Portable, low cost and sensitive cavity enhanced absorption (CEA) detection.

Absorption is a widely used technique for a range of different applications. It has lower sensitivity than many other techniques such as fluorescence which has 100 to 1000 times higher sensitivity than absorption. Optical cavity approaches have been developed where the light passes back and forth, within the sample, between two high reflectivity mirrors to increase the pathlength and sensitivity. These approaches have not yet, however, been widely used for analytical applications and for point-of-care diagnostics. Here we show a portable cavity enhanced absorption (CEA) spectrometer and a low cost point-of-care (POC) reader with CEA detection with mechanical elements fabricated using 3D printing. The CEA spectrometer can be used in both single pass and multi-pass cavity enhanced mode to provide measurements in the visible region that are very sensitive and over a wide dynamic range. The CEA mode was shown for Rhodamine B dye to increase the pathlength 57.8 fold over single pass measurements and an LOD of 7.1 × 10-11 M. The cost of the CEA POC reader was reduced by use of narrow band LEDs, photodiodes and removal of fibre optic coupling and with a 14 fold increase in the pathlength over conventional single pass microplate readers. The CEA POC reader was demonstrated for immunoassay of C-Reactive Protein (CRP), Procalcitonin (PCT) and Interleukin 6 (IL-6), towards a three biomarker panel to aid the diagnosis of sepsis. The CEA POC reader can be integrated with wireless connectivity for cloud based data sharing. We show here the potential for the wider use of optical cavity approaches where there is a need for sensitive absorption measurements and also for low cost point-of-care diagnostics.

Towards More Predictive, Physiological and Animal-free In Vitro Models: Advances in Cell and Tissue Culture 2020 Conference Proceedings.

Experimental systems that faithfully replicate human physiology at cellular, tissue and organ level are crucial to the development of efficacious and safe therapies with high success rates and low cost. The development of such systems is challenging and requires skills, expertise and inputs from a diverse range of experts, such as biologists, physicists, engineers, clinicians and regulatory bodies. Kirkstall Limited, a biotechnology company based in York, UK, organised the annual conference, Advances in Cell and Tissue Culture (ACTC), which brought together people having a variety of expertise and interests, to present and discuss the latest developments in the field of cell and tissue culture and in vitro modelling. The conference has also been influential in engaging animal welfare organisations in the promotion of research, collaborative projects and funding opportunities. This report describes the proceedings of the latest ACTC conference, which was held virtually on 30th September and 1st October 2020, and included sessions on in vitro models in the following areas: advanced skin and respiratory models, neurological disease, cancer research, advanced models including 3-D, fluid flow and co-cultures, diabetes and other age-related disorders, and animal-free research. The roundtable session on the second day was very interactive and drew huge interest, with intriguing discussion taking place among all participants on the theme of replacement of animal models of disease.

Microbioreactor for lower cost and faster optimisation of protein production.

Optimisation of bioprocesses relies on approaches that are either labour intensive or require expensive robotic systems. There is a need for fluidic processing at low volume that can be integrated with existing bioprocess analytics to provide analytical information for the development and optimisation of bioprocesses. We demonstrate a 1 mL polymer inkjet 3D printed (i3DP) microbioreactor with integrated sensing (pH, oxygen and cell density) for optimisation of recombinant protein production with different feeds. A pressurised fluid driving system was used to control flow rates down to 0.7 µL min-1 with fluid switching from four reservoirs using a manifold controlled by solenoid valves. Oxygen transferred from a headspace via a gas-permeable membrane achieved a kLa of up to 90 h-1 at 1500 rpm. Cultivation of E. coli within the microbioreactor was comparable with a 2 L bench scale bioreactor, with optical densities of respectively 7.1 ± 0.4 and 6.5 ± 0.35. Triplicate batch cultivations within the microbioreactor of Pichia pastoris, with diauxic growth on glycerol (0.20 ± 0.02 h-1) and methanol (0.02 ± 0.04 h-1), showed good control of pH and DO and achieved a maximum dry cell weight of 10 ± 1 g L-1. For continuous cultivations, recombinant protein production was higher in pure methanol (314 ± 23) than methanol-sorbitol (202 ± 17) but reduces over time with lower cellular viability for methanol-glucose mixed feed, with less total protein produced and increases in DNA and proteases released. The developed system could be used in different applications including within synthetic biology, cell and gene therapy and organ-on-chips.

Developments in Transduction, Connectivity and AI/Machine Learning for Point-of-Care Testing.

We review some emerging trends in transduction, connectivity and data analytics for Point-of-Care Testing (POCT) of infectious and non-communicable diseases. The patient need for POCT is described along with developments in portable diagnostics, specifically in respect of Lab-on-chip and microfluidic systems. We describe some novel electrochemical and photonic systems and the use of mobile phones in terms of hardware components and device connectivity for POCT. Developments in data analytics that are applicable for POCT are described with an overview of data structures and recent AI/Machine learning trends. The most important methodologies of machine learning, including deep learning methods, are summarised. The potential value of trends within POCT systems for clinical diagnostics within Lower Middle Income Countries (LMICs) and the Least Developed Countries (LDCs) are highlighted.

Cavity enhanced liquid-phase stopped-flow kinetics.

The first application of liquid-phase broadband cavity enhanced spectroscopy (BBCEAS) to the measurement of stopped-flow kinetics is reported. The stopped-flow technique is widely used for the study of the kinetics of fast liquid-phase reactions down to millisecond timescales. UV-visible absorption spectroscopy is commonly used as the detection method. Increased sensitivity can potentially allow reactions which are too fast to be measured, to be studied by slowing down the reaction rate through the use of lower concentration of reactants. A simple low cost BBCEAS experimental setup was coupled to a commercial stopped-flow instrument. Comparative standard absorption measurements were also made using a UV-visible double-beam spectrometer as the detector. Measurements were made on the reaction of potassium ferricyanide with sodium ascorbate under pseudo-first order conditions at pH 8 and pH 9.2 A cavity enhancement factor (CEF) of 78 at 434 nm was obtained whilst the minimum detectable change in the absorption coefficient αmin(t), was 1.35 × 10-5 cm-1 Hz-1/2. The kinetic data at pH 9.2 was too fast to be measured using conventional spectroscopy, whilst the BBCEAS measurements allowed 30 fold lower concentration of reactants to be used which slowed down the reaction rate enough to allow the rate constant to be determined. The BBCEAS results showed a 58 fold improvement in sensitivity over the conventional measurements and also compared favourably with the relatively few previous liquid-phase cavity enhanced kinetic studies which have been performed using significantly more complex and expensive experimental setups.

Cavity-Enhanced Immunoassay Measurements in Microtiter Plates Using BBCEAS.

We report on the first detailed use of broadband cavity enhanced absorption spectroscopy (BBCEAS) as a detection system for immunoassay. A vertical R ≥ 0.99 optical cavity was integrated with a motorized XY stage, which functioned as a receptacle for 96-well microtiter plates. The custom-built cavity enhanced microplate reader was used to make measurements on a commercially available osteocalcin sandwich ELISA kit. A 30-fold increase in path length was obtained with a minimum detectable change in the absorption coefficient, αmin(t), of 5.3 × 10(-5) cm(-1) Hz(-1/2). This corresponded to a 39-fold increase in the sensitivity of measurement when directly compared to measurements in a conventional microplate reader. Separate measurements of a standard STREP-HRP colorimetric reaction in microtiter plates of differing optical quality produced an increase in sensitivity of up to 115-fold compared to a conventional microplate reader. The sensitivity of the developed setup compared favorably with previous liquid-phase cavity enhanced studies and approaches the sensitivity of typical fluorometric ELISAs. It could benefit any biochemical test which uses single pass absorption as a detection method, through either the label free detection of biologically important molecules at lower concentrations or the reduction in the amount of expensive biochemicals needed for a particular test, leading to cheaper tests.

Clarifying the disagreements on various reuse options: Repair, recondition, refurbish and remanufacture.

Earth's natural resources are finite. To be environmentally sustainable, it may not only be necessary to use them 'efficiently' but also 'effectively'. While we consider 'repair', 'recondition', 'refurbish' and 'remanufacture' to be 'reuse' options, not all researchers agree. Also, there is lack of clarity between the different options that are likely to be challenging for both; the policy makers who formulate policies aimed to encourage 'reuse' of 'waste' products and for decision makers to initiate appropriate action for recovering 'reusable resources' from 'waste streams'. This dichotomy could result into more 'waste' to landfill. A systematic analysis of peer reviewed literature is conducted to understand inconsistencies and/or lack of clarity that exist between the definitions or descriptions of identified `reuse' options. This article proposes a 'hierarchy of reuse options' that plots the relative positions of identified 'reuse' options vis-à-vis five variables, namely work content, energy requirement, cost, performance and warranty. Recommendations are made on how to incentivise original equipment manufacturers (OEMs) to 'remanufacture'. Finally, an alternative 'Type II Resource Effective Close-loop Model' is suggested and a conceptual 'Type II/2 Model of Resource Flows' that is restricted to the use of environmentally benign and renewable resources is introduced. These suggestions are likely to help decision makers to prioritise between 'reuse' options, drive resource effectiveness and also environmental sustainability.

An autonomous wheelchair with health monitoring system based on Internet of Thing.

Assistive powered wheelchairs will bring patients and elderly the ability of remain mobile without the direct intervention from caregivers. Vital signs from users can be collected and analyzed remotely to allow better disease prevention and proactive management of health and chronic conditions. This research proposes an autonomous wheelchair prototype system integrated with biophysical sensors based on Internet of Thing (IoT). A powered wheelchair system was developed with three biophysical sensors to collect, transmit and analysis users' four vital signs to provide real-time feedback to users and clinicians. A user interface software embedded with the cloud artificial intelligence (AI) algorithms was developed for the data visualization and analysis. An improved data compression algorithm Minimalist, Adaptive and Streaming R-bit (O-MAS-R) was proposed to achieve a higher compression ratio with minimum 7.1%, maximum 45.25% compared with MAS algorithm during the data transmission. At the same time, the prototype wheelchair, accompanied with a smart-chair app, assimilates data from the onboard sensors and characteristics features within the surroundings in real-time to achieve the functions including obstruct laser scanning, autonomous localization, and point-to-point route planning and moving within a predefined area. In conclusion, the wheelchair prototype uses AI algorithms and navigation technology to help patients and elderly maintain their independent mobility and monitor their healthcare information in real-time.

Discrimination of Sri Lankan black teas using fluorescence spectroscopy and linear discriminant analysis.

BACKGROUND: The quality of teas is currently graded using trained tea tasters, whose evaluation can sometimes be subjective. In this study the simple fluorescence-based technique of total luminescence spectroscopy (TLS) in conjunction with data classification using principal component analysis (PCA) was applied to discriminate between teas from 11 different Sri Lankan plantations. Solvent extraction of the tea samples was followed by TLS to record excitation-emission matrices in the excitation range 250-590 nm and emission range 300-700 nm. RESULTS: The application of PCA and linear discriminant analysis (LDA) allowed the successful classification of all 11 teas using only the first two principal components. LDA demonstrated how the technique was able to discriminate between all teas correctly with 100% classification. CONCLUSION: Further development of this work could lead to a simple device that could be used by tea manufacturers instead of or alongside trained tea tasters to grade teas.

Beyond liquid biopsy: Toward non-invasive assays for distanced cancer diagnostics in pandemics.

Liquid biopsy technologies have seen a significant improvement in the last decade, offering the possibility of reliable analysis and diagnosis from several biological fluids. The use of these technologies can overcome the limits of standard clinical methods, related to invasiveness and poor patient compliance. Along with this there are now mature examples of lab-on-chips (LOC) which are available and could be an emerging and breakthrough technology for the present and near-future clinical demands that provide sample treatment, reagent addition and analysis in a sample-in/answer-out approach. The possibility of combining non-invasive liquid biopsy and LOC technologies could greatly assist in the current need for minimizing exposure and transmission risks. The recent and ongoing pandemic outbreak of SARS-CoV-2, indeed, has heavily influenced all aspects of life worldwide. Ordinary tasks have been forced to switch from "in presence" to "distanced", limiting the possibilities for a large number of activities in all fields of life outside of the home. Unfortunately, one of the settings in which physical distancing has assumed noteworthy consequences is the screening, diagnosis and follow-up of diseases. In this review, we analyse biological fluids that are easily collected without the intervention of specialized personnel and the possibility that they may be used -or not-for innovative diagnostic assays. We consider their advantages and limitations, mainly due to stability and storage and their integration into Point-of-Care diagnostics, demonstrating that technologies in some cases are mature enough to meet current clinical needs.

Fabrication Methods for Microfluidic Devices: An Overview.

Microfluidic devices offer the potential to automate a wide variety of chemical and biological operations that are applicable for diagnostic and therapeutic operations with higher efficiency as well as higher repeatability and reproducibility. Polymer based microfluidic devices offer particular advantages including those of cost and biocompatibility. Here, we describe direct and replication approaches for manufacturing of polymer microfluidic devices. Replications approaches require fabrication of mould or master and we describe different methods of mould manufacture, including mechanical (micro-cutting; ultrasonic machining), energy-assisted methods (electrodischarge machining, micro-electrochemical machining, laser ablation, electron beam machining, focused ion beam (FIB) machining), traditional micro-electromechanical systems (MEMS) processes, as well as mould fabrication approaches for curved surfaces. The approaches for microfluidic device fabrications are described in terms of low volume production (casting, lamination, laser ablation, 3D printing) and high-volume production (hot embossing, injection moulding, and film or sheet operations).

Design and Evaluation of a Flexible Dual-Band Meander Line Monopole Antenna for On- and Off-Body Healthcare Applications.

The human body is an extremely challenging environment for wearable antennas due to the complex antenna-body coupling effects. In this article, a compact flexible dual-band planar meander line monopole antenna (MMA) with a truncated ground plane made of multiple layers of standard off-the-shelf materials is evaluated to validate its performance when worn by different subjects to help the designers who are shaping future complex on-/off-body wireless devices. The antenna was fabricated, and the measured results agreed well with those from the simulations. As a reference, in free-space, the antenna provided omnidirectional radiation patterns (ORP), with a wide impedance bandwidth of 1282.4 (450.5) MHz with a maximum gain of 3.03 dBi (4.85 dBi) in the lower (upper) bands. The impedance bandwidth could reach up to 688.9 MHz (500.9 MHz) and 1261.7 MHz (524.2 MHz) with the gain of 3.80 dBi (4.67 dBi) and 3.00 dBi (4.55 dBi), respectively, on the human chest and arm. The stability in results shows that this flexible antenna is sufficiently robust against the variations introduced by the human body. A maximum measured shift of 0.5 and 100 MHz in the wide impedance matching and resonance frequency was observed in both bands, respectively, while an optimal gap between the antenna and human body was maintained. This stability of the working frequency provides robustness against various conditions including bending, movement, and relatively large fabrication tolerances.

Production of rhamnolipid biosurfactants by Pseudomonas aeruginosa DS10-129 in a microfluidic bioreactor.

A low-cost microBR (microbioreactor) made from PTFE [poly(tetrafluoroethylene)] was used to cultivate a model organism, Pseudomonas aeruginosa DS10-129. The progress of bioprocessing was monitored by comparing the growth of the organism in a microBR, a conventional bench scale bioreactor and a shake flask. Under the microBR conditions, the organism produced 23 mg/ml of pyocyanin that had antimicrobial effects against Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas teessidea and Pseudomonas clemancea. Furthermore, it produced a total of 106 microg/ml of effective biosurfactants consisting of dirhamnolipids (RL2) and monorhamnolipids (RL1). The biosurfactants reduced the surface tension of distilled water from 72 to 27.9 mN/m and emulsified kerosene by 71.30%. The pyocyanin and rhamnolipids were produced during the exponential and stationary phases of growth respectively. The results of the microBR were comparable to those obtained using conventional scale methods.

Biomarkers for Point-of-Care Diagnosis of Sepsis.

Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. In 2017, almost 50 million cases of sepsis were recorded worldwide and 11 million sepsis-related deaths were reported. Therefore, sepsis is the focus of intense research to better understand the complexities of sepsis response, particularly the twin underlying concepts of an initial hyper-immune response and a counter-immunological state of immunosuppression triggered by an invading pathogen. Diagnosis of sepsis remains a significant challenge. Prompt diagnosis is essential so that treatment can be instigated as early as possible to ensure the best outcome, as delay in treatment is associated with higher mortality. In order to address this diagnostic problem, use of a panel of biomarkers has been proposed as, due to the complexity of the sepsis response, no single marker is sufficient. This review provides background on the current understanding of sepsis in terms of its epidemiology, the evolution of the definition of sepsis, pathobiology and diagnosis and management. Candidate biomarkers of interest and how current and developing point-of-care testing approaches could be used to measure such biomarkers is discussed.

Shunting microfluidic PCR device for rapid bacterial detection.

Polymerase chain reaction (PCR) is commonly used for the analysis of nucleic acids in a variety of applications including clinical. There is, however, a need for a low cost portable PCR device that allows rapid identification of pathogenic bacteria. We report a shunting PCR microfluidic device comprising: polycarbonate microfluidic PCR chip; shunting thermal cycler and fluorescence detector. The microfluidic PCR chip - fabricated using micro-milling and thermal fusion bonding for sealing of the cover - was shunted between three double side temperature zones for thermal cycling. Rapid amplification was observed with heating and cooling rates of 1.8 °C/s and 2 °C/s respectively. Lock-in photodetector for fluorescence detection of the microfluidic PCR chip achieved at 95% confidence an LOD of 75pM FITC and 0.7 ng µl-1 of dsDNA using a QuantiFluor assay kit. The device was validated using universal primers - based on chromosomal DNA extracted from non-pathogenic K-12 subtype of Escherichia coli (E. coli) - for amplification of fragments of 250, 552 and 1500 bp. PCR amplification was demonstrated, with annealing temperatures ranging between 54 °C and 68 °C, and confirmed using gel electrophoresis. The developed shunting PCR microfluidic device will allow for low cost and portable nucleic acid amplification for the detection of infectious diseases.

Modulation of Macrophage Function by Lactobacillus-Conditioned Medium.

Probiotics are used as microbial food supplements for health and well-being. They are thought to have immunomodulatory effects although their exact physiological mechanism of action is not clear. This study investigated the influence of probiotic Lactobacillus rhamnosus GG conditioned media (LGG-CM) on macrophage phagocytosis of non-pathogenic Escherichia coli HfrC. The gentamicin protection assay was used to study the bacterial killing phases of phagocytosis. Macrophages co-incubated with E. coli for an hour allowed them to ingest bacteria and then the rate of E. coli killing was monitored for up to 300 min to determine the killing or digestion of the bacteria by recovering them from the macrophage lysate. We found that the LGG-CM significantly increased the bacterial killing by approximately 6-fold when compared with that of controls. By contrast, this killing process was found to be associated with enhanced free radical production via the activation of NADPH oxidase, stimulated by the LGG conditioned medium. We also found that the conditioned medium had small effect on nitric oxide (NO) generation, albeit to a lesser extent. This work suggests that LGG-CM may play an important role in suppressing the total microbial load within the macrophages and hence, the extent to which pro-inflammatory molecules such as free radicals and NO are generated. The modulation of inflammation-promoting signals by LGG-CM may be beneficial as it modulates bacterial killing, and thereby prevents any collateral damage to host.

Broadband cavity enhanced absorption spectroscopy as a detector for HPLC.

The first demonstration of a cavity enhanced absorption spectroscopy (CEAS) based technique, applied to HPLC detection is reported. Broadband cavity enhanced absorption spectroscopy (BBCEAS) has been used for detection in a HPLC system (HPLC-BBCEAS). Measurements were made on the dyes rhodamine 6G and rhodamine B between 450 and 600 nm. The sensitivity of the measurements as determined by the minimum detectable change in the absorption coefficient, alpha(min), were 2.9 x 10(-5) cm(-1) at 527 nm and 1.9 x 10(-5) cm(-1) at 556 nm, the peak absorption wavelengths of rhodamine 6G and rhodamine B, respectively. The limits of detection (LOD) for the two dyes were 426 and 271 pM, respectively. The LOD of the HPLC-BBCEAS setup was found to be between 54 and 77 times lower than with a Perkin-Elmer HPLC (series 200) comprising a 200EP photodiode array detector. The sensitivity of the developed setup also compared favorably with the previous single wavelength HPLC-CRDS studies while using a considerably lower cost experimental setup and simpler experimental methodology. The use of BBCEAS detection also allowed the discrimination following an isocratic HPLC separation of the nearly co-eluting dyes rhodamine 6G and rhodamine B.

Liquid-phase broadband cavity enhanced absorption spectroscopy (BBCEAS) studies in a 20 cm cell.

Sensitive liquid-phase measurements have been made in a 20 cm cell using broadband cavity enhanced absorption spectroscopy (BBCEAS). The cavity was formed by two high reflectivity mirrors which were in direct contact with the liquid-phase analytes. Careful choice of solvent was required to minimise the effect of background solvent absorptions. Measurements were made on the broad absorber Sudan black, dissolved in acetonitrile, using a white LED light source and R > or = 0.99 cavity mirrors, leading to a cavity enhancement factor (CEF) of 82 at 584 nm. The sensitivity as measured by the minimum detectable change in the absorption coefficient (alpha(min)) was 3.4 x 10(-7) cm(-1). Further measurements were made on the strong absorber methylene blue dissolved in acetonitrile at 655 nm. A white LED was used with the R > or = 0.99 cavity mirrors, leading to a CEF of 78 and alpha(min) = 4.4 x 10(-7) cm(-1). The use of a more intense red LED also allowed measurements with higher reflectivity R > or = 0.999 cavity mirrors, leading to a CEF of 429 and alpha(min) = 2.8 x 10(-7) cm(-1). The sensitivity was limited by dark noise from the detector but nevertheless appears to represent the most sensitive liquid-phase absorption measurement to date.

Development of a simple and low cost microbioreactor for high-throughput bioprocessing.

A simple microbioreactor for high-throughput bioprocessing made from low cost polymer polytetrafluoroethylene (PTFE) tubes with a working volume of 1.5 ml is described. We have developed a microfluidic system that handles a small population of cells of a model microorganism, Pseudomonas aeruginosa DS10-129. Under the conditions of the microbioreactor, the organism produced extracellular secondary metabolites by using nutrient broth modified with glycerol. Pyocyanins were isolated from the fermented medium as a metabolite of interest. Antibiotic properties of pyocyanin were effective against a number of microorganisms such as Staphylococcus aureus, S. epidermis, Bacillus subtilis, Micrococcus luteus and Saccharomyces cerevisiae. Batch fermentation of the model organism in the microbioreactor was compared to shake-flask and conventional bench fermenter methods. Results obtained from the microbioreactor compared favourably with the conventional processes.

Impedimetric array in polymer microfluidic cartridge for low cost point-of-care diagnostics.

Deep Vein Thrombosis and pulmonary embolism (DVT/PE) is one of the most common causes of unexpected death for hospital in-patients. D-dimer is used as a biomarker within blood for the diagnosis of DVT/PE. We report a low-cost microfluidic device with a conveniently biofunctionalised interdigitated electrode (IDE) array and a portable impedimetric reader as a point-of-care (POC) device for the detection of D-dimer to aid diagnosis of DVT/PE. The IDE array elements, fabricated on a polyethylenenaphtalate (PEN) substrate, are biofunctionalised in situ after assembly of the microfluidic device by electropolymerisation of a copolymer of polypyrrole to which is immobilised a histidine tag anti-D-Dimer antibody. The most consistent copolymer films were produced using chronopotentiometry with an applied current of 5µA for a period of 50 s using a two-electrode system. The quality of the biofunctionalisation was monitored using optical microscopy, chronopotentiometry curves and impedimetric analysis. Measurement of clinical plasma sample with a D-dimer at concentration of 437 ng/mL with 15 biofunctionalised IDE array electrodes gave a ratiometric percentage of sample reading against the blank with an average value of 124 ±â€¯15 at 95% confidence. We have demonstrated the concept of a low cost disposable microfluidic device with a receptor functionalised on the IDE array for impedimetric detection towards POC diagnostics. Changing the receptor on the IDE array would allow this approach to be used for the direct detection of a wide range of analytes in a low cost manner.