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Digital microfluidics (DM) is an advanced technology in microfluidics where electrowetting force is used to control and manipulate nanolitre and microliter-sized droplets on a set of electrodes.1 DM has gained considerable attention because of fast heat transfer, less reagent volume due to miniaturization of reaction, small footprints and incredible control of droplets. This technique minimizes the analysis time and efforts and offers innovative prospects for process automation and integration in a unique setup. It is very favorable for high throughput applications. The movement of droplets using DM requires a hydrophobic surface on which the processes can be performed easily. SLIPS (Slippery liquid infused porous surface) have been demonstrated to be used as a surface for digital microfluidics because of antifouling properties and fine movement of droplets.2 SLIPS is fabricated by utilizing porous polymeric substrate infused with the slippery fluid. PCR product purification is important step in molecular cloning. The purification is done to remove the enzymes, primers, salts and other non-reacted components from the reaction to get pure amplified band.3 The purity of PCR product is crucial for the gene sequencing, aptamer sequencing and cloning. In addition to this, whole genome sequencing techniques have increased the need for the purity of the nucleic acids used. Purification of DNA using commercially available kits is cumbersome, time-consuming and generates a lot of plastic waste. With digital microfluidics, the high throughput purification of nucleic acids can be achieved. The DM platform used in this study is developed in-house.4 Magnetic nanoparticles have been employed for the purification of PCR product on the DM platform as they have tendency to bind to the DNA. The binding of the DNA to the nanoparticles can be tuned by altering the ratio of DNA and nanoparticles. A pure DNA band, free of primers is obtained on agarose gel after purification via

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.

Lamellar and non‐lamellar liquid crystalline nanodispersions, including liposomes, cubo-somes, and hexosomes are attractive platforms for drug delivery, bio‐imaging, and related pharmaceutical applications. As compared to liposomes, there is a modest number of reports on the continuous production of cubosomes and hexosomes. Using a binary lipid mixture of citrem and soy phosphatidylcholine (SPC), we describe the continuous production of nanocarriers for delivering thymoquinone (TQ, a substance with various therapeutic potentials) by employing a commercial microfluidic hydrodynamic flow‐focusing chip. In this study, nanoparticle tracking analysis (NTA) and synchrotron small‐angle X‐ray scattering (SAXS) were employed to characterize TQ‐free and TQ‐loaded citrem/SPC nanodispersions. Microfluidic synthesis led to formation of TQ‐free and TQ-loaded nanoparticles with mean sizes around 115 and 124 nm, and NTA findings indicated comparable nanoparticle size distributions in these nanodispersions. Despite the attractiveness of the mi-crofluidic chip for continuous production of citrem/SPC nano‐self‐assemblies, it was not efficient as comparable mean nanoparticle sizes were obtained on employing a batch (discontinuous) method based on low‐energy emulsification method. SAXS results indicated the formation of a biphasic feature of swollen lamellar (Lα) phase in coexistence with an inverse bicontinuous cubic Pn3m phase in all continuously produced TQ‐free and TQ‐loaded nanodispersions. Further, a set of SAXS experiments were conducted on samples prepared using the batch method for gaining further insight into the effects of ethanol and TQ concentration on the structural features of citrem/SPC nano‐self-assemblies. We discuss these effects and comment on the need to introduce efficient microfluidic platforms for producing nanocarriers for delivering TQ and other therapeutic agents.

Lamellar and non-lamellar liquid crystalline nanodispersions, including liposomes, cubosomes, and hexosomes are attractive platforms for drug delivery, bio-imaging, and related pharmaceutical applications. As compared to liposomes, there is a modest number of reports on the continuous production of cubosomes and hexosomes. Using a binary lipid mixture of citrem and soy phosphatidylcholine (SPC), we describe the continuous production of nanocarriers for delivering thymoquinone (TQ, a substance with various therapeutic potentials) by employing a commercial microfluidic hydrodynamic flow-focusing chip. In this study, nanoparticle tracking analysis (NTA) and synchrotron small-angle X-ray scattering (SAXS) were employed to characterize TQ-free and TQ-loaded citrem/SPC nanodispersions. Microfluidic synthesis led to formation of TQ-free and TQ-loaded nanoparticles with mean sizes around 115 and 124 nm, and NTA findings indicated comparable nanoparticle size distributions in these nanodispersions. Despite the attractiveness of the microfluidic chip for continuous production of citrem/SPC nano-self-assemblies, it was not efficient as comparable mean nanoparticle sizes were obtained on employing a batch (discontinuous) method based on low-energy emulsification method. SAXS results indicated the formation of a biphasic feature of swollen lamellar (Lα) phase in coexistence with an inverse bicontinuous cubic Pn3m phase in all continuously produced TQ-free and TQ-loaded nanodispersions. Further, a set of SAXS experiments were conducted on samples prepared using the batch method for gaining further insight into the effects of ethanol and TQ concentration on the structural features of citrem/SPC nano-self-assemblies. We discuss these effects and comment on the need to introduce efficient microfluidic platforms for producing nanocarriers for delivering TQ and other therapeutic agents.

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-ethanol, heat and autoclavation-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.

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.

Pyocyanin is a toxin produced by Pseudomonas aeruginosa. Here we describe a novel paper-based electrochemical sensor for pyocyanin detection, manufactured with a simple and inexpensive approach based on electrode printing on paper. The resulting sensors constitute an effective electrochemical method to quantify pyocyanin in bacterial cultures without the conventional time consuming pretreatment of the samples. The electrochemical properties of the paper-based sensors were evaluated by ferri/ferrocyanide as a redox mediator, and showed reliable sensing performance. The paper-based sensors readily allow for the determination of pyocyanin in bacterial cultures with high reproducibility, achieving a limit of detection of 95 nM and a sensitivity of 4.30 μA/μM in standard culture media. Compared to the similar commercial ceramic based sensors, it is a 2.3-fold enhanced performance. The simple in-house fabrication of sensors for pyocyanin quantification allows researchers to understand in vitro adaptation of P. aeruginosa infections via rapid screenings of bacterial cultures that otherwise are expensive and time-consuming.

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'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'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 µM, 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.

A device for concentration of nanoparticles and/or microparticles in liquid flow conditions by dielectrophoresis is disclosed in this invention.

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 SiNWbioFET/ 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 systemwide 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.

Silicon nanowire (SiNW) field effect transistor based biosensors have already been proven to be a promising tool to detect biomolecules. However, the most commonly used fabrication techniques involve expensive Silicon-On-Insulator (SOI) wafers, E-beam lithography and ion-implantation steps. In the work presented here, a top down approach to fabricate SiNW junctionless field effect biosensors using novel in-situ doped polysilicon is demonstrated. The p-type polysilicon is grown with an optimum boron concentration that gives a good metal-silicon electrical contact while maintaining the doping level at a low enough level to provide a good sensitivity for the biosensor. The silicon nanowires are patterned using standard photolithography and a wet etch method. The metal contacts are made from magnetron sputtered TiW and e-beam evaporation of gold. The passivation of electrodes has been done by sputtered Si3N4 which is patterned by a lift-off process. The characterization of the critical fabrication steps is done by Secondary Ion Mass Spectroscopy (SIMS) and by statistical analysis of the measurements made on the width of the SiNWs. The electrical characterization of the SiNW in air is done by sweeping the back gate voltage while keeping the source drain potential to a constant value and surface characterization is done by applying liquid gate in phosphate buffered saline (PBS) solution. The fabricated SiNWs sensors functionalized with (3-aminopropyl)triethoxysilane (APTES) have demonstrated good sensitivity in detecting different pH buffer solutions.

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 apoint-of-care technique for diagnosis of P. aeruginosa infections.

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.

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 µM. 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 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.

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.

Here we present a robust, stable and low-noise experimental set-up for performing electrochemical detection on a centrifugal microfluidic platform. By using a low-noise electronic component (electrical slip-ring) it is possible to achieve continuous, on-line monitoring of electrochemical experiments, even when the microfluidic disc is spinning at high velocities. Automated sample handling is achieved by designing a microfluidic system to release analyte sequentially, utilizing on-disc passive valving. In addition, the microfluidic system is designed to trap and keep the liquid sample stationary during analysis. In this way it is possible to perform cyclic voltammetry (CV) measurements at varying spin speeds, without altering the electrochemical response. This greatly simplifies the interpretation and quantification of data. Finally, real-time and continuous monitoring of an entire electrochemical experiment, including all intermediate sample handling steps, is demonstrated by amperometric detection of on-disc mixing of analytes (PBS and ferricyanide).

The presented microfluidic interconnection system provides an alternative for the individual interfacing of simple microfluidic devices fabricated in polymers such as polymethylmethacrylate, polycarbonate and cyclic olefin polymer. A modification of the device inlet enables the direct attachment of tubing (such as polytetrafluoroethylene tubing) secured and sealed by using a small plug, without the need for additional assembly, glue or o-rings. This provides a very clean connection that does not require additional, potentially incompatible, materials. The tightly sealed connection can withstand pressures above 250 psi and therefore supports applications with high flow rates or highly viscous fluids. The ease of incorporation, configuration, fabrication and use make this interconnection system ideal for the rapid prototyping of simple microfluidic devices or other integrated systems that require microfluidic interfaces. It provides a valuable addition to the toolbox of individual and small arrays of connectors suitable for micromachined or template-based injection molded devices since it does not require protruding, threaded or glued modifications on the inlet and avoids bulky and expensive fittings.