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Vertical flow assays have been developed in recent years addressing limitations of the lateral flow assays, including limited multiplexing capability, quantitation, and hook effect. In the present study, the first passive paper-based vertical flow assay is developed for the detection of the nucleic acid target. Horseradish peroxidase was used as an enzymatic tracer with a high potential for signal amplification. In order to achieve the best signal-to-noise ratio, different parameters of paper-based assays were optimized. The sample is heat denatured and hybridized with a specific probe to form a dual-labeled hybridization complex. A small volume of diluted sample, 12 µl, can be analyzed within 6 min on the assay in a sandwich format. Assay specificity was evaluated by testing different unrelated samples, and also, 1.7 nM was obtained as the limit of detection (LOD) using the 0 + 3SD method, which is equivalent to 8.5 fmol of double-stranded DNA in the 12 µl sample volume. The linear range of 3-194 nM with a 0.978 correlation coefficient was obtained according to the calibration curve. The developed assay was evaluated with 45 hepatitis B virus clinical plasma samples, and the result showed 100% consistency of the assay with the real-time PCR benchmark. In the present study, we sought to develop a mere detection system for nucleic acid targets, and to investigate the possibility of using enzyme reporter in a passive vertical flow assay.

In the future, rapid electrical characterization of cells with impedance flow cytometry promises to be a fast and accurate method for the evaluation of cell properties. In this paper, we investigate how the conductivity of the suspending medium along with the heat exposure time affects the viability classification of heat-treated E. coli. Using a theoretical model, we show that perforation of the bacteria membrane during heat exposure changes the impedance of the bacterial cell from effectively less conducting than the suspension medium to effectively more conducting. Consequently, this results in a shift in the differential argument of the complex electrical current that can be measured with impedance flow cytometry. We observe this shift experimentally through measurements on E. coli samples with varying medium conductivity and heat exposure times. We show that increased exposure time and lower medium conductivity results in improved classification between untreated and heat-treated bacteria. The best classification was achieved with a medium conductivity of 0.045 S/m after 30 min of heat exposure.

Microfluidic biochips have been in the scientific spotlight for over two decades, and although technologically advanced, they still struggle to deliver on the promise for ubiquitous miniaturization and automation for the biomedical sector. One of the most significant challenges hindering the technology transfer is the lack of standardization and the resulting absence of a common infrastructure. Moreover, microfluidics is an interdisciplinary field, but research is often carried out in a cross-disciplinary manner, focused on technology and component level development rather than on a complete future-proof system. This paper aims to raise awareness and facilitate the next evolutionary step for microfluidic biochips: to establish a holistic application-agnostic common microfluidic architecture that allows for gracefully handling changing functional and operational requirements. Allowing a microfluidic biochip to become an integrated part of a highly reconfigurable cyber-fluidic system that adopts the programming and operation model of modern computing will bring unmatched degrees of programmability and design reusability into the microfluidics field. We propose a three-tier architecture consisting of fluidic, instrumentation, and virtual systems that allows separation of concerns and promotes modularity. We also present BiowareCFP as a platform-based implementation of the outlined concepts. The proposed cyber-fluidic architecture and the BiowareCFP facilitate the integration between the virtual and the fluidic domains and pave the way for seamless integration between the cyber-fluidic and biological systems.

In this work we present the results obtained on the simulation and nanofabrication of photonic crystals based on silicon nanopillars. The simulations show the formation of photonic band gaps within 1.31 and 1.89μm, with a gap-to-midgap ratio approaching 40%. The introduction of waveguides and cavities prove the adaptability of these structures to tune the wavelengths allowed to be transmitted through the system within the photonic band gaps. On the other hand, thanks to the use of advanced nanofabrication techniques, the modelled structures have been successfully fabricated.

In this work we have explored the use of a Surface Plasmon resonance (SPR) phenomenon for the detection of interleukin-6 (IL-6), a pro-inflammatory cytokine. It plays an important role in the muscle tissues, having direct relation with muscle contraction and, thus, it is considered a biomarker for some types of muscular dystrophies. Here we show that SPR can be used as a real-time monitoring of the shift of the reflectance dip of a gold diffraction grating in front to the antibody adhesion to gold.

A graphical user interface (GUI) with Tkinter was created in Python to facilitate data analysis for electrochemical techniques in cyclic voltammetry and electrochemical impedance. The GUI lets the user select a technique, measurement(s), and eventually customize the plots.

The ongoing COVID-19 pandemic has shown the importance of having analytical devices that allow a simple, fast, and robust detection of pathogens which cause epidemics and pandemics. The information these devices can collect is crucial for health authorities to make effective decisions to contain the disease's advance. The World Health Organization published a list of primary pathogens that have raised concern as potential causes of future pandemics. Unfortunately, there are no rapid diagnostic tests commercially available and approved by the regulatory bodies to detect most of the pathogens listed by the WHO. This report describes these pathogens, the available detection methods, and highlights areas where more attention is needed to produce rapid diagnostic tests for future pandemic surveillance.

Bacteria detection, counting and analysis is of great importance in several fields. When viability plays a major role in decision making, the counting of colony-forming units grown on agar plates remains the gold standard. However, because plate counts depend on the growth of the bacteria, it is a slow procedure and only works with culturable species. Impedance flow cytometry (IFC) is a promising technology for particle detection, counting and characterization. It relies on the perturbation of an electric field by particles flowing through a microfluidic channel. The perturbation is directly related to the electrical properties of the particles, and therefore provides information about their composition and structure. In this work we investigate whether IFC can be used to differentiate viable cells from inactivated cells. Our findings demonstrate that the specific viability state of the bacteria has to be considered, but that with proper characterization thresholds, IFC can be used to classify bacterial viability states. By using three different inactivation methods&mdash, ethanol, heat and autoclavation&mdash, we have been able to show that the impedance response of Escherichia coli depends on its viability state, but that the specific response depends on the inactivation method. With these findings we expect to be able to optimize IFC for more reliable bacteria detection and counting in the future.

Lateral flow assays (LFAs) are low-cost testing tools widely used for home, point-of-care, or laboratory medical diagnostics. These tests typically use colorimetry to report the presence and the concentration of a certain physical/ biological quantity, showing the result as a color marker. This work presents a computer vision algorithm for the digitalization of LFA readouts, enabling precise and reliable results at low-cost. The algorithm receives as input an image of a sample, identifies the color marker, and computes its average color intensity. In contrast to existing algorithms, the proposed one can detect color markers that are not characterized by a predetermined precise shape, size, and position, since the topology is identified and analyzed by the algorithm itself. The evaluation of the proposed algorithm on a set of LFA strips shows correct functionality and execution time of less than a second.

Primary brain cells cultured on flat surfaces, i.e., in a two-dimensional fashion, have a long history of use as an experimental model system in neuroscience research. However, it is questionable to which extent these cultured brain cells resemble their in vivo counterparts. Mainly, it has been claimed that the non-oxidative glucose metabolism reflected by lactate production is unphysiologically high. Furthermore, it is known that culturing in 2D alters the phenotype of cells. Here we present diphenylalanine peptide nanowires (PNWs) as a culturing substrate for primary neocortical neurons from mice. The topology of the PNWs leads to neuronal cultures developing in 2.5D environment and hence improved culturing conditions. We investigate the effect of different concentrations of PNWs and different cell densities of neurons on the culturing conditions. The neocortical neurons were examined through scanning electron microscopy in order to study the effect of PNW concentrations and neuron densities on the structural appearance of the cells. Then employing the optimal combination of neuron density and PNW concentration, the neurons were evaluated functionally and metabolically by comparison with neocortical neurons standard culturing methods in 2D. Specifically, we tested neuronal viability, capacity for vesicular release of neurotransmitter GABA, as well as oxidative and non-oxidative glucose metabolism. It was evident that neurons cultured on PNWs exhibited increased viability combined with an increased capacity for neurotransmitter release and a lower fraction of non-oxidative metabolism than neurons cultured in 2D. Hence, neocortical neurons cultured in 2.5D on PNWs appear to be healthier and less glycolytic than neurons cultured in 2D.

Pseudomonas aeruginosa (PA) is a pathogen that is recognized for its advanced antibiotic resistance and its association with serious diseases such as ventilator-associated pneumonia and cystic fibrosis. The ability to rapidly detect the presence of pathogenic bacteria in patient samples is crucial for the immediate eradication of the infection. Pyocyanin is one of PA’s virulence factors used to establish infections. Pyocyanin promotes virulence by interfering in numerous cellular functions in host cells due to its redox-activity. Fortunately, the redox-active nature of pyocyanin makes it ideal for detection with simple electrochemical techniques without sample pretreatment or sensor functionalization. The previous decade has seen an increased interest in the electrochemical detection of pyocyanin either as an indicator of the presence of PA in samples or as a tool for quantifying PA virulence. This review provides the first overview of the advances in electrochemical detection of pyocyanin and offers an input regarding the future directions in the field.

Next‐generation genome sequencing machines and Point‐of‐Care (PoC) in vitro diagnostics devices are precursors of an emerging class of Cyber‐Physical Systems (CPS), one that harnesses biomolecular‐scale mechanisms to enable novel "wet‐technology" applications in medicine, biotechnology, and environmental science. Although many such applications exist, testifying the importance of innovative life‐sciences instrumentation, recent events have highlighted the difficulties that designing organizations face in their attempt to guarantee safety, reliability, and performance of this special class of CPS. New regulations and increasing competition pressure innovators to rethink their design and engineering practices, and to better address the above challenges. The pace of innovation will be determined by how organizations manage to ensure the satisfaction of aforementioned constraints while also streamlining product development, maintaining high cost‐efficiency and shortening time‐to‐market. Model‐Based Systems Engineering provides a valuable framework for addressing these challenges. In this paper, we demonstrate that existing and readily available model‐based development frameworks can be adopted early in the life‐sciences instrumentation design process. Such frameworks are specifically helpful in describing and characterizing CPS including elements of a biological nature both at the architectural and performance level. We present the SysML model of a smartphone‐based PoC diagnostics system designed for detecting a particular molecular marker. By modeling components and behaviors spanning across the biological, physical‐nonbiological, and computational domains, we were able to characterize the important systemic relations involved in the specification of our system's Limit of Detection. Our results illustrate the suitability of such an approach and call for further work toward formalisms enabling the formal verification of systems including biomolecular components.

As a result of biological sciences becoming more quantitative together with the growing economical and societal challenges of improving our health care system, we have witnessed an increasing interest in developing new technologies for the biomedical field. Cyber-medical systems are the fusion of computational and medical technologies aimed to support health management for diagnosis, monitoring, and prevention of diseases. With the advent of miniaturized biochemical laboratories, the classical bench-sized lab robots have transformed into chip-sized complex biochemical laboratories. This development has allowed integration of classical computation with biochemical processes to a degree where computations are moving small amounts of liquids. In this chapter, we survey state-of-the-art microfluidic technologies and argue that even though their applications are still limited to simple passive and fixed structures, they hold the promises of solving many challenging and complex problems related to healthcare. We argue that liquid handling technologies have the potential to scale, but this will require utilization of active components and the ability to abstract their basic operations to a level similar to that of classical computation, i.e., we need to build the equivalent of a general purpose processor—a lab-on-chip (or a biochip) which can be programmed and re-programmed.

Bentazone is one of the most problematic pesticides polluting groundwater resources. It is on the list of pesticides that are mandatory to analyze at water work controls. The current pesticide measuring approach includes manual water sampling and time-consuming chromatographical quantification of the bentazone content at centralized laboratories. Here, we report the use of an electrochemical approach for analytical determination of bentazone that takes 10 s. The electrochemical electrodes were manually screen printed, resulting in the low-cost fabrication of the sensors. The current response was linearly proportional to the bentazone concentration with a R2 ~ 0.999. We demonstrated a sensitivity of 0.0987 μA/μM and a limit of detection of 0.034 μM, which is below the U.S. Health Advisory level. Furthermore, the sensors have proved to be reusable and stable with a drop of only 2% after 15 times reuse. The sensors have been applied to successfully quantify bentazone spiked in real groundwater and lake water. The sensing method presented here is a step towards on-site application of electrochemical detection of pesticides in water sources. Keywords: Bentazone, Sensor, Groundwater, Electrochemistry, Pesticide, Contamination

Pyocyanin is a virulence factor solely produced by the pathogen Pseudomonas aeruginosa. Pyocyanin is also a redox active molecule that can be directly detected by electrochemical sensing. A nanograss (NG) based sensor for sensitive quantification of pyocyanin in sputum samples from cystic fibrosis (CF) patients is presented here. The NG sensors were custom made in a cleanroom environment by etching nanograss topography on the electrode surface followed by depositing 200 nm gold. The NG sensors were utilized for amperometric quantification of pyocyanin in spiked hypertonic saline samples, resulting in a linear calibration curve with a R2 value of 0.9901 and a limit of detection of 172 nM. The NG sensors were applied in a small pilot test on five airway samples from five CF patients. The NG sensor was capable of identifying P. aeruginosa in the airway samples in 60 s without any sample pretreatment.

The micro flow cytometer presented here is a combination of a set of coplanar electrodes and a microfluidic channel to test cell viability based on Single-cell Electrical Impedance Spectroscopy analysis. The device was tested both with a control and with a treated HeLa cells suspension. Impedance signals acquired were filtered and processed to obtain the data, classifying and clustering the results by dead and living cells to make the cell viability test. Results were compared with the standards of MTT and Neubauer Chamber viability tests, showing that $0.83\mu \mathbf{L}$ of sample in the prototype designed and fabricated in this work was enough to obtain an approximate response of them.

Monitoring of bacteria concentrations is of great importance in drinking water management. Continuous real-time monitoring enables better microbiological control of the water and helps prevent contaminated water from reaching the households. We have developed a microfluidic sensor with the potential to accurately assess bacteria levels in drinking water in real-time. Multi frequency electrical impedance spectroscopy is used to monitor a liquid sample, while it is continuously passed through the sensor. We investigate three aspects of this sensor: First we show that the sensor is able to differentiate Escherichia coli (Gram-negative) bacteria from solid particles (polystyrene beads) based on an electrical response in the high frequency phase and individually enumerate the two samples. Next, we demonstrate the sensor&rsquo, s ability to measure the bacteria concentration by comparing the results to those obtained by the traditional CFU counting method. Last, we show the sensor&rsquo, s potential to distinguish between different bacteria types by detecting different signatures for S. aureus and E. coli mixed in the same sample. Our investigations show that the sensor has the potential to be extremely effective at detecting sudden bacterial contaminations found in drinking water, and eventually also identify them.

Glyphosate (Gly) is one of the most problematic pesticides that repeatedly appears in drinking water. Continuous on-site detection of Gly in water supplies can provide an early warning in incidents of contamination, before the pesticide reaches the drinking water. Here, we report the first direct detection of Gly in tap water with electrochemical sensing. Gold working electrodes were used to detect the pesticide in spiked tap water without any supporting electrolyte, sample pretreatment or electrode modifications. Amperometric measurements were used to quantify Gly to a limit of detection of 2 &mu, M, which is below the regulation limit of permitted contamination of drinking water in the United States. The quantification of Gly was linearly proportional with the measured signal. The selectivity of this method was evaluated by applying the same technique on a Gly Metabolite, AMPA, and on another pesticide, omethoate, with a chemical structure similar to Gly. The testing revealed no interfering electrochemical activity at the potential range used for Gly detection. The simple detection of Gly presented in this work may lead to direct on-site monitoring of Gly contamination at drinking water sources.

Candidemia and invasive candidiasis is a cause of high mortality and morbidity rates among hospitalized patients worldwide. The occurrence of the infections increases due to the complexity of the patients and overuse of the antifungal therapy. The current Candida detection method includes blood culturing which is a lengthy procedure and thus delays the administration of the antifungal therapy. Even though the results are available after 48 h it is still the gold standard in pathogen detection in a hospital setting. In this work we present an electrochemical impedance sensor that is capable of detecting Candida albicans yeast. The yeast cells are captured on electrodes specifically functionalized with anti-Candida antibodies and detection is achieved by electrochemical impedance spectroscopy. The sensor allows for detection of the yeast cells at clinically relevant concentrations in less than 1 h.

Miniaturizing the traditional wet lab processes and realizing the full potential of lab-on-a-chip technology (LOC) has been a great challenges for scientists and engineers in the quest of providing health care where such does not exists, or to increase lab efficiency and lower the operational cost. A range of practical LOC fabrication technologies has already been demonstrated, and as a result, the research of real life applications has been steadily increasing. However, due to the variety of used technologies and LOC form factors, it often requires a modification or even a complete redesign of the available instrumentation systems. Standardization and flexible instrumentation has seen limited development in the LOC field even though developing on all application levels has been proven as an effective way to stimulate a growth in a research field. To address this, we present a modular digital microfluidics (DMF) instrumentation platform and a concept of integrated chip-instrument co-design that has the potential to solve a wide range of LOC instrumentation challenges.

Based on a modified version of standard chips for fast differential scanning calorimetry, DSC of liquid samples has been performed at temperature scan rates of up to 1000 °C/s. This paper describes experimental results with the protein lysozyme, bovine serum, and olive oil. The heating and cooling rate of the sensor is measured for temperature scan rates of up to 1300 °C/s with water and 2-butanol, in the temperature range of −90 °C/s to +130 °C/s. The lysozyme is measured at temperature scan rates varying from 10 °C/s to 400 °C/s and in concentrations between 0.1% and 10% protein by weight. The bovine serum measurements show two main peaks, in good agreement with standard DSC measurements. Olive oil has been measured, with good agreement for the cooling curve and qualitative agreement for the heater curve, compared to DSC measurements.

This work presents a versatile, membrane based electrochemical sensor with thin film electrodes fabricated through E-beam evaporation directly on porous materials (membranes). Here, the fabrication of the electrodes is described along with possible methods for integration in fluidic systems and characterisation of the electrodes through cyclic voltammetry (CV). The continued porous nature of the membranes after metal deposition is documented and its robustness and stability is investigated. Furthermore, amperometric sensing of dopamine is demonstrated as a proof of concept to validate the usability of the membrane sensor.

Although silicon nanowire biological field-effect transistors (SiNW-bioFETs) have steadily demonstrated their ability to detect biological markers at ultra-low concentration, they have not yet translated into routine diagnostics applications. One of the challenges inherent to the technology is that it requires an instrumentation capable of recovering ultra-low signal variations from sensors usually designed and operated in a highly resistive configuration. Often overlooked, the SiNW-bioFET/instrument interactions are yet critical factors in determining overall system biodetection performances. Here, we carry out, for the first time, the system-level sensitivity analysis of a generic SiNW-bioFET model coupled to a custom-design instrument based on the lock-in amplifier. By investigating a large parametric space spanning over both sensor and instrumentation specifications, we demonstrate that system-wide investigations can be instrumental in identifying the design trade-offs that will ensure the lowest limits of detection. The generic character of our analytical model allows us to elaborate on the most general SiNW-bioFET/instrument interactions and their overall implications on detection performances. Our model can be adapted to better match specific sensor or instrument designs to either ensure that ultra-high sensitivity SiNW-bioFETs are coupled with an appropriately sensitive and noise-rejecting instrumentation, or to best tailor SiNW-bioFET design to the specifications of an existing instrument.

This article presents a novel membrane-based sensor for real-time electrochemical investigations of cellular- or tissue cultures. The membrane sensor enables recording of electrical signals from a cell culture without any signal dilution, thus avoiding loss of sensitivity. Moreover, the porosity of the membrane provides optimal culturing conditions similar to existing culturing techniques allowing more efficient nutrient uptake and molecule release. The patterned sensor electrodes were fabricated on a porous membrane by electron-beam evaporation. The electrochemical performance of the membrane electrodes was characterized by cyclic voltammetry and chronoamperometry, and the detection of synthetic dopamine was demonstrated down to a concentration of 3.1 pM. Furthermore, to present the membrane-sensor functionality the dopamine release from cultured PC12 cells was successfully measured. The PC12 cells culturing experiments showed that the membrane-sensor was suitable as a cell culturing substrate for bio-applications. Real-time measurements of dopamine exocytosis in cell cultures were performed, where the transmitter release was recorded at the point of release. The developed membrane-sensor provides a new functionality to the standard culturing methods, enabling sensitive continuous in vitro monitoring and closely mimicking the in vivo conditions.

Metaphase chromosome spreading is the most crucial step required for successful karyotyping and FISH analysis. These two techniques are routinely used in cytogenetics to assess the chromosome abnormalities. The spreading process has been studied for years but it is still considered an art more than a science. The chromosome spreading greatly depends on the environmental conditions such as humidity and temperature, which govern the evaporation of fixative, in which the cells are suspended. The spreading is normally performed manually in ambient conditions on glass slides, which are hydrophilic, and thus allow for better quality spreads. Further cytogenetic analysis depends on the quality of the spreads, which is dependent on the skills of the personnel and is thus limited to laboratory settings. Here, we present a semi-closed microfluidic chip for preparation of the metaphase spreads on a glass and a Topasr substrate rendered more hydrophilic by oxygen plasma treatment coupled with photografting. The device consists of a microfluidic chamber with perfusion holes that facilitate the evaporation of fixative and reliable formation of the spreads. The usability of the chromosome spreads formed on the glass and the Topasr slide is tested by performing FISH analysis.

A modified and improved photografting procedure of Topas® surface hydrophilization is investigated in order to obtain stable modification of the polymer for long term storage. The achieved hydrophilicity and monitoring of the wettability during one month of storage are presented as well as a description of the optimal cleaning procedure and storage conditions to maintain the modified surface. Three minutes of oxygen plasma activation followed by 4 min of acrylic acid UV-photografting at 50 °C leads to the most stable hydrophilicity that was characterized by an initial water contact angle of 53.5° ± 1.2°. Storage of the modified material in cold water at 4 °C and refraining from ultrasonic cleaning limit water contact angle increase to 5° over 30 days. In comparison with pristine hydrophobic Topas, the proposed treatment improves chromosome spreading ability significantly.

A new method for measuring specific protein concentrations in solutions has been developed. The technique makes use of the Coulter effect for detecting and sizing of micro-scaled objects suspended in a buffer fluid. The method is completely label-free as it is only based on the electrical readout when a suspension of microscopic beads flows over a set of electrodes in a microfluidic device. Since no electrode functionalization is needed the same device can be used in a number of different assays. Using goat-anti-rat IgG functionalized polystyrene beads we have shown proof of principle detecting rat IgG in solution. When the analyte (rat IgG) is present oligomers of beads are formed. The electrical readout of the oligomers is different compared to a zero control sample with no rat IgG. Detection of the protein has been performed in a concentration as small as 14 ng/mL. The dynamic range of the system has been demonstrated to be relatively large, ranging from 1 μg/mL to 14 ng/mL. The microfluidic system is made from polymer and glass and very little volume of sample (

Downscaling of microfluidic cell culture and detection devices for electrochemical monitoring has mostly focused on miniaturization of the microfluidic chips which are often designed for specific applications and therefore lack functional flexibility. We present a compact microfluidic cell culture and electrochemical analysis platform with in-built fluid handling and detection, enabling complete cell based assays comprising on-line electrode cleaning, sterilization, surface functionalization, cell seeding, cultivationand electrochemical real-time monitoring of cellular dynamics. To demonstrate the versatility and multifunctionality of the platform, we explored amperometric monitoring of intracellular redox activity in yeast (Saccharomyces cerevisiae) and detection of exocytotically released dopamine from rat pheochromocytoma cells (PC12). Electrochemical impedance spectroscopy was used in bothapplications for monitoring cell sedimentation and adhesion as well as proliferation in the case of PC12 cells. The influence of flow rate on the signal amplitude in the detection of redox metabolism as well as the effect of mechanical stimulation on dopamine release were demonstrated using the programmable fluid handling capability. The here presented platform is aimed at applications utilizing cell based assays, ranging from e.g. monitoring of drug effects in pharmacological studies, characterization of neural stem cell differentiation, and screening of genetically modified microorganisms to environmental monitoring.

Protein nanofibrils and nanotubes are now widely accepted as having potential for use in the field of bionanotechnology. For this to be a feasible alternative to existing technologies, there is a need for a commercially viable source. Previous work has identified amyloid fibrils formed from crude crystallin proteins as such a source, since these fibrils can be produced in large quantities at a low cost. Applications include use of fibrils as templates for the formation of nanowires or as biosensing scaffolds. There remains a number of practical considerations, such as stability and the ability to control their arrangement. In this study, crude crystallin amyloid fibrils are shown to be stable in a range of biological and clean room solvents, with the fibril presence confirmed by transmission electron microscopy and the thioflavin T fluorescent assay. The fibrils were also immobilised between microelectrodes using dielectrophoresis, which enabled the recording of I-V curves for small numbers of fibrils. This investigation showed the fibrils to have low conductivity, with current values in the range of 10(-10) A recorded. This low conductivity could be increased through modification, or alternately, the fibrils could be used unmodified for applications where they can act as templates or high surface area nanoscaffolds.

This Letter involved the preparation of a conjugate between single-walled carbon nanotubes and folic acid that was obtained without covalent chemical functionalization using a simple ‘one pot’ synthesis method. Subsequently, the conjugate was investigated by a computational hybrid method: our own N-layered Integrated Molecular Orbital and Molecular Mechanics (B3LYP(6–31G(d):UFF)). The results confirmed that the interaction occurred via hydrogen bonding between protons of the glutamic moiety from folic acid and π electrons from the carbon nanotubes. The single-walled carbon nanotube-folic acid conjugate presented herein is believed to lead the way to new potential applications as carbon nanotube-based drug delivery systems.

The purpose of this work is to develop novel 3D micro- and nanoelectrodes and pipettes by use of carefully optimised standard microfabrication techniques such as wet (by KOH) and dry silicon etching. Two types of electrodes have been fabricated and characterized: small nanoelectrodes to be used for localised measurements on cell cultures and high aspect ratio scalloped microelectrodes for measurements in brain slices. This paper presents improved fabrication processes for both types of electrodes and the pipettes, as well as the electrical and electrochemical characterization of the small electrodes in order to confirm their functionality. Although functional, an increase in the electrode surface area is needed if they are to be used for electrophysiological measurements. Finally, the pipettes fabricated have openings of the order of 500nm, which makes them ideal candidates for localised stimulation of cell or brain slice cultures.

The association of smart mobile devices and lab-on-chip technologies offers unprecedented opportunities for the emergence of direct-to-consumer in vitro medical diagnostics applications. Despite their clear transformative potential, obstacles remain to the large-scale disruption and long-lasting success of these systems in the consumer market. For instance, the increasing level of complexity of instrumented lab-on-chip devices, coupled to the sporadic nature of point-of-care testing, threatens the viability of a business model mainly relying on disposable/consumable lab-on-chips. We argued recently that system evolvability, defined as the design characteristic that facilitates more manageable transitions between system generations via the modification of an inherited design, can help remedy these limitations. In this paper, we discuss how platform-based design can constitute a formal entry point to the design and implementation of evolvable smart device/lab-on-chip systems. We present both a hardware/software design framework and the implementation details of a platform prototype enabling at this stage the interfacing of several lab-on-chip variants relying on current- or impedance-based biosensors. Our findings suggest that several change-enabling mechanisms implemented in the higher abstraction software layers of the system can promote evolvability, together with the design of change-absorbing hardware/software interfaces. Our platform architecture is based on a mobile software application programming interface coupled to a modular hardware accessory. It allows the specification of lab-on-chip operation and post-analytic functions at the mobile software layer. We demonstrate its potential by operating a simple lab-on-chip to carry out the detection of dopamine using various electroanalytical methods.

Scalability is a design principle often valued for the engineering of complex systems. Scalability is the ability of a system to change the current value of one of its specification parameters. Although targeted frameworks are available for the evaluation of scalability for specific digital systems, methodologies enabling scalability analysis of multidomain, complex systems, are still missing. In acknowledgment of the importance for complex systems to present the ability to change or evolve, we present in this work a systemlevel model-based methodology allowing the multidisciplinary parametric evaluation of scalability. Our approach can be used to determine how a set of limited changes to targeted system modules could affect design specifications of interest. It can also help predict and trace system bottlenecks over several product generations, offering system designers the chance to to better plan re-engineering efforts for scaling a system specification efficaciously. We demonstrate the value of our methodology by investigating a smartphone-based biosensing instrumentation platform. Specifically, we carry out scalability analysis for the system’s bandwidth specification: the maximum analog voltage waveform excitation frequency the system could output while allowing continuous acquisition and wireless streaming of bioimpedance measurements. We rely on several SysML modelling tools, including dependency matrices, as well as a fault-detection Simulink Stateflow executable model to conclude on how the successive re-engineering of 5 independent system modules, from the replacement of a wireless Bluetooth interface, to the revision of the ADC sample-and-hold operation could help increase system bandwidth.

Aim: Pseudomonas aeruginosa is a pathogen that is prevalent in serious infections in compromised patients worldwide. A unique virulence factor of this bacterium is the redox-active molecule pyocyanin, which is a potential biomarker for the identification of P. aeruginosa infections. Here we report a direct, selective and rapid detection technique of pyocyanin. Materials & methods: Pyocyanin was detected by amperometry at a relatively high potential where the pyocyanin signal was unaffected by background contributions. Results & conclusion: Pyocyanin was detected at concentrations down to 125 nM in a 50 µM mixture of interfering compounds with a reproducibility of r2 = 0.999 (n = 5) within 200 s. The results document a step toward a point-of-care technique for diagnosis of P. aeruginosa infections.

Lab-on-Chip technologies offer great opportunities for the democratization of in-vitro medical diagnostics to the consumer-market. Despite the limitations set by the strict instrumentation and control requirements of certain families of these devices, new solutions are emerging. Smartphones now routinely demonstrate their potential as an interface of choice for operating complex, instrumented Lab-on-Chips. The sporadic nature of home-based in-vitro medical diagnostics testing calls for the development of systems capable of evolving with new applications or new technologies for Lab-on-Chip devices. We present in this work how we evolved the first generation of a smartphone/Lab-on-Chip platform designed for evolvability. We demonstrate how reengineering efforts can be confined to the mobile-software layer and illustrate some of the benefits of building evolvable systems. We implement electrochemical capabilities on our platform prototype and carry out cyclic voltammetry to measure dopamine concentrations over several orders of magnitude.

Pyocyanin is a virulence factor uniquely produced by the pathogen Pseudomonas aeruginosa. The fast and selective detection of pyocyanin in clinical samples can reveal important information about the presence of this microorganism in patients. Electrochemical sensing of the redox-active pyocyanin is a route to directly quantify pyocyanin in real time and in situ in hospitals and clinics. The selective quantification of pyocyanin is, however, limited by other redox-active compounds existing in human fluids and by other metabolites produced by pathogenic bacteria. Here we present a direct selective method to detect pyocyanin in a complex electroactive environment using commercially available electrodes. It is shown that cyclic voltammetry measurements between –1.0 V to 1.0 V reveal a potential detection window of pyocyanin of 0.58–0.82 V that is unaffected by other redox-active interferents. The linear quantification of pyocyanin has an R2 value of 0.991 across the clinically relevant concentration range of 2–100 ηM. The proposed method was tested on human saliva showing a standard deviation of 2.5% ±1% (n = 5) from the known added pyocyanin concentration to the samples. This inexpensive procedure is suggested for clinical use in monitoring the presence and state of P. aeruginosa infection in patients.

The integration of two-photon polymerization technology with standard microfabrication techniques is imperative for the use of this tool in micro- and nanotechnology and especially for the future commercialization of the technology. In this work, we report a novel method for the fabrication of 3D polymeric structures via a two-photon polymerization based system. The method consists of combining a two-photon polymerization system with conventional photolithography techniques in order to create 3D polymer electrodes. The functionality of the final structures was confirmed by electrochemical characterization techniques.

A conjugate between single-walled carbon nanotubes, chitosan and folic acid has been prepared. It was characterized by diffusion ordered two-dimensional hydrogen-1 nuclear magnetic resonance and hydrogen-1 nuclear magnetic resonance spectroscopy which revealed the presence of a conjugate that was generated by the linkage between the carboxyl moiety of the folic acid and the amino group of the chitosan, which in turn was non-covalently bound to the single-walled carbon nanotubes. The obtained diffusion coefficient values demonstrated that free folic acid diffused more rapidly than the folic acid conjugated to single-walled carbon nanotubes–chitosan. The values of the proton signal of hydrogen-1 nuclear magnetic resonance spectroscopy and two-dimensional hydrogen-1 nuclear magnetic resonance spectroscopy further confirmed that the folic acid was conjugated to the chitosan, wrapping the single-walled carbon nanotubes.

Cytogenetic and molecular cytogenetic analyses, which aim to detect chromosome abnormalities, are routinely performed in cytogenetic laboratories all over the world. Traditional cytogenetic studies are performed by analyzing the banding pattern of chromosomes, and are complemented by molecular cytogenetic techniques such as fluorescent in situ hybridization (FISH). To improve FISH application in cytogenetic analysis the issues with long experimental time, high volumes of expensive reagents and requirement for trained technicians need to be addressed. The protocol has recently evolved towards on chip detection of chromosome abnormalities with the development of microsystems for FISH analysis. The challenges addressed by the developed microsystems are mainly the automation of the assay performance, reduction in probe volume, as well as reduction of assay time. The recent focus on the development of automated systems for performing FISH on chip is summarized in this review.

In this paper some of the technological platforms developed in our group for biological cell analysis will be highlighted. The paper first presents a short introduction pinpointing the advantages of using micro and nano technology in cellular studies. The issues of requiring transient analysis while working in a biological environment maintaining the cells viability and adding analyte are addressed and discussed. An example of a cell culturing chamber useful for both adherent and non-adherent cells, with the capability of adding analyte is given, a small discussion of in vitro cellular studies mimicking the in vivo situation is presented and an example of surface modification for cellular growth is described. Then novel electronic sensor platforms are discussed and an example of a nanosensor with electronic readout is given utilizing both micro- and nanotechnology. Finally an example of sorting cells using dielectrophoresis will be given, aiming at early cancer detection.

This study describes an integrated micro ''factory'' for the preparation of biological self-assembled peptide nanotubes and nanoparticles on a polymer chip, yielding controlled growth conditions. Self-assembled peptides constitute attractive building blocks for the fabrication of biological nanostructures due to the mild conditions of their synthesis process. This biological material can form nanostructures in a rapid way and the synthesis method is less expensive as compared to that of carbon nanotubes or silicon nanowires. The present article thus reports on the on-chip fabrication of self-assembled peptide nanostructures by means of a microfluidic device that is able to resist the harsh conditions imposed by the solvent used during the nanostructure synthesis. This on-chip fabrication was found to be simple, rapid, and convenient.

Microfluidic bioreactors (μBR) are becoming increasingly popular for cell culture, sample preparation and analysis in case of routine genetic and clinical diagnostics. We present a novel μBR for non-adherent cells designed to mimic in vivo perfusion of cells based on diffusion of media through a sandwiched membrane. The culture chamber and perfusion chamber are separated by a sandwiched membrane and each chamber has separate inlet/outlets for easy loading/unloading of cells and perfusion of the media. The perfusion of media and exchange of nutrients occur through the sandwiched membrane, which was also verified with simulations. Finally, we present the application of this device for cytogenetic sample preparation, whereby we culture and arrest peripheral T-lymphocytes in metaphase and later fix them in the μBR. The expansion of T-lymphocytes from an unknown patient sample was quantified by means of CFSE staining and subsequent counting in a flow cytometer. To conclude on the applicability of μBR for genetic diagnostics, we prepare chromosome spreads on glass slides from the cultured samples, which is the primary step for metaphase FISH analysis.

The development of new drug therapies relies on studies of cell transmigration in in vitro systems. Migration has traditionally been studied using two methods, the Boyden chamber and a shear flow chamber assay. Though, commonly applied in cell transmigration studies, they are far from imitating a natural migration process. Here we describe a novel in vitro cell transmigration microfluidic assay, which mimicks physiological shear flow conditions in blood vessels. The device was designed to incorporate the principles of both the Boyden chamber and the shear flow chamber assay, i.e. migration through the membrane under flow conditions. The 3D environment of migrating cells is imitated by injecting cell adhesion proteins to coat the membrane in the device. We tested the developed device with Jurkat cells migration towards medium supplemented with serum, and with chemokine induced lymphocytes migration. The applied continuous flow of cell suspension and chemoattractant ensures that the concentration gradient is maintained in time and space. The cell adhesion proteins used to enhance cell migration in the device were fibronectin and VCAM-1. We successfully observed a multistep transmigration process by means of the developed microfluidic migration assay. The presented device is inexpensive, easy to fabricate and disposable, having a potential to be applied in basic research as well as in the drug development process.

This study has evaluated self-assembled peptide nanotubes (PNTS) and nanowires (PNWS) as etching mask materials for the rapid and low-cost fabrication of silicon wires using reactive ion etching (RIE). The self-assembled peptide structures were fabricated under mild conditions and positioned on clean silicon wafers, after which these biological nanostructures were exposed to an RIE etching process. Following this treatment, the structure of the remaining nanotubes and nanowires was analyzed by scanning electron microscopy (SEM). Important differences in the behavior of the nanotubes and the nanowires were observed after the RIE process. The nanotubes remained intact while the nanowires were destroyed by the RIE process. The instability of the peptide nanowires during this process was further confirmed with focused ion beam milling experiments. The PNTS could stand energetic argon ions for around 32 s while the PNWS resisted only 4 s before becoming milled. Based on these results, self-assembled PNTS were further used as an etching mask to fabricate silicon wires in a rapid and low-cost manner. The obtained silicon wires were subjected to structural and electrical characterization by SEM and I–V measurements. Additionally, the fabricated silicon structures were functionalized with fluorescent molecules via a biotin–streptavidin interaction in order to probe their potential in the development of biosensing devices.

Hydrophobins are small fungal proteins with amphipatic properties and the ability to self-assemble on a hydrophobic/hydrophilic interface; thus, many technical applications for hydrophobins have been suggested. The pathogenic fungus Aspergillus fumigatus expresses the hydrophobins RodA and RodB on the surface of its conidia. RodA is known to be of importance to the pathogenesis of the fungus, while the biological role of RodB is currently unknown. Here, we report the successful expression of both hydrophobins in Pichia pastoris and present fed-batch fermentation yields of 200-300 mg/l fermentation broth. Protein bands of expected sizes were detected by SDS-PAGE and western blotting, and the identity was further confirmed by tandem mass spectrometry. Both proteins were purified using his-affinity chromatography, and the high level of purity was verified by silver-stained SDS-PAGE. Recombinant RodA as well as rRodB were able to convert a glass surface from hydrophilic to hydrophobic similar to native RodA, but only rRodB was able to decrease the hydrophobicity of a Teflon-like surface to the same extent as native RodA, while rRodA showed this ability to a lesser extent. Recombinant RodA and native RodA showed a similar ability to emulsify air in water, while recombinant RodB could also emulsify oil in water better than the control protein bovine serum albumin (BSA). This is to our knowledge the first successful expression of hydrophobins from A. fumigatus in a eukaryote host, which makes it possible to further characterize both hydrophobins. Furthermore, the expression strategy and fed-batch production using P. pastoris may be transferred to hydrophobins from other species.