In nanobiotechnology, the importance of controlling interactions between biological molecules and surfaces is paramount. In recent years, many devices based on nanostructured silicon materials have been presented, such as nanopores and nanochannels. However, there is still a clear lack of simple, reliable, and efficient protocols for preventing and controlling biomolecule adsorption in such structures. In this work, we show a simple method for passivation or selective biofunctionalization of silica, without the need for polymerization reactions or vapor-phase deposition. The surface is simply exposed stepwise to three different chemicals over the course of ∼1 h. First, the use of aminopropylsilatrane is used to create a monolayer of amines, yielding more uniform layers than conventional silanization protocols. Second, a cross-linker layer and click chemistry are used to make the surface reactive toward thiols. In the third step, thick and dense poly(ethylene glycol) brushes are prepared by a grafting-to approach. The modified surfaces are shown to be superior to existing options for silica modification, exhibiting ultralow fouling (a few ng/cm2) after exposure to crude serum. In addition, by including a fraction of biotinylated polymer end groups, the surface can be functionalized further. We show that avidin can be detected label-free from a serum solution with a selectivity (compared to nonspecific binding) of more than 98% without the need for a reference channel. Furthermore, we show that our method can passivate the interior of 150 nm × 100 nm nanochannels in silica, showing complete elimination of adsorption of a sticky fluorescent protein. Additionally, our method is shown to be compatible with modifications of solid-state nanopores in 20 nm thin silicon nitride membranes and reduces the noise in the ion current. We consider these findings highly important for the broad field of nanobiotechnology, and we believe that our method will be very useful for a great variety of surface-based sensors and analytical devices.
Exosomes, nanovesicles derived from cells, contain a variety of biomolecules that can be considered biomarkers for disease diagnosis, including microRNAs (miRNAs). Given knowledge and demand, inexpensive, robust, and easy-to-use tools that are compatible with downstream nucleic acid detection should be developed to replace traditional methodologies for point-of-care testing (POCT) applications. This study deploys a paper-based extraction kit for exosome and exosomal miRNA analytical system with some quantifying methods to serve as an easy sample preparation for a possible POCT process. Exosomes concentrated from HCT116 cell cultures were arrested on paper-based immunoaffinity devices, which were produced by immobilizing anti-CD63 antibodies on Whatman filter paper, before being subjected to paper-based silica devices for nucleic acids to be trapped by silica nanoparticles adsorbed onto Whatman filter paper. Concentrations of captured exosomes were quantified by enzyme-linked immunosorbent assay (ELISA), demonstrating that paper-based immunoaffinity devices succeeded in capturing and determining exosome levels from cells cultured in both neutral and acidic microenvironments, whereas microRNA 21 (miR-21), a biomarker for various types of cancers and among the nucleic acids absorbed onto the silica devices, was determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) to prove that paper-based silica devices were capable of trapping exosomal nucleic acids. The developed paper-based kit and the devised procedure was successfully exploited to isolate exosomes and exosomal nucleic acids from different biological samples (platelet-poor plasma and lesion fluid) as clinical applications.
Emerging evidence has shown that microRNAs play pivotal roles in wound healing. MicroRNA-21 (miR-21) was previously found to upregulate in order to fulfill an anti-inflammation role for wounds. Exosomal miRNAs have been identified and explored as essential markers for diagnostic medicine. However, the role of exosomal miR-21 in wounds has yet to be well studied. In order to facilitate the early management of poorly healing wounds, we developed an easy-to-use, rapid, paper-based microfluidic-exosomal miR-21 extraction device to determine wound prognosis in a timely manner. We isolated and then quantitatively examined exosomal miR-21 in wound fluids from normal tissues and acute and chronic wounds. Eight improving wounds displayed lower levels of exosomal miR-21 expression after wound debridement. However, four instances of increased exosomal miR-21 expression levels were notably associated with patients with poor healing wounds despite aggressive wound debridement, indicating a predictive role of tissue exosomal miR-21 for wound outcome. Paper-based nucleic acid extraction device provides a rapid and user-friendly approach for evaluating exosomal miR-21 in wound fluids as a means of monitoring wounds. Our data suggest that tissue exosomal miR-21 is a reliable marker for determining current wound status.
Emerging evidence has shown that microRNAs play pivotal roles in wound healing. MicroRNA-21 (miR-21) was previously found to upregulate in order to fulfill an anti-inflammation role for wounds. Exosomal miRNAs have been identified and explored as essential markers for diagnostic medicine. However, the role of exosomal miR-21 in wounds has yet to be well studied. In order to facilitate the early management of poorly healing wounds, we developed an easy-to-use, rapid, paper-based microfluidic-exosomal miR-21 extraction device to determine wound prognosis in a timely manner. We isolated and then quantitatively examined exosomal miR-21 in wound fluids from normal tissues, acute and chronic wounds. Eight improving wounds displayed lower levels of exosomal miR-21 expression after wound debridement. However, four instances of increased exosomal miR-21 expression levels were notably associated with patients with poor healing wounds despite aggressive wound debridement, indicating a predictive role of tissue exosomal miR-21 for wound outcome. Paper-based nucleic acid extraction device provides a rapid and user-friendly approach for evaluating exosomal miR-21 in wound fluids as a means of monitoring wounds. Our data suggest that tissue exosomal miR-21 is a reliable marker for determining current wound status.
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