Employing electrochemically derived pH gradients for Lab-on-PCB protein preconcentration devices.

Protein preconcentration is an essential sample preparation step for analysis in which the targeted proteins exist in low concentrations, such as bodily fluids, water, or wastewater. Nonetheless, very few practical implementations of miniaturized protein preconcentration devices have been demonstrated in practice, and even fewer have been integrated with other microanalytical steps. Existing approaches rely heavily on additional chemicals and reagents and introduce complexity to the overall assay. In this paper, we propose a novel miniaturized isoelectric focusing-based protein preconcentration screening device based on electrochemically derived pH gradients rather than existing chemical reagent approaches. In this way, we reduce the need for additional chemical reagents to zero while enabling device incorporation in a seamlessly integrated full protein analysis microsystem via Lab-on-PCB technology. We apply our previously presented Lab-on-PCB approach to quantitatively control the pH of a solution in the vicinity of planar electrodes using electrochemical acid generation through redox-active self-assembled monolayers. The presented device comprises a printed circuit board with an array of gold electrodes that were functionalized with 4-aminothiophenol; this formed a self-assembled monolayer that was electropolymerized to improve its electrochemical reversibility. Protein preconcentration was performed in two configurations. The first was open and needed the use of a holder to suspend a well of fluid above the electrodes; the second used microfluidic channels to enclose small volumes of fluid. Reported here are the resulting data for protein preconcentration in both these forms, with a quantitative concentration factor shown for the open form and qualitative proof shown for the microfluidic.

LoCKAmp: lab-on-PCB technology for <3 minute virus genetic detection.

The recent COVID-19 outbreak highlighted the need for lab-on-chip diagnostic technology fit for real-life deployment in the field. Existing bottlenecks in multistep analytical microsystem integration and upscalable, standardized fabrication techniques delayed the large-scale deployment of lab-on-chip solutions during the outbreak, throughout a global diagnostic test shortage. This study presents a technology that has the potential to address these issues by redeploying and repurposing the ubiquitous printed circuit board (PCB) technology and manufacturing infrastructure. We demonstrate the first commercially manufactured, miniaturised lab-on-PCB device for loop-mediated isothermal amplification (LAMP) genetic detection of SARS-CoV-2. The system incorporates a mass-manufactured, continuous-flow PCB chip with ultra-low cost fluorescent detection circuitry, rendering it the only continuous-flow µLAMP platform with off-the-shelf optical detection components. Ultrafast, SARS-CoV-2 RNA amplification in wastewater samples was demonstrated within 2 min analysis, at concentrations as low as 17 gc µL-1. We further demonstrate our device operation by detecting SARS-CoV-2 in 20 human nasopharyngeal swab samples, without the need for any RNA extraction or purification. This renders the presented miniaturised nucleic-acid amplification-based diagnostic test the fastest reported SARS-CoV-2 genetic detection platform, in a practical implementation suitable for deployment in the field. This technology can be readily extended to the detection of alternative pathogens or genetic targets for a very broad range of applications and matrices. LoCKAmp lab-on-PCB chips are currently mass-manufactured in a commercial, ISO-compliant PCB factory, at a small-scale production cost of £2.50 per chip. Thus, with this work, we demonstrate a high technology-readiness-level lab-on-chip-based genetic detection system, successfully benchmarked against standard analytical techniques both for wastewater and nasopharyngeal swab SARS-CoV-2 detection.

Innovative Quantification of Critical Quality Attributes.

Potency testing is an important part of the evaluation of cellular therapy products. In vitro quantification of identified quality-related biomarkers is a technique often used at the laboratory. Nonetheless, the limited stability of most cellular therapy products, the lot variability and the limited time within which to perform testing are currently hindering their widespread use. Fortunately, within the last two decades, the evolution of material technology and miniaturisation processes has enabled the research community to shift the spotlight of attention towards the Lab-on-Chip concept for diagnostic applications. Such devices enable portable, rapid, sensitive, automated and affordable biochemical analyses aiming to advance the healthcare services across a broad application spectrum. However, it could be argued that the aspirations on their affordability are far from being exceeded, mainly due to the lack of a practical manufacturing technology. The Lab-on-Printed Circuit Board (Lab-on-PCB) approach has demonstrated enormous potential for developing economical diagnostic platforms leveraging the advantage provided by economy of scale manufacturing of the long-standing PCB industry. The integration capabilities that the PCB platform introduces to the Lab-on-Chip concept concerning the electronics and microfluidics seem to be unique. In this chapter, we will be reviewing the progress of Lab-on-PCB prototypes quantifying within miniaturised microchips a range of critical quality attributes with potential in potency testing. We will focus on their technology and applications whilst addressing the potential of this approach in practical use and commercialisation.

Electrochemical sensors based on metal nanoparticles with biocatalytic activity.

Biosensors have attracted a great deal of attention, as they allow for the translation of the standard laboratory-based methods into small, portable devices. The field of biosensors has been growing, introducing innovations into their design to improve their sensing characteristics and reduce sample volume and user intervention. Enzymes are commonly used for determination purposes providing a high selectivity and sensitivity; however, their poor shelf-life is a limiting factor. Researchers have been studying the possibility of substituting enzymes with other materials with an enzyme-like activity and improved long-term stability and suitability for point-of-care biosensors. Extra attention is paid to metal and metal oxide nanoparticles, which are essential components of numerous enzyme-less catalytic sensors. The bottleneck of utilising metal-containing nanoparticles in sensing devices is achieving high selectivity and sensitivity. This review demonstrates similarities and differences between numerous metal nanoparticle-based sensors described in the literature to pinpoint the crucial factors determining their catalytic performance. Unlike other reviews, sensors are categorised by the type of metal to study their catalytic activity dependency on the environmental conditions. The results are based on studies on nanoparticle properties to narrow the gap between fundamental and applied research. The analysis shows that the catalytic activity of nanozymes is strongly dependent on their intrinsic properties (e.g. composition, size, shape) and external conditions (e.g. pH, type of electrolyte, and its chemical composition). Understanding the mechanisms behind the metal catalytic activity and how it can be improved helps designing a nanozyme-based sensor with the performance matching those of an enzyme-based device.

A flow-through microfluidic chip for continuous dielectrophoretic separation of viable and non-viable human T-cells.

Effective methods for rapid sorting of cells according to their viability are critical in T cells based therapies to prevent any risk to patients. In this context, we present a novel microfluidic device that continuously separates viable and non-viable T-cells according to their dielectric properties. A dielectrophoresis (DEP) force is generated by an array of castellated microelectrodes embedded into a microfluidic channel with a single inlet and two outlets; cells subjected to positive DEP forces are drawn toward the electrodes array and leave from the top outlet, those subjected to negative DEP forces are repelled away from the electrodes and leave from the bottom outlet. Computational fluid dynamics is used to predict the device separation efficacy, according to the applied alternative current (AC) frequency, at which the cells move from/to a negative/positive DEP region and the ionic strength of the suspension medium. The model is used to support the design of the operational conditions, confirming a separation efficiency, in terms of purity, of 96% under an applied AC frequency of 1.5 × 106  Hz and a flow rate of 20 µl/h. This work represents the first example of effective continuous sorting of viable and non-viable human T-cells in a single-inlet microfluidic chip, paving the way for lab-on-a-chip applications at the point of need.

Utilising Commercially Fabricated Printed Circuit Boards as an Electrochemical Biosensing Platform.

Printed circuit boards (PCBs) offer a promising platform for the development of electronics-assisted biomedical diagnostic sensors and microsystems. The long-standing industrial basis offers distinctive advantages for cost-effective, reproducible, and easily integrated sample-in-answer-out diagnostic microsystems. Nonetheless, the commercial techniques used in the fabrication of PCBs produce various contaminants potentially degrading severely their stability and repeatability in electrochemical sensing applications. Herein, we analyse for the first time such critical technological considerations, allowing the exploitation of commercial PCB platforms as reliable electrochemical sensing platforms. The presented electrochemical and physical characterisation data reveal clear evidence of both organic and inorganic sensing electrode surface contaminants, which can be removed using various pre-cleaning techniques. We demonstrate that, following such pre-treatment rules, PCB-based electrodes can be reliably fabricated for sensitive electrochemical biosensors. Herein, we demonstrate the applicability of the methodology both for labelled protein (procalcitonin) and label-free nucleic acid (E. coli-specific DNA) biomarker quantification, with observed limits of detection (LoD) of 2 pM and 110 pM, respectively. The proposed optimisation of surface pre-treatment is critical in the development of robust and sensitive PCB-based electrochemical sensors for both clinical and environmental diagnostics and monitoring applications.

Multiplexed Prostate Cancer Companion Diagnostic Devices.

Prostate cancer (PCa) remains one of the most prominent forms of cancer for men. Since the early 1990s, Prostate-Specific Antigen (PSA) has been a commonly recognized PCa-associated protein biomarker. However, PSA testing has been shown to lack in specificity and sensitivity when needed to diagnose, monitor and/or treat PCa patients successfully. One enhancement could include the simultaneous detection of multiple PCa-associated protein biomarkers alongside PSA, also known as multiplexing. If conventional methods such as the enzyme-linked immunosorbent assay (ELISA) are used, multiplexed detection of such protein biomarkers can result in an increase in the required sample volume, in the complexity of the analytical procedures, and in adding to the cost. Using companion diagnostic devices such as biosensors, which can be portable and cost-effective with multiplexing capacities, may address these limitations. This review explores recent research for multiplexed PCa protein biomarker detection using optical and electrochemical biosensor platforms. Some of the novel and potential serum-based PCa protein biomarkers will be discussed in this review. In addition, this review discusses the importance of converting research protocols into multiplex point-of-care testing (xPOCT) devices to be used in near-patient settings, providing a more personalized approach to PCa patients' diagnostic, surveillance and treatment management.

Microfluidics Integration into Low-Noise Multi-Electrode Arrays.

Organ-on-Chip technology is commonly used as a tool to replace animal testing in drug development. Cells or tissues are cultured on a microchip to replicate organ-level functions, where measurements of the electrical activity can be taken to understand how the cell populations react to different drugs. Microfluidic structures are integrated in these devices to replicate more closely an in vivo microenvironment. Research has provided proof of principle that more accurate replications of the microenvironment result in better micro-physiological behaviour, which in turn results in a higher predictive power. This work shows a transition from a no-flow (static) multi-electrode array (MEA) to a continuous-flow (dynamic) MEA, assuring a continuous and homogeneous transfer of an electrolyte solution across the measurement chamber. The process through which the microfluidic system was designed, simulated, and fabricated is described, and electrical characterisation of the whole structure under static solution and a continuous flow rate of 80 µL/min was performed. The latter reveals minimal background disturbance, with a background noise below 30 µVpp for all flow rates and areas. This microfluidic MEA, therefore, opens new avenues for more accurate and long-term recordings in Organ-on-Chip systems.

Printable graphene BioFETs for DNA quantification in Lab-on-PCB microsystems.

Lab-on-Chip is a technology that aims to transform the Point-of-Care (PoC) diagnostics field; nonetheless a commercial production compatible technology is yet to be established. Lab-on-Printed Circuit Board (Lab-on-PCB) is currently considered as a promising candidate technology for cost-aware but simultaneously high specification applications, requiring multi-component microsystem implementations, due to its inherent compatibility with electronics and the long-standing industrial manufacturing basis. In this work, we demonstrate the first electrolyte gated field-effect transistor (FET) DNA biosensor implemented on commercially fabricated PCB in a planar layout. Graphene ink was drop-casted to form the transistor channel and PNA probes were immobilized on the graphene channel, enabling label-free DNA detection. It is shown that the sensor can selectively detect the complementary DNA sequence, following a fully inkjet-printing compatible manufacturing process. The results demonstrate the potential for the effortless integration of FET sensors into Lab-on-PCB diagnostic platforms, paving the way for even higher sensitivity quantification than the current Lab-on-PCB state-of-the-art of passive electrode electrochemical sensing. The substitution of such biosensors with our presented FET structures, promises further reduction of the time-to-result in microsystems combining sequential DNA amplification and detection modules to few minutes, since much fewer amplification cycles are required even for low-abundance nucleic acid targets.

Ultra stable, inkjet-printed pseudo reference electrodes for lab-on-chip integrated electrochemical biosensors.

Lab-on-Chip technology comprises one of the most promising technologies enabling the widespread adoption of Point-of-Care testing in routine clinical practice. However, until now advances in Lab-on-Chip have not been translated to the anticipated degree to commercialized tools, with integrated device mass manufacturing cost still not at a competitive level for several key clinical applications. Lab-on-PCB is currently considered as a candidate technology addressing this issue, owing to its intuitive compatibility with electronics, seamless integration of electrochemical biosensors and the extensive experience regarding industrial manufacturing processes. Inkjet-printing in particular is a compatible fabrication method, widening the range of electronic materials available and thus enabling seamlessly integrated ultrasensitive electronic detection. To this end, in this work stable pseudo-reference electrodes are fabricated for the first time by means of commercial inkjet-printing on a PCB-integrated electrochemical biosensing platform. SEM and XPS analysis are employed to characterize the electrodes' structure and composition and identify any special characteristics, compared to published work on alternative substrates. Additionally, this paper analyzes integrated reference electrodes from a new perspective, focusing mainly on their characteristics in real-life operation: chemical sintering as opposed to high budget thermal one, stability under continuous flow, pH dependency and bias stress effects on electrode instability, a parameter often overlooked in electrochemical biosensors.

Integration of paper microfluidic sensors into contact lenses for tear fluid analysis.

In this article, using the integration of paper microfluidics within laser-inscribed commercial contact lenses, we demonstrate the multiplexed detection of clinically relevant analytes including hydrogen ions, proteins, glucose, nitrites and l-ascorbic acid, all sampled directly from model tears. In vitro measurements involved the optimization of colorimetric assays, with readouts collected, stored and analyzed using a bespoke Tears Diagnostics smartphone application prototype. We demonstrate the potential of the device to perform discrete measurements either for medical diagnosis or disease screening in the clinic or at the point-of-care (PoC), with future applications including monitoring of ocular infections, uveitis, diabetes, keratopathies and assessing oxidative stress.

Enzyme-assisted glucose quantification for a painless Lab-on-PCB patch implementation.

In the context of an integrated Lab-on-PCB wearable patch extracting interstitial fluid from the patient via integrated microneedles, the requirements from the integrated biosensing part are quite special compared to static glucose electrochemical biosensors. Hence, in this study, a fully PCB-integrated enzymatic glucose quantification Lab-on-Chip device is presented and evaluated considering these special requirements for such a patch implementation: a) range and limit of detection compatible with interstitial fluid glucose levels of diabetic patients and b) effect of sample flow rate on the biosensing platform performance. This work employs a chronoamperometric approach for glucose detection based on covalently immobilized glucose oxidase on PCB-integrated electrodes. The chronoamperometric measurements show that this platform exhibits µM range sensitivity, high specificity, and good reproducibility, and the assay can detect glucose from 10 µM to 9 mM with a lower limit of detection of 10 µM. The demonstrated detection range under continuous flow proved compatible with interstitial fluid glucose levels of diabetic patients. The sample-to-answer time of our Lab-on-PCB device is less than 1 min (sample delivery of few seconds and 20 s for electrochemical measurement), employing sample volumes of 50 µL in this instance. Increased flow rates substantially improve the platform sensitivity (1.1 µA/mM @0 µL/min to 6.2 µA/mM @10 µL/min), with the measured current increasing exponentially to the flow rate, as opposed to the theoretically expected much lower dependence. This work demonstrates the feasibility of Lab-on-PCB patches in terms of biosensing performance, paving the way for the first cost-effective, painless diabetes management microsystem.

Correction: Development and characterisation of acoustofluidic devices using detachable electrodes made from PCB.

Correction for 'Development and characterisation of acoustofluidic devices using detachable electrodes made from PCB' by Roman Mikhaylov et al., Lab Chip, 2020, 20, 1807-1814, DOI: 10.1039/C9LC01192G.

Development and characterisation of acoustofluidic devices using detachable electrodes made from PCB.

Acoustofluidics has been increasingly applied in biology, medicine and chemistry due to its versatility in manipulating fluids, cells and nano-/micro-particles. In this paper, we develop a novel and simple technology to fabricate a surface acoustic wave (SAW)-based acoustofluidic device by clamping electrodes made using a printed circuit board (PCB) with a piezoelectric substrate. The PCB-based SAW (PCB-SAW) device is systematically characterised and benchmarked with a SAW device made using the conventional photolithography process with the same specifications. Microparticle manipulations such as streaming in droplets and patterning in microchannels were demonstrated in the PCB-SAW device. In addition, the PCB-SAW device was applied as an acoustic tweezer to pattern lung cancer cells to form three or four traces inside the microchannel in a controllable manner. Cell viability of ∼97% was achieved after acoustic manipulation using the PCB-SAW device, which proved its ability as a suitable tool for acoustophoretic applications.

Integrated Electrochemical Biosensors for Detection of Waterborne Pathogens in Low-Resource Settings.

More than 783 million people worldwide are currently without access to clean and safe water. Approximately 1 in 5 cases of mortality due to waterborne diseases involve children, and over 1.5 million cases of waterborne disease occur every year. In the developing world, this makes waterborne diseases the second highest cause of mortality. Such cases of waterborne disease are thought to be caused by poor sanitation, water infrastructure, public knowledge, and lack of suitable water monitoring systems. Conventional laboratory-based techniques are inadequate for effective on-site water quality monitoring purposes. This is due to their need for excessive equipment, operational complexity, lack of affordability, and long sample collection to data analysis times. In this review, we discuss the conventional techniques used in modern-day water quality testing. We discuss the future challenges of water quality testing in the developing world and how conventional techniques fall short of these challenges. Finally, we discuss the development of electrochemical biosensors and current research on the integration of these devices with microfluidic components to develop truly integrated, portable, simple to use and cost-effective devices for use by local environmental agencies, NGOs, and local communities in low-resource settings.

Label-Free Electrochemical Detection of S. mutans Exploiting Commercially Fabricated Printed Circuit Board Sensing Electrodes.

This paper reports for the first time printed-circuit-board (PCB)-based label-free electrochemical detection of bacteria. The demonstrated immunosensor was implemented on a PCB sensing platform which was designed and fabricated in a standard PCB manufacturing facility. Bacteria were directly captured on the PCB sensing surface using a specific, pre-immobilized antibody. Electrochemical impedance spectra (EIS) were recorded and used to extract the charge transfer resistance (Rct) value for the different bacteria concentrations under investigation. As a proof-of-concept, Streptococcus mutans (S. mutans) bacteria were quantified in a phosphate buffered saline (PBS) buffer, achieving a limit of detection of 103 CFU/mL. Therefore, the proposed biosensor is an attractive candidate for the development of a simple and robust point-of-care diagnostic platform for bacteria identification, exhibiting good sensitivity, high selectivity, and excellent reproducibility.

Extracellular Electrophysiology in the Prostate Cancer Cell Model PC-3.

Although prostate cancer is one of the most common cancers in the male population, its basic biological function at a cellular level remains to be fully understood. This lack of in depth understanding of its physiology significantly hinders the development of new, targeted and more effective treatment strategies. Whilst electrophysiological studies can provide in depth analysis, the possibility of recording electrical activity in large populations of non-neuronal cells remains a significant challenge, even harder to address in the picoAmpere-range, which is typical of cellular level electrical activities. In this paper, we present the measurement and characterization of electrical activity of populations of prostate cancer cells PC-3, demonstrating for the first time a meaningful electrical pattern. The low noise system used comprises a multi-electrode array (MEA) with circular gold electrodes on silicon oxide substrates. The extracellular capacitive currents present two standard patterns: an asynchronous sporadic pattern and a synchronous quasi-periodic biphasic spike pattern. An amplitude of ±150 pA, a width between 50⁻300 ms and an inter-spike interval around 0.5 Hz characterize the quasi-periodic spikes. Our experiments using treatment of cells with Gd³âº, known as an inhibitor for the Ca²âº exchanges, suggest that the quasi-periodic signals originate from Ca²âº channels. After adding the Gd³âº to a population of living PC-3 cells, their electrical activity considerably decreased; once the culture was washed, thus eliminating the Gd³âº containing medium and addition of fresh cellular growth medium, the PC-3 cells recovered their normal electrical activity. Cellular viability plots have been carried out, demonstrating that the PC-3 cells remain viable after the use of Gd³âº, on the timescale of this experiment. Hence, this experimental work suggests that Ca²âº is significantly affecting the electrophysiological communication pattern among PC-3 cell populations. Our measuring platform opens up new avenues for real time and highly sensitive investigations of prostate cancer signalling pathways.

A PNA-based Lab-on-PCB diagnostic platform for rapid and high sensitivity DNA quantification.

We report the development of a Lab-on-PCB DNA diagnostic platform, exploiting peptide nucleic acid (PNA) sequences as probes. The study demonstrates the optimization and characterization of two commercial PCB manufacturing gold electroplating processes for biosensing applications. Using an optimized ratio of PNA with a spacer molecule (MCH), the lowest limit of detection (LoD) to date for PCB-based DNA biosensors of 57 fM is reported. The study also showcases a fully integrated Lab-on-PCB microsystem designed for rapid detection, which employs PCB-integrated sample delivery, achieving DNA quantification in the 0.1-100 pM range for 5 µL samples analyzed within 5 min under continuous flow. The demonstrated biosensor proves the capability of PCB-based DNA biosensors for high sensitivity and paves the way for their integration in Lab-on-PCB DNA diagnostic microsystems.

The lab-on-PCB approach: tackling the µTAS commercial upscaling bottleneck.

Commercialization of lab-on-a-chip devices is currently the "holy grail" within the µTAS research community. While a wide variety of highly sophisticated chips which could potentially revolutionize healthcare, biology, chemistry and all related disciplines are increasingly being demonstrated, very few chips are or can be adopted by the market and reach the end-users. The major inhibition factor lies in the lack of an established commercial manufacturing technology. The lab-on-printed circuit board (lab-on-PCB) approach, while suggested many years ago, only recently has re-emerged as a very strong candidate, owing to its inherent upscaling potential: the PCB industry is well established all around the world, with standardized fabrication facilities and processes, but commercially exploited currently only for electronics. Owing to these characteristics, complex µTASs integrating microfluidics, sensors, and electronics on the same PCB platform can easily be upscaled, provided more processes and prototypes adapted to the PCB industry are proposed. In this article, we will be reviewing for the first time the PCB-based prototypes presented in the literature to date, highlighting the upscaling potential of this technology. The authors believe that further evolution of this technology has the potential to become a much sought-after standardized industrial fabrication technology for low-cost µTASs, which could in turn trigger the projected exponential market growth of µTASs, in a fashion analogous to the revolution of Si microchips via the CMOS industry establishment.

Amperometric IFN-γ immunosensors with commercially fabricated PCB sensing electrodes.

Lab-on-a-Chip (LoC) technology has the potential to revolutionize medical Point-of-Care diagnostics. Currently, considerable research efforts are focused on innovative production technologies that will make commercial upscaling of lab-on-chip products financially viable. Printed circuit board (PCB) manufacturing techniques have several advantages in this field. In this paper we focus on transferring a complete IFN-γ enzyme-linked immune-sorbent assay (ELISA) onto a commercial PCB electrochemical biosensing platform, We adapted a commercially available ELISA to detect the enzyme product TMB/H2O2 using amperometry, successfully reproducing the colorimetry-obtained ELISA standard curve. The results demonstrate the potential for the integration of these components into an automated, disposable, electronic ELISA Lab-on-PCB diagnostic platform.