microenvironment (TME). This approach enables the self‐organized formation of a vascular barrier around tumor spheroids and comprehensively evaluates drug efficacy across different conditions of TME models.
One of the challenges in tissue engineering is to provide adequate supplies of oxygen and nutrients to cells within the engineered tissue construct. Soft-lithographic techniques have allowed the ...generation of hydrogel scaffolds containing a network of fluidic channels, but at the cost of complicated and often time-consuming manufacturing steps. We report a three-dimensional (3D) direct printing technique to construct hydrogel scaffolds containing fluidic channels. Cells can also be printed on to and embedded in the scaffold with this technique. Collagen hydrogel precursor was printed and subsequently crosslinked via nebulized sodium bicarbonate solution. A heated gelatin solution, which served as a sacrificial element for the fluidic channels, was printed between the collagen layers. The process was repeated layer-by-layer to form a 3D hydrogel block. The printed hydrogel block was heated to 37°C, which allowed the gelatin to be selectively liquefied and drained, generating a hollow channel within the collagen scaffold. The dermal fibroblasts grown in a scaffold containing fluidic channels showed significantly elevated cell viability compared to the ones without any channels. The on-demand capability to print fluidic channel structures and cells in a 3D hydrogel scaffold offers flexibility in generating perfusable 3D artificial tissue composites. Biotechnol. Bioeng. 2010;105: 1178-1186.
PDF
