Active microfluidic reactor-assisted controlled synthesis of nanoparticles and related potential biomedical applications.

Fabricating high-performance nanoparticles (NPs) is currently a focus of researchers due to their manipulative size-dependent unique properties required to develop next-generation advanced systems. To harness the unique properties of NPs, maintaining identical characteristics throughout the processing and application process system is crucial to producing uniform-sized, or monodisperse, NPs. In this direction, mono-dispersity can be achieved by exerting extreme control over the reaction conditions during the NP synthesis process. Microfluidic technology offers a unique approach to control fluid conditions at the microscale and is thus well-positioned as an alternative strategy to synthesize NPs in reactors demonstrating micrometric dimensions and advanced size-controlled nanomaterial production. These microfluidic reactors can be broadly classified as active or passive based on their dependence on external energy sources. Passive microfluidic reactors, despite their lack of reliance on external energy, are frequently constrained in terms of their mixing efficacy when compared to active systems. However, despite several fundamental and technological advantages, this area of research as well as its application to the biological sciences is not well-discussed. To fill this gap, this review for the first time discusses various strategies for synthesizing NPs using active microfluidic reactors including acoustic, pressure, temperature, and magnetic assisted microfluidic reactors. Various established ways for achieving size control on NP synthesis in microfluidic reactors representing the applicability of micro-reaction technology in developing novel nanomaterials suitable for potential biomedical applications are presented in this review along with a comprehensive discussion about the challenges and prospects.

Bio-acceptability of wearable sensors: a mechanistic study towards evaluating ionic leaching induced cellular inflammation.

The recent need for remote health wellness monitoring has led to the extensive use of wearable sensors. Owing to their increased use, these sensors are required to exhibit both functionality and safety to the user. A major component in the fabrication of these sensors and their associated circuitry is the use of metallic/organic conductive inks. However, very less is known about the interfacial and molecular interactions of these inks with biological matter as they can result in an inflammatory reaction to the user. Significant efforts are thus needed to explore and improve the bio-acceptability of such conductive ink-based wearable sensors. The present study investigates the biocompatibility of encapsulated and non-encapsulated wearable electrochemical sensors used for sensing uric acid as a biomarker for wound healing fabricated using screen-printing technique. Ionic release of metallic ions was investigated first to understand the susceptibility of the conductive inks towards ionic leaching when in contact with a fluid. Time-lapse investigation using ICPS (inductive couple plasma spectroscopy) shows a high concentration (607.31 ppb) of leached silver (Ag+) ions from the non-encapsulated sensors. The cell viability data suggests a 2.5-fold improvement in the sensor biocompatibility for an encapsulated sensor. While the carbon ink shows negligible effect on cell viability, the silver ink elicits significant decrease (< 50%) in cell viability at concentrations higher than 2 mg ml-1. The toxicity pathway of these sensors was further determined to be through the generation of reactive oxygen species resulting in over 20% apoptotic cell death. Our results show that the lower biocompatibility of the non-encapsulated sensor attributes to the higher leaching of Ag+ ions from the printed inks which elicits several different inflammatory pathways. This work highlights the importance biocompatibility evaluation of the material used in sensor fabrication to develop safe and sustainable sensors for long-term applications.

New dynamic microreactor system to mimic biofilm formation and test anti-biofilm activity of nanoparticles.

Microbial biofilms are composed of surface-adhered microorganisms enclosed in extracellular polymeric substances. The biofilm lifestyle is the intrinsic drug resistance imparted to bacterial cells protected by the matrix. So far, conventional drug susceptibility tests for biofilm are reagent and time-consuming, and most of them are in static conditions. Rapid and easy-to-use methods for biofilm formation and antibiotic activity testing need to be developed to accelerate the discovery of new antibiofilm strategies. Herein, a Lab-On-Chip (LOC) device is presented that provides optimal microenvironmental conditions closely mimicking real-life clinical biofilm status. This new device allows homogeneous attachment and immobilization of Pseudomonas aeruginosa PA01-EGFP cells, and the biofilms grown can be monitored by fluorescence microscopy. P. aeruginosa is an opportunistic pathogen known as a model for drug screening biofilm studies. The influence of flow rates on biofilms growth was analyzed by flow simulations using COMSOL® 5.2. Significant cell adhesion to the substrate and biofilm formation inside the microchannels were observed at higher flow rates > 100 µL/h. After biofilm formation, the effectiveness of silver nanoparticles (SNP), chitosan nanoparticles (CNP), and a complex of chitosan-coated silver nanoparticles (CSNP) to eradicate the biofilm under a continuous flow was explored. The most significant loss of biofilm was seen with CSNP with a 65.5% decrease in average live/dead cell signal in biofilm compared to the negative controls. Our results demonstrate that this system is a user-friendly tool for antibiofilm drug screening that could be simply applied in clinical laboratories.Key Points• A continuous-flow microreactor that mimics real-life clinical biofilm infections was developed.• The antibiofilm activity of three nano drugs was evaluated in dynamic conditions.• The highest biofilm reduction was observed with chitosan-silver nanoparticles.

A facile one-step method for cell lysis and DNA extraction of waterborne pathogens using a microchip.

Globally, waterborne organisms are the primary causative agents for the transmission of various forms of diarrheal diseases. For accurate diagnosis, molecular tools have gained considerable attention in the recent past. Molecular tools require DNA as the starting material for diagnosis, and hence, a prerequisite is the quality and integrity of DNA. To obtain high quality DNA rapidly, we have fabricated a microchip in poly(dimethyl siloxane) (PDMS) by soft lithography process. The microchip facilitated in-flow coating of chitosan on the magnetic nanoparticles, which under external mechanical vibration caused cell lysis and released DNA in the supernatant. The released DNA was captured by the nanoparticles owing to its positive charge (chitosan coating). The magnetic nanoparticle-DNA complex was then isolated from the in-flow matrix using permanent magnet, Further, removal of the cell debris, proteins, and carbohydrates was done using wash buffer. DNA extracted using the microchip was pure with absorbance (260/280) ratio of 1.77±0.04, as compared to 1.79±0.03 obtained by TRIzol method. The complete isolation of the DNA using the microchip took ~ 15min as against>2h with a TRIzol method. Six gram-negative waterborne pathogens were used to demonstrate the efficacy of the microchip based DNA extraction process. The integrity of the isolated DNA was assessed by amplifying the 16S rRNA gene using Com1 and Com2 universal primers. The presence of a band at 407bp on gel electrophoresis confirmed the amplified product. Further, the gel image was used for quantification of the amplified product using ImageJ software. Higher regression values obtained using microchip confirmed better quality and integrity of the extracted DNA as opposed to the conventional method. The lower (<2%) relative standard deviation values obtained from the data suggested that the microchip process was reproducible. The quality and integrity of the obtained DNA proved the simplicity, rapidity, and sensitivity of the microchip-assisted DNA extraction process.

Chitosan nanoparticles synthesis caught in action using microdroplet reactions.

The ionic gelation process for the synthesis of chitosan nanoparticles was carried out in microdroplet reactions. The synthesis could be stopped instantaneously at different time points by fast dilution of the reaction mixture with DI water. Using this simple technique, the effect of temperature and reactant concentrations on the size and distribution of the nanoparticles formed, as a function of time, could be investigated by DLS and SEM. Results obtained indicated very early (1-5 s) nucleation of the particles followed by growth. The concentration of reactants, reaction temperature as well as time, were found to (severally and collectively) determine the size of nanoparticles and their distribution. Nanoparticles obtained at 4 °C were smaller (60-80 nm) with narrower size distribution. Simulation experiments using Comsol software showed that at 4 °C 'droplet synthesis' of nanoparticles gets miniaturised to 'droplet-core synthesis', which is being reported for the first time.

Synthesis of Monodisperse Chitosan Nanoparticles and in Situ Drug Loading Using Active Microreactor.

Chitosan nanoparticles are promising drug delivery vehicles. However, the conventional method of unregulated mixing during ionic gelation limits their application because of heterogeneity in size and physicochemical properties. Therefore, a detailed theoretical analysis of conventional and active microreactor models was simulated. This led to design and fabrication of a polydimethylsiloxane microreactor with magnetic micro needles for the synthesis of monodisperse chitosan nanoparticles. Chitosan nanoparticles synthesized conventionally, using 0.5 mg/mL chitosan, were 250 ± 27 nm with +29.8 ± 8 mV charge. Using similar parameters, the microreactor yielded small size particles (154 ± 20 nm) at optimized flow rate of 400 µL/min. Further optimization at 0.4 mg/mL chitosan concentration yielded particles (130 ± 9 nm) with higher charge (+39.8 ± 5 mV). The well-controlled microreactor-based mixing generated highly monodisperse particles with tunable properties including antifungal drug entrapment (80%), release rate, and effective activity (MIC, 1 µg/mL) against Candida.