Rapid Concentration and Detection of Bacteria in Milk Using a Microfluidic Surface Acoustic Wave Activated Nanosieve.

Rapid detection of microbes is a key feature for monitoring food quality. Unfortunately, current detection systems rely on labor-intensive and time-consuming lab-based processes that are not suitable for point-of-interest applications and typically require several days before results are available. Here, we demonstrate a microfluidic system capable of rapidly concentrating, fluorescent staining, and detecting bacteria in unprocessed complex biological media such as milk. This concentration is done using a surface acoustic wave-driven microfluidic device which operates based on the Bjerknes force, a force generated on one particle by another in its close proximity. We exploit this effect by exciting a tightly packed bed of 50 µm polystyrene microparticles temporarily with surface acoustic waves within a microfluidic device to capture and release bacterial cells on demand. The bacterial cells are fluorescently stained during capture and then detected using fluorescence microscopy upon release. This device offers a high capturing efficiency (>80%) and a 34 Colony Forming Units (CFU)/mL limit of detection, which is 1 order of magnitude below that of plate counting at 30 CFU per standard 100 µL plate (or 300 CFU/mL). This can be attained in just 1 h of processing at 10 µL/min. With this system, we demonstrate that bacterial detection from extremely low concentration samples down to the order of ∼10 CFU/mL is possible without requiring any additional external pre- or postprocessing.

Acousto-optofluidic 3D single cell imaging of macrophage phagocytosis of Pseudomonas Aeruginosa.

Understanding how immune cells such as monocytes or macrophages within our blood and tissue engulf and destroy foreign organisms is important for developing new therapies. The process undertaken by these cells, called phagocytosis, has yet to be observed in real-time at the single cell level. Microfluidic-based imaging platforms offer a wide range of tools for precise fluid control and biomolecule manipulation that makes regulating long term experiments and data collection possible. With the compatibility between acoustofluidics and light-sheet fluorescent microscopy (LSFM) previously demonstrated, here an acousto-optfluidic device with on-chip fluid flow direction control was developed. The standing surface acoustic waves (SSAWs) were used to trap, load and safeguard individual cells within a highly controllable fluid loop, created via the triggering of on-chip PDMS valves, to demonstrate multiple rounds of live single cell imaging. The valves allowed for the direction of the fluid flow to be changed (between forward and reverse operation) without altering the inlet flow rate, an important factor for performing reproducible and comparable imaging of samples over time. With this high-resolution imaging system, volumetric reconstructions of phagocytosed bacteria within macrophages could be resolved over a total of 9 rounds of imaging: totalling 19 reconstructed images of the cell membrane with visible intracellular bacteria.

A novel droplet-based approach to study phase transformations in lyotropic liquid crystalline systems.

HYPOTHESIS: Lyotropic liquid crystals (LLC) and their phase transformations in response to stimuli have gathered much interest for controlled and 'on-demand' drug applications. Bulk methods of preparation impose limitations on studying the transformations, especially induced by compositional changes, such as enzymatic changes to lipid structure. Here we hypothesise that controlled microfluidic production and coalescence of dissimilar aqueous and lipid droplets emulsified in a third mutually immiscible liquid will provide a new approach to the spatio-temporal study of structure formation in lyotropic liquid crystalline materials. EXPERIMENTS: Separate lipid and aqueous droplets, dispersed in a fluorocarbon oil were generated using a microfluidic format. The chip, prepared as a hybrid polydimethylsiloxane (PDMS) and glass microfluidic device, was constructed to enable in-situ acquisition of time-resolved synchrotron small angle X-ray scattering (SAXS) and crossed polarised light microscopy of the coalesced droplets to determine the structures present during aging. FINDINGS: Janus-like droplets formed upon coalesce, with distinct lipid and aqueous portions with a gradient between the two sides of the merged droplet. SAXS and polarised light microscopy revealed a progression of mesophases as the lipid portion was hydrated by the aqueous portion via the diffusion limited interface which separated the portions. Thus demonstrating, on a droplet scale, a new approach for studying the phase transformation kinetics and identification of non-equilibrium phase in droplet-based lyotropic liquid systems.

Glass-embedded PDMS microfluidic device for enhanced concentration of nanoparticles using an ultrasonic nanosieve.

Surface acoustic wave (SAW) driven devices typically employ polymeric microfluidic channels of low acoustic impedance mismatch to the fluid in contact, to allow precise control of the wave field. Several of these applications, however, can benefit from the implementation of an acoustically reflective surface at the microfluidic channel's ceiling to increase energy retention within the fluid and hence, performance of the device. In this work, we embed a glass insert at the ceiling of the PDMS microfluidic channel used in a SAW activated nanosieve, which utilises a microparticle resonance for enrichment of nanoparticles. Due to the system's independence of performance on channel geometry and wave field pattern, the glass-inserted device allowed for a 30-fold increase in flow rate, from 0.05 µl min-1 to 1.5 µL min-1, whilst maintaining high capture efficiencies of >90%, when compared to its previously reported design. This effectively enables the system to process larger volume samples, which typically is a main limitation of these type of devices. This work demonstrates a simple way to increase the performance and throughput of SAW-based devices, especially within systems that can benefit from the energy retention.

Wearable microneedle array-based sensor for transdermal monitoring of pH levels in interstitial fluid.

Microneedle-based wearable sensors offer an alternative approach to traditional invasive blood-based health monitoring and disease diagnostics techniques. Instead of blood, microneedle-based sensors target the skin interstitial fluid (ISF), in which the biomarker type and concentration profile resemble the one found in the blood. However, unlike blood, interstitial fluid does not have the same pH-buffering capacity causing deviation of pH levels from the physiological range. Information about the skin ISF pH levels can be used as a biomarker for a wide range of pathophysiological conditions and as a marker for the calibration of a wearable sensor. The ISF pH can significantly affect the detection accuracy of other biomarkers as it influences enzyme activity, aptamer affinity, and antibody-antigen interaction. Herein, we report the fabrication of a high-density polymeric microneedle array-based (PMNA) sensing patch and its optimization for the potentiometric transdermal monitoring of pH levels in ISF. The wearable sensor utilizes a polyaniline-coated PMNA having a density of ∼10,000 microneedles per cm2, containing individual microneedles with a height of ∼250 µm, and a tip diameter of ∼2 µm. To prevent interference from other body fluids like sweat, an insulating layer is deposited at the base of the PMNA. The wearable pH sensor operates from pH 4.0 to 8.6 with a sensitivity of 62.9 mV per pH unit and an accuracy of ±0.036 pH units. Furthermore, testing on a mouse demonstrates the ability of the PMNA to provide a real-time reading of the transdermal pH values. This microneedle-based system will significantly contribute to advancing transdermal wearable sensors technology, simplifying the fabrication process, and improving the cost-effectiveness of such devices.

Emerging biomaterials and technologies to control stem cell fate and patterning in engineered 3D tissues and organoids.

The ability to create complex three-dimensional cellular models that can effectively replicate the structure and function of human organs and tissues in vitro has the potential to revolutionize medicine. Such models could facilitate the interrogation of developmental and disease processes underpinning fundamental discovery science, vastly accelerate drug development and screening, or even be used to create tissues for implantation into the body. Realization of this potential, however, requires the recreation of complex biochemical, biophysical, and cellular patterns of 3D tissues and remains a key challenge in the field. Recent advances are being driven by improved knowledge of tissue morphogenesis and architecture and technological developments in bioengineering and materials science that can create the multidimensional and dynamic systems required to produce complex tissue microenvironments. In this article, we discuss challenges for in vitro models of tissues and organs and summarize the current state-of-the art in biomaterials and bioengineered systems that aim to address these challenges. This includes both top-down technologies, such as 3D photopatterning, magnetism, acoustic forces, and cell origami, as well as bottom-up patterning using 3D bioprinting, microfluidics, cell sheet technology, or composite scaffolds. We illustrate the varying ways that these can be applied to suit the needs of different tissues and applications by focussing on specific examples of patterning the bone-tendon interface, kidney organoids, and brain cancer models. Finally, we discuss the challenges and future prospects in applying materials science and bioengineering to develop high-quality 3D tissue structures for in vitro studies.

Acoustofluidic cell micro-dispenser for single cell trajectory control.

The precise manipulation of individual cells is a key capability for the study of single cell physiological characteristics or responses to stimuli. Currently, only large cell populations can be transferred with certainty using expensive and laborious flow cytometry platforms. However, when approaching small populations of cells, this task becomes increasingly challenging. Here, we report an effective acoustofluidic micro-dispenser, utilising surface acoustic waves (SAWs), with the ability to trap and release cells on demand, which when combined with an external valve can guide the trajectory of individual cells. We demonstrate single cell trap and release with a single cell trapping effectiveness of 74%, enabling the capability of dispensing a highly controlled amount of cells without any harmful effects. This device has the potential to be easily integrated into a wide range of analytical platforms for applications such as single cell fluorescent imaging and single cell proteomic studies.

High-Frequency Ultrasound Boosts Bull and Human Sperm Motility.

Sperm motility is a significant predictor of male fertility potential and is directly linked to fertilization success in both natural and some forms of assisted reproduction. Sperm motility can be impaired by both genetic and environmental factors, with asthenozoospermia being a common clinical presentation. Moreover, in the setting of assisted reproductive technology clinics, there is a distinct absence of effective and noninvasive technology to increase sperm motility without detriment to the sperm cells. Here, a new method is presented to boost sperm motility by increasing the intracellular rate of metabolic activity using high frequency ultrasound. An increase of 34% in curvilinear velocity (VCL), 10% in linearity, and 32% in the number of motile sperm cells is shown by rendering immotile sperm motile, after just 20 s exposure. A similar effect with an increase of 15% in VCL treating human sperm with the same setting is also identified. This cell level mechanotherapy approach causes no significant change in cell viability or DNA fragmentation index, and, as such, has the potential to be applied to encourage natural fertilization or less invasive treatment choices such as in vitro fertilization rather than intracytoplasmic injection.

The Bumpy Road to Stem Cell Therapies: Rational Design of Surface Topographies to Dictate Stem Cell Mechanotransduction and Fate.

Cells sense and respond to a variety of physical cues from their surrounding microenvironment, and these are interpreted through mechanotransductive processes to inform their behavior. These mechanisms have particular relevance to stem cells, where control of stem cell proliferation, potency, and differentiation is key to their successful application in regenerative medicine. It is increasingly recognized that surface micro- and nanotopographies influence stem cell behavior and may represent a powerful tool with which to direct the morphology and fate of stem cells. Current progress toward this goal has been driven by combined advances in fabrication technologies and cell biology. Here, the capacity to generate precisely defined micro- and nanoscale topographies has facilitated the studies that provide knowledge of the mechanotransducive processes that govern the cellular response as well as knowledge of the specific features that can drive cells toward a defined differentiation outcome. However, the path forward is not fully defined, and the "bumpy road" that lays ahead must be crossed before the full potential of these approaches can be fully exploited. This review focuses on the challenges and opportunities in applying micro- and nanotopographies to dictate stem cell fate for regenerative medicine. Here, key techniques used to produce topographic features are reviewed, such as photolithography, block copolymer lithography, electron beam lithography, nanoimprint lithography, soft lithography, scanning probe lithography, colloidal lithography, electrospinning, and surface roughening, alongside their advantages and disadvantages. The biological impacts of surface topographies are then discussed, including the current understanding of the mechanotransductive mechanisms by which these cues are interpreted by the cells, as well as the specific effects of surface topographies on cell differentiation and fate. Finally, considerations in translating these technologies and their future prospects are evaluated.

Colorimetric Detection of Extracellular Hydrogen Peroxide Using an Integrated Microfluidic Device.

It is well known that hydrogen peroxide (H2O2) is a signaling molecule essential for vital physiological reactions in mammalian cells, such as cell survival, intercellular communication, and cancer metabolism. However, to fully understand the function of H2O2, it is critical to monitor its intracellular and/or extracellular concentrations. Current techniques implemented to address this need require large sample volumes, expensive instrumentation, and long sample preparation and analysis times, inapplicable to inline or online monitoring. In this paper, a new integrated microfluidic device capable of overcoming these limitations is demonstrated for the colorimetric detection of extracellular hydrogen peroxide H2O2. The device contains an optical waveguide to determine absorbance changes and micromixers to enable complete mixing of reagents using a passive approach. This novel H2O2-sensing device has allowed the detection of H2O2 in the range of 0.5-60 µM with a detection limit of 167 ± 5.8 nM and a sensitivity of 13.5 ± 0.1 AU/mM. Proof of concept of the device was demonstrated by quantifying H2O2 release from benign prostatic epithelial (BPH-1) cells upon stimulation with phorbol 12-myristate 13-acetate (PMA). Results show that this integrated device can be potentially utilized to continuously monitor cell-released metabolites autonomously without constant human supervision during the process. Furthermore, this can be achieved without interfering with the cell culture conditions, as only a very small volume of conditioned media (less than 0.4 µL), and not the cells, is required.

Three-dimensional imaging on a chip using optofluidics light-sheet fluorescence microscopy.

Volumetric, sub-micron to micron level resolution imaging is necessary to assay phenotypes or characteristics at the sub-cellular/organelle scale. However, three-dimensional fluorescence imaging of cells is typically low throughput or compromises on the achievable resolution in space and time. Here, we capitalise on the flow control capabilities of microfluidics and combine it with microoptics to integrate light-sheet based imaging directly into a microfluidic chip. Our optofluidic system flows suspended cells through a sub-micrometer thick light-sheet formed using micro-optical components that are cast directly in polydimethylsiloxane (PDMS). This design ensures accurate alignment, drift-free operation, and easy integration with conventional microfluidics, while providing sufficient spatial resolution, optical sectioning and volumetric data acquisition. We demonstrate imaging rates of 120 ms per cell at sub-µm resolution, that allow extraction of complex cellular phenotypes, exemplified by imaging of cell clusters, receptor distribution, and the analysis of endosomal size changes.

Three-Dimensional Micropatterning Deters Early Bacterial Adherence and Can Eliminate Colonization.

Developing strategies to prevent bacterial infections that do not rely on the use of drugs is regarded globally as an important means to stem the tide of antimicrobial resistance, as argued by the World Health Organization (WHO) (Mendelson, M.; Matsoso, M. P. The World Health Organization Global Action Plan for Antimicrobial Resistance. S. Afr. Med. J. 2015, 105 (5), 325-325. DOI: 10.7196/SAMJ.9644). Given that many antimicrobial-resistant infections are caused by the bacterial colonization of indwelling medical devices such as catheters and ventilators, the use of microengineered surfaces to prevent the initial attachment of microbes to these devices is a promising solution. In this work, it is demonstrated that 3D engineered surfaces can inhibit the initial phases of surface colonization for Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, representing the three most common catheter-associated urinary tract bacterial infections, identified by the WHO as urgent threats. A variety of designs including 11 different topographies and configurations that exhibited random distributions, sharp protrusions, and/or curvilinear shapes with dimensions ranging between 500 nm and 2 µm were tested to better understand the initial stages of surface colonization and how to optimize the design of fabricated surfaces for improved inhibition. These topographies were fabricated in two configurations to obtain either a standard 2D cross section or a 3D engineered topography using a novel UV lithography process enabling cost-efficient high-throughput manufacturing. Evaluating both the number of adhered bacteria and microcolonies formed by all three bacterial pathogens on the different surfaces provides insight into the initial colonization phase of bacterial growth on the various surfaces. The results demonstrate that both initial attachment and subsequent colonization can be significantly reduced on concrete 3D engineered patterns when compared to flat substrates and standard 2D micropatterns. Thus, this technology has great potential to reduce the colonization of bacteria on surfaces in clinical settings without the need for chemical treatments that might enhance antimicrobial resistance.

Precision Surface Microtopography Regulates Cell Fate via Changes to Actomyosin Contractility and Nuclear Architecture.

Cells are able to perceive complex mechanical cues from their microenvironment, which in turn influences their development. Although the understanding of these intricate mechanotransductive signals is evolving, the precise roles of substrate microtopography in directing cell fate is still poorly understood. Here, UV nanoimprint lithography is used to generate micropillar arrays ranging from 1 to 10 µm in height, width, and spacing to investigate the impact of microtopography on mechanotransduction. Using mesenchymal stem cells (MSCs) as a model, stark pattern-specific changes in nuclear architecture, lamin A/C accumulation, chromatin positioning, and DNA methyltransferase expression, are demonstrated. MSC osteogenesis is also enhanced specifically on micropillars with 5 µm width/spacing and 5 µm height. Intriguingly, the highest degree of osteogenesis correlates with patterns that stimulated maximal nuclear deformation which is shown to be dependent on myosin-II-generated tension. The outcomes determine new insights into nuclear mechanotransduction by demonstrating that force transmission across the nuclear envelope can be modulated by substrate topography, and that this can alter chromatin organisation and impact upon cell fate. These findings have potential to inform the development of microstructured cell culture substrates that can direct cell mechanotransduction and fate for therapeutic applications in both research and clinical sectors.

Microfluidic Electrochemical Sensor for Cerebrospinal Fluid and Blood Dopamine Detection in a Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder involving dopaminergic neurons from the substantia nigra. The loss of dopaminergic neurons results in decreased dopamine (DA) release in the striatum and thus impaired motor functions. DA is one of the key neurotransmitters monitored for the diagnosis and during the progression and treatment of PD. Therefore, sensitive and selective DA detection methods are of high clinical relevance. In this study, a new microfluidic device utilized for electrochemical DA detection is reported. The microfluidic sensing device operates in the range of 0.1-1000 nM DA requiring only ∼2.4 µL sample volume, which corresponds to detectable 240 amol of DA. Using this sensor, we were able to monitor the changes in DA levels in cerebrospinal fluid and plasma of a mouse model of PD and following the treatment of drug l-3,4-dihydroxyphenylalanine.

High-Aspect-Ratio SU-8-Based Optofluidic Device for Ammonia Detection in Cell Culture Media.

Miniaturization of sensing technology has led to the development of multifunctional micro total analysis systems (µTAS) that benefit from microfluidic technology. Optical sensing is one of the most commonly used sensing approaches integrated into µTAS devices and features high sensitivity and low detection limits. Different materials have been used for the fabrication of µTAS devices, each having their advantages and disadvantages. Herein, a high-aspect-ratio optofluidic waveguide fabricated from SU-8 is presented for the first time. The suitable optical properties and chemical inertness of SU-8 provide a durable device made by a flexible and cost-efficient fabrication process. The optofluidic device was used for colorimetric ammonia (NH3) sensing with a dynamic range of 3-70 µM, a detection limit of 2.5 µM, a response time of 8 min, and close to 10 times better analytical performance compared to using a standard microplate reader. The µTAS device was capable of monitoring NH3 accumulating in the cell culture media of prostatic epithelial cell (BPH-1) culture.

The emerging role of microfluidics in multi-material 3D bioprinting.

To assist the transition of 3D bioprinting technology from simple lab-based tissue fabrication, to fully functional and implantable organs, the technology must not only provide shape control, but also functional control. This can be accomplished by replicating the cellular composition of the native tissue at the microscale, such that cell types interact to provide the desired function. There is therefore a need for precise, controllable, multi-material printing that could allow for high, possibly even single cell, resolution. This paper aims to draw attention to technological advancements made in 3D bioprinting that target the lack of multi-material, and/or multi cell-type, printing capabilities of most current devices. Unlike other reviews in the field, which largely focus on variations in single-material 3D bioprinting involving the standard methods of extrusion-based, droplet-based, laser-based, or stereolithographic methods; this review concentrates on sophisticated multi-material 3D bioprinting using multi-cartridge printheads, co-axial nozzles and microfluidic-enhanced printing nozzles.

Enhanced electrochemical sensing performance by insitu electrocopolymerization of pyrrole and thiophene-grafted chitosan.

Nowadays, there is increasing number of electrochemical biosensors which utilize chitosan (Ch); as an enzyme immobilization matrix, and conductive nanomaterials; as electron carriers improving sensitivity of the biosensor. However, the challenge these sensors face is the lack of uniform dispersion of nanomaterials throughout the Ch film, which can negatively affect analytical performance of the biosensor. In this study, we report the development of an enzyme immobilization matrix that displays enhanced electrochemical performance thanks to a novel conductive thin film prepared via in situ electrocopolymerization of pyrrole (Py) and thiophene-grafted chitosan (Th-Ch). This is a simple thin film preparation method that can help overcome aforementioned challenges by providing a uniformly distributed conductive layer on the electrode. We are also for the first time reporting the synthesis and characterization of Th-Ch, where grafted Th plays an essential role as a linking group between Ch and Py. The resulting conductive Ch-based thin film was modified with glucose oxidase (GOx) which served as a model enzyme. In situ electrocopolymerization of Py with Th-Ch resulted in a highly conductive thin film enabling approximately 40% higher sensitivity when compared to a Py-Ch composite. This new type of composite thin film is promising in biosensor technology due to its biocompatibility, the chemically and physically modifiable structure, as well as its electrical conductivity.

Recent Progress in Lab-On-a-Chip Systems for the Monitoring of Metabolites for Mammalian and Microbial Cell Research.

Lab-on-a-chip sensing technologies have changed how cell biology research is conducted. This review summarises the progress in the lab-on-a-chip devices implemented for the detection of cellular metabolites. The review is divided into two subsections according to the methods used for the metabolite detection. Each section includes a table which summarises the relevant literature and also elaborates the advantages of, and the challenges faced with that particular method. The review continues with a section discussing the achievements attained due to using lab-on-a-chip devices within the specific context. Finally, a concluding section summarises what is to be resolved and discusses the future perspectives.

Reversible Light-Switching of Enzymatic Activity on Orthogonally Functionalized Polymer Brushes.

Copolymer brushes, composed of glycidyl methacrylate and a furan-protected maleimide-containing monomer, were grafted from radical initiators at the surface of irradiation-activated fluoropolymer foils. After postpolymerization modification with enzymatically active microperoxidase-11 and photochromic spiropyran moieties, the polymer brushes catalyzed the oxidation of 3,3'5,5'-tetramethylbenzidine. Exposure to either UV or visible-light allowed switching the turnover by more than 1 order of magnitude, as consequence of the reversible, light-induced spiropyran-merocyanine transition. The modified samples were integrated into an optofluidic device that allowed the reversible switching of enzymatic activity for several cycles under flow, validating the potential for application in smart lab-on-a-chip systems.

High-aspect-ratio nanoimprint process chains.

Different methods capable of developing complex structures and building elements with high-aspect-ratio nanostructures combined with microstructures, which are of interest in nanophotonics, are presented. As originals for subsequent replication steps, two families of masters were developed: (i) 3.2 µm deep, 180 nm wide trenches were fabricated by silicon cryo-etching and (ii) 9.8 µm high, 350 nm wide ridges were fabricated using 2-photon polymerization direct laser writing. Both emerging technologies enable the vertical smooth sidewalls needed for a successful imprint into thin layers of polymers with aspect ratios exceeding 15. Nanoridges with high aspect ratios of up to 28 and no residual layer were produced in Ormocers using the micromoulding into capillaries (MIMIC) process with subsequent ultraviolet-curing. This work presents and balances the different fabrication routes and the subsequent generation of working tools from masters with inverted tones and the combination of hard and soft materials. This provides these techniques with a proof of concept for their compatibility with high volume manufacturing of complex micro- and nanostructures.