Multifunctional nanocoating for enhanced titanium implant osseointegration.

Preventing bacterial infection and promoting osseointegration are essential for the long-term success of titanium (Ti) implants. In this study, we developed a multifunctional nanocoating on Ti mini-implants to simultaneously address these challenges. The nanocoating consists of self-assembled antimicrobial peptides GL13K and silver nanoparticles, referred to as Ag-GL. Our results showed that the Ag-GL coating did not alter the surface morphology of the mini-implants. Ag-GL coated mini-implants demonstrated a two orders of magnitude reduction in colony-forming unit (CFU) values compared to the noncoated eTi group, resulting in minimal inflammation and no apparent bone destruction in a bacterial infection in vivo model. When evaluating osseointegration properties, micro-CT analysis, histomorphometric analysis, and pull-out tests revealed that the Ag-GL coating significantly enhanced osseointegration and promoted new bone formation in vivo.

Integration of RT-LAMP and Microfluidic Technology for Detection of SARS-CoV-2 in Wastewater as an Advanced Point-of-Care Platform.

Development of lab-on-a-chip (LOC) system based on integration of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and microfluidic technology is expected to speed up SARS-CoV-2 diagnostics allowing early intervention. In the current work, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and RT-LAMP assays were performed on extracted RNA of seven wastewater samples from COVID-19 hotspots. RT­LAMP assay was also performed on wastewater samples without RNA extraction. Current detection of SARS-CoV-2 is mainly by RT-qPCR of ORF (ORF1ab) and N genes so we targeted both to find the best target gene for SARS-CoV-2 detection. We also performed RT-LAMP with/without RNA extraction inside microfluidic device to target both genes. Positivity rates of RT-qPCR and RT-LAMP performed on extracted RNA were 100.0% (7/7) and 85.7% (6/7), respectively. RT-qPCR results revealed that all 7 wastewater samples were positive for N gene (Ct range 37-39), and negative for ORF1ab, suggesting that N gene could be the best target gene for SARS-CoV-2 detection. RT-LAMP of N and ORF (ORF1a) genes performed on wastewater samples without RNA extraction indicated that all 7 samples remains pink (negative). The color remains pink in all microchannels except microchannels which subjected to RT-LAMP for targeting N region after RNA extraction (yellow color) in 6 out of 7 samples. This study shows that SARS-CoV-2 was successfully detected from wastewater samples using RT-LAMP in microfluidic chips. This study brings the novelty involving the use of wastewater samples for detection of SARS-CoV-2 without previous virus concentration and with/without RNA extraction.

Plant-inspired TransfOrigami microfluidics.

The healthy functioning of the plants' vasculature depends on their ability to respond to environmental changes. In contrast, synthetic microfluidic systems have rarely demonstrated this environmental responsiveness. Plants respond to environmental stimuli through nastic movement, which inspires us to introduce transformable microfluidics: By embedding stimuli-responsive materials, the microfluidic device can respond to temperature, humidity, and light irradiance. Furthermore, by designing a foldable geometry, these responsive movements can follow the preset origami transformation. We term this device TransfOrigami microfluidics (TOM) to highlight the close connection between its transformation and the origami structure. TOM can be used as an environmentally adaptive photomicroreactor. It senses the environmental stimuli and feeds them back positively into photosynthetic conversion through morphological transformation. The principle behind this morphable microsystem can potentially be extended to applications that require responsiveness between the environment and the devices, such as dynamic artificial vascular networks and shape-adaptive flexible electronics.

Co-doping optimized hydrogel-elastomer micro-actuators for versatile biomimetic motions.

Hydrogels can respond to changes in humidity or temperature, while elastomers can resist structural collapse due to dehydration or external force application. A hybrid bilayer of hydrogel-elastomers while retaining the merits of both the hydrogels and elastomers has emerged as a promising stimuli-responsive micro-actuator. However, the preparation of a hydrogel-elastomer micro-actuator requires multiple steps, mainly due to the differences in the surface properties of these two materials. Among them, the steps to surface-treat the elastomer and functionalize the material of each layer involve intricate processes and excessive consumption of resources. In this work, we introduce a co-doping method to optimize the preparation of a stimuli-responsive hydrogel-elastomer micro-actuator. The surface treatment and functionalization processes are combined into one step by directly doping the polymerization initiator and functional nanomaterials into the hybrid bilayer. The thermo-responsive hydrogel is combined with a photothermal elastomer to fabricate a soft micro-actuator that can bend and unbend in response to changes in humidity and light. Based on this actuator, a set of biomimetic soft micro-robots were developed, demonstrating a series of motions, such as grabbing, crawling, and jumping. This strategy of stimuli-responsive micro-actuator preparation can benefit the hydrogel-elastomer hybrid micro-robot designs for applications ranging from self-locomotive robots in environmental monitoring to drug delivery in biomedical engineering.

Interferon α2-Thymosin α1 Fusion Protein (IFNα2-Tα1): A Genetically Engineered Fusion Protein with Enhanced Anticancer and Antiviral Effect.

Human interferon α2 (IFNα2) and thymosin α1 (Tα1) are therapeutic proteins used for the treatment of viral infections and different types of cancer. Both IFNα2 and Tα1 show a synergic effect in their activities when used in combination. Furthermore, the therapeutic fusion proteins produced through the genetic fusion of two genes can exhibit several therapeutic functions in one molecule. In this study, we determined the anticancer and antiviral effect of human interferon α2-thymosin α1 fusion protein (IFNα2-Tα1) produced in our laboratory for the first time. The cytotoxic and genotoxic effect of IFNα2-Tα1 was evaluated in HepG2 and MDA-MB-231 cells. The in vitro assays confirmed that IFNα2-Tα1 inhibited the growth of cells more effectively than IFNα2 alone and showed an elevated genotoxic effect. The expression of proapoptotic genes was also significantly enhanced in IFNα2-Tα1-treated cells compared to IFNα2-treated cells. Furthermore, the HCV RNA level was significantly reduced in IFNα2-Tα1-treated HCV-infected Huh7 cells compared to IFNα2-treated cells. The quantitative PCR analysis showed that the expression of various genes, the products of which inhibit HCV replication, was significantly enhanced in IFNα2-Tα1-treated cells compared to IFNα2-treated cells. Our findings demonstrate that IFNα2-Tα1 is more effective than single IFNα2 as an anticancer and antiviral agent.

A portable droplet microfluidic device for cortisol measurements using a competitive heterogeneous assay.

Point-of-care monitoring of chemical biomarkers in real-time holds great potential in rapid disease diagnostics and precision medicine. However, monitoring is still rare in practice, as the measurement of biomarkers often requires time consuming and labour intensive assay procedures such as enzyme linked immunosorbent assay (ELISA), which pose a challenge to an autonomous point-of-care device. This paper describes a prototype device capable of performing ELISA autonomously and repeatedly in a high frequency using droplet microfluidics. Driven by a specially designed peristaltic pump, the device can collect liquid samples from a reservoir, produce trains of droplets, complete magnetic bead based ELISA protocols and provide readouts with colourimetric measurement. Here, cortisol was chosen as a target analyte as its concentration in the human body varies on a circadian rhythm which may be perturbed by disease. The prototype device draws in and analyses 350 nL of the sample containing free bioactive cortisol every 10 seconds, with a sample-to-signal time of 10 minutes, and measures favourably in the analytical range of 3.175-100 ng ml-1, with reliably lower variability compared with the well plate based assay. As most ELISA type assays share similar procedures, we envisage that this approach could form a platform technology for measurement or even continuous monitoring of biomarkers in biological fluids at the point-of-care.

COVID-19 Crisis Creates Opportunity towards Global Monitoring & Surveillance.

The spectrum of emerging new diseases as well as re-emerging old diseases is broadening as infectious agents evolve, adapt, and spread at enormous speeds in response to changing ecosystems. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recent phenomenon and may take a while to understand its transmission routes from less traveled territories, ranging from fomite exposure routes to wastewater transmission. The critical challenge is how to negotiate with such catastrophic pandemics in high-income countries (HICs ~20% of the global population) and low-and middle-income countries (LMICs ~ 80% of the global population) with a total global population size of approximately eight billion, where practical mass testing and tracing is only a remote possibility, particularly in low-and middle-income countries (LMICs). Keeping in mind the population distribution disparities of high-income countries (HICs) and LMICs and urbanisation trends over recent years, traditional wastewater-based surveillance such as that used to combat polio may help in addressing this challenge. The COVID-19 era differs from any previous pandemics or global health challenges in the sense that there is a great deal of curiosity within the global community to find out everything about this virus, ranging from diagnostics, potential vaccines/therapeutics, and possible routes of transmission. In this regard, the fact that the gut is the common niche for both poliovirus and SARS-CoV-2, and due to the shedding of the virus through faecal material into sewerage systems, the need for long-term wastewater surveillance and developing early warning systems for better preparedness at local and global levels is increasingly apparent. This paper aims to provide an insight into the ongoing COVID-19 crisis, how it can be managed, and what measures are required to deal with a current global international public health concern. Additionally, it shed light on the importance of using wastewater surveillance strategy as an early warning practical tool suitable for massive passive screening, as well as the urgent need for microfluidic technology as a rapid and cost-effective approach tracking SARS-CoV-2 in wastewater.

A Review of Biodegradable Natural Polymer-Based Nanoparticles for Drug Delivery Applications.

Biodegradable natural polymers have been investigated extensively as the best choice for encapsulation and delivery of drugs. The research has attracted remarkable attention in the pharmaceutical industry. The shortcomings of conventional dosage systems, along with modified and targeted drug delivery methods, are addressed by using polymers with improved bioavailability, biocompatibility, and lower toxicity. Therefore, nanomedicines are now considered to be an innovative type of medication. This review critically examines the use of natural biodegradable polymers and their drug delivery systems for local or targeted and controlled/sustained drug release against fatal diseases.

Glycoprotein- and Lectin-Based Approaches for Detection of Pathogens.

Infectious diseases alone are estimated to result in approximately 40% of the 50 million total annual deaths globally. The importance of basic research in the control of emerging and re-emerging diseases cannot be overemphasized. However, new nanotechnology-based methodologies exploiting unique surface-located glycoproteins or their patterns can be exploited to detect pathogens at the point of use or on-site with high specificity and sensitivity. These technologies will, therefore, affect our ability in the future to more accurately assess risk. The critical challenge is making these new methodologies cost-effective, as well as simple to use, for the diagnostics industry and public healthcare providers. Miniaturization of biochemical assays in lab-on-a-chip devices has emerged as a promising tool. Miniaturization has the potential to shape modern biotechnology and how point-of-care testing of infectious diseases will be performed by developing smart microdevices that require minute amounts of sample and reagents and are cost-effective, robust, and sensitive and specific. The current review provides a short overview of some of the futuristic approaches using simple molecular interactions between glycoproteins and glycoprotein-binding molecules for the efficient and rapid detection of various pathogens at the point of use, advancing the emerging field of glyconanodiagnostics.

Capillary-Driven Flow Microfluidics Combined with Smartphone Detection: An Emerging Tool for Point-of-Care Diagnostics.

Point-of-care (POC) or near-patient testing allows clinicians to accurately achieve real-time diagnostic results performed at or near to the patient site. The outlook of POC devices is to provide quicker analyses that can lead to well-informed clinical decisions and hence improve the health of patients at the point-of-need. Microfluidics plays an important role in the development of POC devices. However, requirements of handling expertise, pumping systems and complex fluidic controls make the technology unaffordable to the current healthcare systems in the world. In recent years, capillary-driven flow microfluidics has emerged as an attractive microfluidic-based technology to overcome these limitations by offering robust, cost-effective and simple-to-operate devices. The internal wall of the microchannels can be pre-coated with reagents, and by merely dipping the device into the patient sample, the sample can be loaded into the microchannel driven by capillary forces and can be detected via handheld or smartphone-based detectors. The capabilities of capillary-driven flow devices have not been fully exploited in developing POC diagnostics, especially for antimicrobial resistance studies in clinical settings. The purpose of this review is to open up this field of microfluidics to the ever-expanding microfluidic-based scientific community.

Design and Fabrication of Capillary-Driven Flow Device for Point-Of-Care Diagnostics.

Point-of-care (POC) diagnostics enables the diagnosis and monitoring of patients from the clinic or their home. Ideally, POC devices should be compact, portable and operatable without the requirement of expertise or complex fluid mechanical controls. This paper showcases a chip-and-dip device, which works on the principle of capillary-driven flow microfluidics and allows analytes' detection by multiple microchannels in a single microchip via smartphone imaging. The chip-and-dip device, fabricated with inexpensive materials, works by simply dipping the reagents-coated microchip consisting of microchannels into a fluidic sample. The sample is loaded into the microchannels via capillary action and reacts with the reagents to produce a colourimetric signal. Unlike dipstick tests, this device allows the loading of bacterial/pathogenic samples for antimicrobial testing. A single device can be coated with multiple reagents, and more analytes can be detected in one sample. This platform could be used for a wide variety of assays. Here, we show the design, fabrication and working principle of the chip-and-dip flow device along with a specific application consisting in the determination of ß-lactamase activity and cortisol. The simplicity, robustness and multiplexing capability of the chip-and-dip device will allow it to be used for POC diagnostics.

Design and Fabrication of Optical Flow Cell for Multiplex Detection of ß-lactamase in Microchannels.

Miniaturized quantitative assays offer multiplexing capability in a microfluidic device for high-throughput applications such as antimicrobial resistance (AMR) studies. The detection of these multiple microchannels in a single microfluidic device becomes crucial for point-of-care (POC) testing and clinical diagnostics. This paper showcases an optical flow cell for detection of parallel microchannels in a microfluidic chip. The flow cell operates by measuring the light intensity from the microchannels based on Beer-Lambert law in a linearly moving chip. While this platform could be tailored for a wide variety of applications, here we show the design, fabrication and working principle of the device. ß-lactamase, an indicator of bacterial resistance to ß-lactam antibiotics, especially in milk, is shown as an example. The flow cell has a small footprint and uses low-powered, low-cost components, which makes it ideally suited for use in portable devices that require multiple sample detection in a single chip.

Easily fabricated monolithic fluoropolymer chips for sensitive long-term absorbance measurement in droplet microfluidics.

Maintaining a hydrophobic channel surface is critical to ensuring long-term stable flow in droplet microfluidics. Monolithic fluoropolymer chips ensure robust and reliable droplet flow as their native fluorous surfaces naturally preferentially wet fluorocarbon oils and do not deteriorate over time. Their fabrication, however, typically requires expensive heated hydraulic presses that make them inaccessible to many laboratories. Here we describe a method for micropatterning and bonding monolithic fluoropolymer flow cells from a commercially available melt-processable fluoropolymer, Dyneon THV 500GZ, that only requires a standard laboratory oven. Using this technique, we demonstrate the formation of complex microstructures, specifically the fabrication of sensitive absorbance flow cells for probing droplets in flow, featuring path lengths up to 10 mm. The native fluorous channel surface means the flow cells can be operated over extended periods, demonstrated by running droplets continuously through a chip for 16 weeks.

A Droplet Microfluidic-Based Sensor for Simultaneous in Situ Monitoring of Nitrate and Nitrite in Natural Waters.

Microfluidic-based chemical sensors take laboratory analytical protocols and miniaturize them into field-deployable systems for in situ monitoring of water chemistry. Here, we present a prototype nitrate/nitrite sensor based on droplet microfluidics that in contrast to standard (continuous phase) microfluidic sensors, treats water samples as discrete droplets contained within a flow of oil. The new sensor device can quantify the concentrations of nitrate and nitrite within each droplet and provides high measurement frequency and low fluid consumption. Reagent consumption is at a rate of 2.8 mL/day when measuring every ten seconds, orders of magnitude more efficient than those of the current state-of-the-art sensors. The sensor's capabilities were demonstrated during a three-week deployment in a tidal river. The accurate and high frequency data (6% error relative to spot samples, measuring at 0.1 Hz) elucidated the influence of tidal variation, rain events, diurnal effects, and anthropogenic input on concentrations at the deployment site. This droplet microfluidic-based sensor is suitable for a wide range of applications such as monitoring of rivers, lakes, coastal waters, and industrial effluents.

Monitoring biomolecule concentrations in tissue using a wearable droplet microfluidic-based sensor.

Knowing how biomarker levels vary within biological fluids over time can produce valuable insight into tissue physiology and pathology, and could inform personalised clinical treatment. We describe here a wearable sensor for monitoring biomolecule levels that combines continuous fluid sampling with in situ analysis using wet-chemical assays (with the specific assay interchangeable depending on the target biomolecule). The microfluidic device employs a droplet flow regime to maximise the temporal response of the device, using a screw-driven push-pull peristaltic micropump to robustly produce nanolitre-sized droplets. The fully integrated sensor is contained within a small (palm-sized) footprint, is fully autonomous, and features high measurement frequency (a measurement every few seconds) meaning deviations from steady-state levels are quickly detected. We demonstrate how the sensor can track perturbed glucose and lactate levels in dermal tissue with results in close agreement with standard off-line analysis and consistent with changes in peripheral blood levels.

Micromachined optical flow cell for sensitive measurement of droplets in tubing.

Here a micromachined flow cell with enhanced optical sensitivity is presented that allows high-throughput analysis of microdroplets. As a droplet flows through multiple concatenated measurement points, the rate of enzymatic reaction in the droplet can be fully characterized without stopping the flow. Since there is no cross-talk between the droplets, the flow cell is capable of continuously measuring biochemical assays in a droplet flow and thus is suitable to be used for continuous point-of-care diagnostics monitoring. This paper describes the design and operation of the device and its validation by application to the accurate and continuous quantification of glucose concentrations using an oxidase enzymatic assay. The flow cell forms an important component in the miniaturization of chemical and bio analyzers into portable or wearable devices.

Nitrate measurement in droplet flow: gas-mediated crosstalk and correction.

In droplet microfluidics, droplets have traditionally been considered discrete self-contained reaction chambers, however recent work has shown that dissolved solutes can transfer into the oil phase and migrate into neighbouring droplets under certain conditions. The majority of reports on such inter-droplet "crosstalk" have focused on surfactant-driven mechanisms, such as transport within micelles. While trialling a droplet-based system for quantifying nitrate in water, we encountered crosstalk driven by a very different mechanism: conversion of the analyte to a gaseous intermediate which subsequently diffused between droplets. Importantly we found that the crosstalk occurred predictably, could be experimentally quantified, and measurements rationally post-corrected. This showed that droplet microfluidic systems susceptible to crosstalk such as this can nonetheless be used for quantitative analysis.

Phased peristaltic micropumping for continuous sampling and hardcoded droplet generation.

Droplet microfluidics has recently emerged as a new engineering tool for biochemical analysis of small sample volumes. Droplet generation is most commonly achieved by introducing aqueous and oil phases into a T-junction or a flow focusing channel geometry. This method produces droplets that are sensitive to changes in flow conditions and fluid composition. Here, we present an alternative approach using a simple peristaltic micropump to deliver the aqueous and oil phases in antiphase pulses resulting in a robust "chopping"-like method of droplet generation. This method offers controllable droplet dynamics, with droplet volumes solely determined by the pump design, and is insensitive to liquid properties and flow rates. Importantly, sequences of droplets with controlled composition can be hardcoded into the pump, allowing chemical operations such as titrations and dilutions to be easily achieved. The push-pull pump is compact and can continuously collect samples, generating droplets close to the sampling site and with short stabilisation time. We envisage that this robust droplet generation method is highly suited for continuous in situ sampling and chemical measurement, allowing droplet microfluidics to step out of the lab and into field-deployable applications.

Continuous measurement of enzymatic kinetics in droplet flow for point-of-care monitoring.

Droplet microfluidics is ideally suited to continuous biochemical analysis, requiring low sample volumes and offering high temporal resolution. Many biochemical assays are based on enzymatic reactions, the kinetics of which can be obtained by probing droplets at multiple points over time. Here we present a miniaturised multi-detector flow cell to analyse enzyme kinetics in droplets, with an example application of continuous glucose measurement. Reaction rates and Michaelis-Menten kinetics can be quantified for each individual droplet and unknown glucose concentrations can be accurately determined (errors <5%). Droplets can be probed continuously giving short sample-to-result time (∼30 s) measurement. In contrast to previous reports of multipoint droplet measurement (all of which used bulky microscope-based setups) the flow cell presented here has a small footprint and uses low-powered, low-cost components, making it ideally suited for use in field-deployable devices.

Droplet interfaced parallel and quantitative microfluidic-based separations.

High-throughput, quantitative, and rapid microfluidic-based separations has been a long-sought goal for applications in proteomics, genomics, biomarker discovery, and clinical diagnostics. Using droplet-interfaced microchip electrophoresis (MCE) techniques, we have developed a novel parallel MCE platform, based on the concept of combining the Slipchip principle with a newly developed "Gelchip". The platform consists of two plastic plates, with droplet wells on one plate and separation channels with preloaded/cured gel in the other. A single relative movement of one plate enables generation and then loading of multiple sample droplets in parallel into the separation channels, allowing electrophoretic separation of biomolecules in the droplets in parallel and with high-throughput. As proof of concept, we demonstrated the separation of 30 sub-nL sample droplets containing fluorescent dyes or DNA fragments.