Smartphone-Enabled Personalized Diagnostics: Current Status and Future Prospects.

Smartphones are becoming increasingly versatile thanks to the wide variety of sensor and actuator systems packed in them. Mobile devices today go well beyond their original purpose as communication devices, and this enables important new applications, ranging from augmented reality to the Internet of Things. Personalized diagnostics is one of the areas where mobile devices can have the greatest impact. Hitherto, the camera and communication abilities of these devices have been barely exploited for point of care (POC) purposes. This short review covers the recent evolution of mobile devices in the area of POC diagnostics and puts forward some ideas that may facilitate the development of more advanced applications and devices in the area of personalized diagnostics. With this purpose, the potential exploitation of wireless power and actuation of sensors and biosensors using near field communication (NFC), the use of the screen as a light source for actuation and spectroscopic analysis, using the haptic module to enhance mass transport in micro volumes, and the use of magnetic sensors are discussed.

A self-powered skin-patch electrochromic biosensor.

One of the limitations of many skin-patch wearable sensors today is their dependence on silicon-based electronics, increasing their complexity and unit cost. Self-powered sensors, in combination with electrochromic materials, allow simplifying the construction of these devices, leading to powerful analytical tools that remove the need for external detection systems. This work describes the construction, by screen-printing, of a self-powered electrochromic device that can be adapted for the determination of metabolites in sweat by the naked eye in the form of a 3 × 15 mm colour bar. The device comprises a lactate oxidase and osmium-polymer -based anode connected to a coplanar 3 × 15 mm Prussian Blue, PB, cathode printed over a transparent poly(3,4-ethylenedioxythiophene) polystyrene sulfonate, PEDOT:PSS electrode. An ion-gel composed of Poly(vinylidene fluoride-co-hexafluoropropylene), PVDF-co-HFP, a gelling agent, and ionic liquid 1-Ethyl-3-methylimidazolium trifluoromethanesulfonate, EMIM-Tf, effectively separates the cathode display from the biosensing anode, protecting it from the sample. Despite its cathodic electrochromism, the PEDOT:PSS has a transmission above 90% and does not mask the Prussian Blue colour change because the cathode does not operate below 0 V vs Ag/AgCl at any time. The sensor displays lactate concentrations in the range of 0-10 mM over the length of the electrochromic display, which has a contrast ratio of 1.43. Although full response takes up to 24 min, 85% of the colour change is displayed within 10 min.

Electrochemical POC device for fast malaria quantitative diagnosis in whole blood by using magnetic beads, Poly-HRP and microfluidic paper electrodes.

Malaria, a parasitic infection caused by Plasmodium parasites and transmitted through the bite of infected female Anopheles mosquitos, is one of the main causes of mortality in many developing countries. Over 200 million new infections and nearly half a million deaths are reported each year, and more than three billion people are at risk of acquiring malaria worldwide. Nevertheless, most malaria cases could be cured if detected early. Malaria eradication is a top priority of the World Health Organisation. However, achieving this goal will require mass population screening and treatment, which will be hard to accomplish with current diagnostic tools. We report an electrochemical point-of-care device for the fast, simple and quantitative detection of Plasmodiumfalciparum lactate dehydrogenase (PfLDH) in whole blood samples. Sample analysis includes 5-min lysis to release intracellular parasites, and stirring for 5 more min with immuno-modified magnetic beads (MB) along with an immuno-modified signal amplifier. The rest of the magneto-immunoassay, including sample filtration, MB washing and electrochemical detection, is performed at a disposable paper electrode microfluidic device. The sensor provides PfLDH quantitation down to 2.47 ng mL-1 in spiked samples and for 0.006-1.5% parasitemias in Plasmodium-infected cultured red blood cells, and discrimination between healthy individuals and malaria patients presenting parasitemias >0.3%. Quantitative malaria diagnosis is attained with little user intervention, which is not achieved by other diagnostic methods.

Detection of plasma MMP-9 within minutes. Unveiling some of the clues to develop fast and simple electrochemical magneto-immunosensors.

Magnetic beads (MB) have been extensively used to produce sensitive and efficient electrochemical magneto-immunosensors. However, MB effective handling requires training, and MB washing after each incubation step is time consuming and contributes to raise result variability. Consequently, most of the electrochemical magneto-immunosensors reported to date, which entailed relatively long and complex multi-step procedures, would be difficult to carry out at point-of-care (POC) settings or by laypersons. For this reason, here we targeted the development of a simplified detection path, which is fast and simple enough to be operated at a POC setting, sufficiently efficient to provide analyte quantitation comparable to classical diagnostic methods, and dependent on minimal technical requirements to facilitate method global exploitation. As a proof-of-concept, we optimized an extremely simple, fast and efficient electrochemical magneto-immunosensor for detection of matrix metalloproteinase 9 (MMP-9). To accomplish this, we optimized MB immunomodification, produced an immunomodified Poly-HRP signal amplifier, developed a single-step magneto-immunoassay, and optimized electrochemical detection using a multiplexed magnetic holder and a ready-to-use commercial substrate solution. The sensor was finally calibrated by detecting MMP-9 in clinical samples. This electrochemical magneto-immunosensor detected MMP-9 in just 12-15 min, displaying linear response between 0.03 and 2 ng mL-1 of MMP-9, limits of detection (LOD) and quantification (LOQ) of 13 pg mL-1 and 70 pg mL-1, respectively, %CV< 6%, and accurate quantification of MMP-9 in patient plasma samples. These results were comparable to those afforded by a 5-h reference ELISA that used the same antibodies, confirming the applicability of our simplified method.

Paper-based microfluidic biofuel cell operating under glucose concentrations within physiological range.

This work addresses the development of a compact paper-based enzymatic microfluidic glucose/O2 fuel cell that can operate using a very limited sample volume (≈35µl) and explores the energy generated by glucose at concentrations typically found in blood samples at physiological conditions (pH 7.4). Carbon paper electrodes combined with a paper sample absorption substrate all contained within a plastic microfluidic casing are used to construct the paper-based fuel cell. The anode catalysts consist of glucose dehydrogenase and [Os(4,4'-dimethoxy-2,2'-bipyridine)2(poly-vinylimidazole)10Cl]+ as mediator, while the cathode catalysts were bilirubin oxidase and [Os(2,2'-bipyridine)2(poly-vinylimidazole)10Cl]+ as mediator. The fuel cell delivered a linear power output response to glucose over the range of 2.5-30mM, with power densities ranging from 20 to 90µWcm-2. The quantification of the available electrical power as well as the energy density extracted from small synthetic samples allows planning potential uses of this energy to power different sensors and analysis devices in a wide variety of in-vitro applications.

Rapid prototyping of electrochemical lateral flow devices: stencilled electrodes.

A straightforward and very cost effective method is proposed to prototype electrodes using pressure sensitive adhesives (PSA) and a simple cutting technique. Two cutting methods, namely blade cutting and CO2 laser ablation, are compared and their respective merits are discussed. The proposed method consists of turning the protective liner on the adhesive into a stencil to apply screen-printing pastes. After the electrodes have been printed, the liner is removed and the PSA can be used as a backing material for standard lateral flow membranes. We present the fabrication of band electrodes down to 250 µm wide, and their characterization using microscopy techniques and cyclic voltammetry. The prototyping approach presented here facilitates the development of new electrochemical devices even if very limited fabrication resources are available. Here we demonstrate the fabrication of a simple lateral-flow device capable of determining glucose in blood. The prototyping approach presented here is highly suitable for the development of novel electroanalytical tools.

Dual chronoamperometric detection of enzymatic biomarkers using magnetic beads and a low-cost flow cell.

In this work we report on the production of a low cost microfluidic device for the multiplexed electrochemical detection of magneto bioassays. As a proof of concept, the device has been used to detect myeloperoxidase (MPO), a cardiovascular biomarker. With this purpose, two bioassays have been optimized in parallel onto magnetic beads (MBs) for the simultaneous detection of MPO endogenous peroxidase activity and quantification of total MPO. Since the two bioassays produced signals of different magnitude for each concentration of MPO tested, two detection strategies have been compared, which entailed registering steady state currents (Iss) under substrate flow, and measuring the peak currents (Ip) produced in a stopped flow approach. As it will be shown, appropriate tuning of the detection and flow conditions can provide extremely sensitive detection, but also allow simultaneous detection of assays or parameters that would produce signals of different orders of magnitude when measured by a single detection strategy. In order to demonstrate the feasibility of the detection strategy reported, a dual MPO mass and activity assay has been finally applied to the study of 10 real plasma samples, allowing patient classification according to the risk of suffering a cardiovascular event.

Scanning electrochemical microscopy for study of aptamer-thrombin interfacial interactions on gold disk microelectrodes.

A feasibility for the determination of thrombin on gold disk microelectrodes (GDMs) using scanning electrochemical microscopy (SECM) is reported. The assembly process step-by-step of thrombin aptasensor on GDMs is monitored by SECM. SECM analysis reveals the immobilization of thrombin aptamers on GDMs. The interaction between thrombin aptamers and thrombin on GDMs is imaged by SECM with feedback mode using ferrocenemethanol as an electrochemical mediator. The formation of thrombin/thrombin aptamer complex on GDMs results in a decrease in the tip peak current on spatial SECM images. This method is able to linearly and selectively detect thrombin over a linear range from 10(-12) to 10(-5)M with a detection limit of 6.07 fM.

Membraneless glucose/O2 microfluidic enzymatic biofuel cell using pyrolyzed photoresist film electrodes.

Biofuel cells typically yield lower power and are more difficult to fabricate than conventional fuel cells using inorganic catalysts. This work presents a glucose/O2 microfluidic biofuel cell (MBFC) featuring pyrolyzed photoresist film (PPF) electrodes made on silicon wafers using a rapid thermal process, and subsequently encapsulated by rapid prototyping techniques into a double-Y-shaped microchannel made entirely of plastic. A ferrocenium-based polyethyleneimine polymer linked to glucose oxidase (GOx/Fc-C6-LPEI) was used in the anode, while the cathode contained a mixture of laccase, anthracene-modified multi-walled carbon nanotubes, and tetrabutylammonium bromide-modified Nafion (MWCNTs/laccase/TBAB-Nafion). The cell performance was studied under different flow-rates, obtaining a maximum open circuit voltage of 0.54 ± 0.04 V and a maximum current density of 290 ± 28 µA cm(-2) at room temperature under a flow rate of 70 µL min(-1) representing a maximum power density of 64 ± 5 µW cm(-2). Although there is room for improvement, this is the best performance reported to date for a bioelectrode-based microfluidic enzymatic biofuel cell, and its materials and fabrication are amenable to mass production.

Sensitive electrochemical thrombin aptasensor based on gold disk microelectrodearrays.

The construction of a sensitive electrochemical aptamer sensor (aptasensor) for thrombin detection is described. Among the advantages of using microelectrode-based devices are the possibility to work with small sample volumes and enjoying faster mass transport rates and lower interfacial capacitance than at macroelectrodes. Therefore, gold disk microelectrode arrays are an attractive transducer option for aptasensors. The morphology of the gold disk microelectrode arrays was inspected by scanning electron microscope. The interaction between a thrombin aptamer and thrombin on gold disk microelectrode arrays was demonstrated by differential pulse voltammetry using methylene blue (MB) as an electrochemical indicator. MB adsorbed to aptamers via their guanine base. When thrombin was introduced, it displaced the MB adsorbed to the aptamers and bound to them. This resulted in a decrease of MB peak current which correlated to the concentration of thrombin over a dynamic range spanning from 10⁻5 to 10⁻¹² M. This method was able to linearly and selectively detect thrombin with a detection limit of 0.143 pM.

Cancer prognostics by direct detection of p53-antibodies on gold surfaces by impedance measurements.

The identification and measurement of biomarkers is critical to a broad range of methods that diagnose and monitor many diseases. Serum auto-antibodies are rapidly becoming interesting targets because of their biological and medical relevance. This paper describes a highly sensitive, label-free approach for the detection of p53-antibodies, a prognostic indicator in ovarian cancer as well as a biomarker in the early stages of other cancers. This approach uses impedance measurements on gold microelectrodes to measure antibody concentrations at the picomolar level in undiluted serum samples. The biosensor shows high selectivity as a result of the optimization of the epitopes responsible for the detection of p53-antibodies and was validated by several techniques including microcontact printing, self-assembled-monolayer desorption ionization (SAMDI) mass spectrometry, and adhesion pull-off force by atomic force microscopy (AFM). This transduction method will lead to fast and accurate diagnostic tools for the early detection of cancer and other diseases.

Electrochemical detection of testosterone by use of three-dimensional disc-ring microelectrode sensing platforms: application to doping monitoring.

Testosterone is one of the androgenic steroid hormones, the consumption of which is considered doping in most sports. Here, we present powerful 3D sensing platforms using novel disc-ring microelectrode array devices and exploit them for the competitive immunosensing of testosterone. Each device contains a microelectrode array that consists of a large number of individual microdiscs and is used as the substrate for immunofunctionalization and assay performance. One micrometer above it, a second microelectrode array, this time consisting of microrings, is used as the working electrode for electrochemical monitoring. The physical separation of these two functions allows the incorporation of relatively thick biocomponent layers during immunofunctionalization of the microdiscs without negatively affecting electrochemical detection at the rings. Moreover, it permits electrochemical activation of the latter immediately before substrate addition and hence enables optimal electrode performance. The optimized assay showed a linear range between 0.01 and 10 ng/mL and a limit of detection of 12.5 pg/mL testosterone with detection times of 45 min.

Toward membrane-free amperometric gas sensors: a microelectrode array approach.

Room temperature ionic liquids (RTILs) have been applied to a microelectrode array and been demonstrated to form effective, membrane-free amperometric gas sensors. Determining the RTIL [P(6,6,6,14)][FAP] as the most appropriate choice for extended use, the amperometric quantification of oxygen has been demonstrated. The response of the sensor was quantified by both cyclic voltammetry and chronoamperometry. A range of O(2) contents (2-13% v/v) and RTIL layer thicknesses (from ca. 6 to 125 mum) have been investigated. The combination of microelectrode array and RTIL, as well as the absence of membrane and volatile solvent, results in an elegant, easy to calibrate gas sensor with potential utility in standard and nonstandard conditions.

Microarrays of ring-recessed disk electrodes in transient generator-collector mode: theory and experiment.

The fabrication, characterization, and use of arrays of ring-recessed disk microelectrodes are reported. These devices are operated in generator-collector mode with a disk acting as the generator and the ring as the collector. We report experiments and simulations relating to time-of-flight experiments in which material electrogenerated at a disk is diffusionally transported to the ring. Analysis of the current transient measured at the latter when it is potentiostatted at a value to ensure diffusionally controlled "collection" is shown to sensitively reflect the diffusion coefficients of the species forming the redox couple being driven at the generator electrode. The method is applied to the ferrocene/ferrocenium couple in the room temperature ionic liquid [N(6, 2, 2, 2)][NTf(2)], and the results are found to agree with independent measurements.

Why 'the bigger the better' is not always the case when utilising microelectrode arrays: high density vs. low density arrays for the electroanalytical sensing of chromium(VI).

We demonstrate, with the example of the electroanalytical sensing of chromium(vi) using ultra-microelectrode arrays, that a larger number of microelectrodes comprising an array do not necessarily provide improved electroanalytical performance. Using a low density array, which consists of 256 microdiscs where each microdisc comprising the array has a radius of 10 microns in a cubic arrangement separated from their nearest neighbour by 100 microns, the electroanalytical sensing of chromium(vi) is shown to be possible over the range 13-428 microM with a limit of detection of 3.4 microM readily achievable. Using a high density microelectrode, consisting of 2597 microdiscs where each microdisc has a radius of 2.5 microns in a hexagonal pattern which are separated from their nearest neighbour by 55 microns, the electroanalytical performance, in terms of linear range and sensitivity, is considerably lower going against the misconception that a high density array should produce a superior analytical response. The reason for this disparity is discussed and it is shown that the arrangement of the microelectrodes on the array is critical due to the interaction of diffusion zones between neighbouring electrodes allowing analysts to make informed decisions on the conscientious choice of microelectrode arrays.

Fast electrochemical detection of anti-HIV antibodies: coupling allosteric enzymes and disk microelectrode arrays.

Here a novel electrochemical method for the rapid detection of anti-HIV antibodies in serum is presented. The novelty lies in the combination of allosteric enzymes and coulometry to yield a fast, simple and reliable HIV diagnostic method. We have used a previously developed beta-galactosidase enzyme that is efficiently activated by anti-HIV antibodies directed against a major B-cell epitope of the gp41 glycoprotein. When these antibodies bind the enzyme, the 3D conformation changes positively affecting the performance of the active site and, consequently, the enzyme activity is stimulated. Using 4-aminophenyl beta-D-galactopyranoside (PAPG) as substrate yields p-aminophenol (PAP), which is reversibly oxidised at a very mild potential, ca. 0.37 V vs. Ag/AgCl over a range of electrode materials within the working pH range of beta-galactosidase. In the present case, photolithographically produced microelectrode arrays resulted in a detection limit of 4 microM for 4-aminophenol (PAP). The presence of anti-HIV antibodies results in enzyme activity increases above 50% which, combined with the sensitivity and response time afforded by the microelectrode arrays, allowed for the diagnosis of HIV in sera samples within an hour.

Investigating the concept of diffusional independence. Potential step transients at nano- and micro-electrode arrays: theory and experiment.

Microelectrode arrays find broad application in electroanalysis offering the enhanced sensitivity associated with microelectrodes, but with a high total current output. Such arrays are often constructed to make the electrodes 'diffusionally independent'. To emphasize that this is a time dependent property, a two-dimensional simulation, in conjunction with the diffusional domain approach, is used to model potential step transient currents at microelectrode arrays. Two types of array, hexagonal and cubic, are considered. In both cases the absolute (not relative) microelectrode separation distance has a significant effect on transient current. Three different regimes of transient current versus time can be observed at microelectrode arrays. At short times the transient response of isolated microelectrodes is seen, then at intermediate times the steady-state response of independent electrodes can be observed. At longer times planar diffusion to the entire array takes over. It follows that only at timescales corresponding to the first two regimes can the electrodes be considered as diffusionally independent. To verify the theory the potential step experiment is performed at a regularly spaced hexagonal iridium microdisk array. Theory is found to be in a good agreement with the experimental results.

Immunofunctionalisation of gold transducers for bacterial detection by physisorption.

Functionalisation of the sensing surface is a key factor in immunosensor fabrication as it allows target-selective capture and prevents nonspecific adsorption of undesired components. Gold immunofunctionalisation using self-assembled monolayers (SAM) has been widely exploited to this end for the detection of small targets. However, we recently demonstrated that this strategy fails when detecting whole bacteria cells (Baldrich et al., Anal Bioanal Chem 390:1557-1562, 2008). We now investigate different physisorption-based alternatives using E. coli as the target organism. Our results demonstrate that physisorption generates the appropriate substrate for the specific detection of bacteria on gold surfaces, providing detection limits down to 10(5) cells mL(-1) in an ELISA-type colorimetric assay. Additionally, surface coverage is highly reproducible when assayed by impedance spectroscopy and the inter- and intra-assay coefficients of variation are below 10-15% in all cases. These surfaces were stable, retained functionality and did not suffer from significant biomolecule desorption after 10 days storage in PBS at 37 degrees C, hence confirming physisorption as a cheap, simple and efficient strategy for the detection of bacteria.

Self-assembled monolayers as a base for immunofunctionalisation: unequal performance for protein and bacteria detection.

Biosensor development strongly depends on the optimisation of surface functionalisation strategies. When gold surfaces are considered, immunofunctionalisation by modification of self-assembled monolayers (SAMs) is one of the preferred approaches. In this respect, SAM-based antibody (Ab) incorporation has shown better performance than Ab physisorption for the detection of proteins and small targets. Reports on bacteria detection are less frequent. In this work, we assess the performance of various SAM-based gold immunofunctionalisation strategies, currently applied to protein detection, in the field of bacteria determination. We present the results for Ab chemical conjugation on mercaptopropanoic acid and mercaptoundecanoic acid SAMs, as well as on a dextranized cysteamine SAM. All the modified surfaces studied were shown to be appropriate for the direct detection of an enzyme-labelled protein, but none succeeded in detecting a bacterial target in a sandwich assay format. Conversely, gold functionalised by Ab physisorption allowed E. coli detection when a sandwich enzyme-linked assay was carried out. The implications of bacteria size and wall complexity are discussed. These results indicate that immunofunctionalisation strategies appropriate for protein detection are not necessarily transferable to work with more complex targets such as bacteria. In this respect, Ab physisorption appears to be a suitable alternative to SAM-based gold functionalisation for bacteria detection.

Pathogen detection: a perspective of traditional methods and biosensors.

The detection of pathogenic bacteria is key to the prevention and identification of problems related to health and safety. Legislation is particularly tough in areas such as the food industry, where failure to detect an infection may have terrible consequences. In spite of the real need for obtaining analytical results in the shortest time possible, traditional and standard bacterial detection methods may take up to 7 or 8 days to yield an answer. This is clearly insufficient, and many researchers have recently geared their efforts towards the development of rapid methods. The advent of new technologies, namely biosensors, has brought in new and promising approaches. However, much research and development work is still needed before biosensors become a real and trustworthy alternative. This review not only offers an overview of trends in the area of pathogen detection but it also describes main techniques, traditional methods, and recent developments in the field of pathogen bacteria biosensors.