Reusable Colorimetric Biosensors on Sustainable Silk-Based Platforms.

In biosensor development, silk fibroin is advantageous for providing transparent, flexible, chemically/mechanically stable, biocompatible, and sustainable substrates, where the biorecognition element remains functional for long time periods. These properties are employed here in the production of point-of-care biosensors for resource-limited regions, which are able to display glucose levels without the need for external instrumentation. These biosensors are produced by photopatterning silk films doped with the enzymes glucose oxidase and peroxidase and photoelectrochromic molecules from the dithienylethene family acting as colorimetric mediators of the enzymatic reaction. The photopatterning results from the photoisomerization of dithienylethene molecules in the silk film from its initial uncolored opened form to its pink closed one. The photoisomerization is dose-dependent, and colored patterns with increasing color intensities are obtained by increasing either the irradiation time or the light intensity. In the presence of glucose, the enzymatic cascade reaction is activated, and peroxidase selectively returns closed dithienylethene molecules to their initial uncolored state. Color disappearance in the silk film is proportional to glucose concentration and used to distinguish between hypoglycemic (below 4 mM), normoglycemic (4-6 mM), and hyperglycemic levels (above 6 mM) by visual inspection. After the measurement, the biosensor can be regenerated by irradiation with UV light, enabling up to five measurement cycles. The coupling of peroxidase activity to other oxidoreductases opens the possibility to produce long-life reusable smart biosensors for other analytes such as lactate, cholesterol, or ethanol.

Fiber-Optic-Based System for High-Resolution Monitoring of Stretch in Excised Tissues.

Cardiovascular diseases cause a high number of deaths nowadays. To improve these statistics, new strategies to better understand the electrical and mechanical abnormalities underlying them are urgently required. This study focuses on the development of a sensor to measure tissue stretch in excised tissues, enabling improved knowledge of biomechanical properties and allowing greater control in real time. A system made of biocompatible materials is described, which is based on two cantilevered platforms that integrate an optical fiber inside them to quantify the amount of stretch the tissues are exposed to with a precision of µm. The operating principle of the sensor is based on the variation of the optical path with the movement of the platforms onto which the samples are fixed. The conducted tests highlight that this system, based on a simple topology and technology, is capable of achieving the desired purpose (a resolution of ∼1 µm), enabling the tissue to be bathed in any medium within the system.

Ultrasensitive bacterial sensing using a disposable all-in-one amperometric platform with self-noise cancellation.

The early detection of very low bacterial concentrations is key to minimize the healthcare and safety issues associated with microbial infections, food poisoning or water pollution. In amperometric integrated circuits for electrochemical sensors, flicker noise is still the main bottleneck to achieve ultrasensitive detection with small footprint, cost-effective and ultra-low power instrumentation. Current strategies rely on autozeroing or chopper stabilization causing negative impacts on chip size and power consumption. This work presents a 27-µW potentiostatic-amperometric Delta-Sigma modulator able to cancel its own flicker noise and provide a 4-fold improvement in the limit of detection. The 2.3-mm2 all-in-one CMOS integrated circuit is glued to an inkjet-printed electrochemical sensor. Measurements show that the limit of detection is 15 pArms, the extended dynamic range reaches 110 dB and linearity is R2 = 0.998. The disposable device is able to detect, in less than 1h, live bacterial concentrations as low as 102 CFU/mL from a 50-µL droplet sample, which is equivalent to 5 microorganisms.

Advances in bacterial concentration methods and their integration in portable detection platforms: A review.

Early detection and identification of microbial contaminants is crucial in many sectors, including clinical diagnostics, food quality control and environmental monitoring. Biosensors have recently gained attention among other bacterial detection technologies due to their simplicity, rapid response, selectivity, and integration/miniaturization potential in portable microfluidic platforms. However, biosensors are limited to the analysis of small sample volumes, and pre-concentration steps are necessary to reach the low sensitivity levels of few bacteria per mL required in the analysis of real clinical, industrial or environmental samples. Many platforms already exist where bacterial detection and separation/accumulation systems are integrated in a single platform, but they have not been compiled and critically analysed. This review reports on most recent advances in bacterial concentration/detection platforms with emphasis on the concentration strategy. Systems based on five concentration strategies, i.e. centrifugation, filtration, magnetic separation, electric separation or acoustophoresis, are here presented and compared in terms of processed sample volume, concentration efficiency, concentration time, ability to work with different types of samples, and integration potential, among others. The critical evaluation presented in the review is envision to facilitate the development of future platforms for fast, sensitive and in situ bacterial detection in real sample.

Bacteria Detection at a Single-Cell Level through a Cyanotype-Based Photochemical Reaction.

The detection of living organisms at very low concentrations is necessary for the early diagnosis of bacterial infections, but it is still challenging as there is a need for signal amplification. Cell culture, nucleic acid amplification, or nanostructure-based signal enhancement are the most common amplification methods, relying on long, tedious, complex, or expensive procedures. Here, we present a cyanotype-based photochemical amplification reaction enabling the detection of low bacterial concentrations up to a single-cell level. Photocatalysis is induced with visible light and requires bacterial metabolism of iron-based compounds to produce Prussian Blue. Bacterial activity is thus detected through the formation of an observable blue precipitate within 3 h of the reaction, which corresponds to the concentration of living organisms. The short time-to-result and simplicity of the reaction are expected to strongly impact the clinical diagnosis of infectious diseases.

Electrochromogenic Detection of Live Bacteria Using Soluble and Insoluble Prussian Blue.

Microbial detection is crucial for the control and prevention of infectious diseases, being one of the leading causes of mortality worldwide. Among the techniques developed for bacterial detection, those based on metabolic indicators are progressively gaining interest due to their simplicity, adaptability, and, most importantly, their capacity to differentiate between live and dead bacteria. Prussian blue (PB) may act as a metabolic indicator, being reduced by bacterial metabolism, producing a visible color change from blue to colorless. This molecule can be present in two main forms, namely, the soluble and the insoluble, having different properties and structures. In the current work, the bacterial-sensing capacity of soluble and insoluble PB will be tested and compared both in suspensions as PB-NPs and after deposition on transparent indium tin oxide-poly(ethylene terephthalate) (ITO-PET) electrodes. In the presence of live bacteria, PB-NPs are metabolized and completely reduced to the Prussian white state in less than 10 h for soluble and insoluble forms. However, when electrodeposited on ITO-PET substrates, less than 1 h of incubation with bacteria is required for both forms, although the soluble one presents faster metabolic reduction kinetics. This study paves the way to the use of Prussian blue as a metabolic indicator for the early detection of bacterial infection in fields like microbial diagnostics, surface sterilization, food and beverage contamination, and environmental pollution, among others.

Toward Rapid Detection of Viable Bacteria in Whole Blood for Early Sepsis Diagnostics and Susceptibility Testing.

Sepsis is a serious bloodstream infection where the immunity of the host body is compromised, leading to organ failure and death of the patient. In early sepsis, the concentration of bacteria is very low and the time of diagnosis is very critical since mortality increases exponentially with every hour after infection. Common culture-based methods fail in fast bacteria determination, while recent rapid diagnostic methods are expensive and prone to false positives. In this work, we present a sepsis kit for fast detection of bacteria in whole blood, here achieved by combining selective cell lysis and a sensitive colorimetric approach detecting as low as 103 CFU/mL bacteria in less than 5 h. Homemade selective cell lysis buffer (combination of saponin and sodium cholate) allows fast processing of whole blood in 5 min while maintaining bacteria alive (100% viability). After filtration, retained bacteria on filter paper are incubated under constant illumination with the electrochromic precursors, i.e., ferricyanide and ferric ammonium citrate. Viable bacteria metabolically reduce iron(III) complexes, initiating a photocatalytic cascade toward Prussian blue formation. As a proof of concept, we combine this method with antibiotic susceptibility testing to determine the minimum inhibitory concentration (MIC) using two antibiotics (ampicillin and gentamicin). Although this kit is used to demonstrate its applicability to sepsis, this approach is expected to impact other key sectors such as hygiene evaluation, microbial contaminated food/beverage, or UTI, among others.

Solid Multiresponsive Materials Based on Nitrospiropyran-Doped Ionogels.

The application of molecular switches for the fabrication of multistimuli-responsive chromic materials and devices still remains a challenge because of the restrictions imposed by the supporting solid matrices where these compounds must be incorporated: they often critically affect the chromic response as well as limit the type and nature of external stimuli that can be applied. In this work, we propose the use of ionogels to overcome these constraints, as they provide a soft, fluidic, transparent, thermally stable, and ionic-conductive environment where molecular switches preserve their solution-like properties and can be exposed to a number of different stimuli. By exploiting this strategy, we herein pioneer the preparation of nitrospiropyran-based materials using a single solid platform that exhibit optimal photo-, halo-, thermo-, and electrochromic switching behaviors.

Nanoporous silk films with capillary action and size-exclusion capacity for sensitive glucose determination in whole blood.

In optical biosensing, silk fibroin (SF) appears as a promising alternative where other materials, such as paper, find limitations. Besides its excellent optical properties and unmet capacity to stabilize biomacromolecules, SF in test strips exhibits additional functions, i.e. capillary pumping activity of 1.5 mm s-1, capacity to filter blood cells thanks to its small, but tuneable, porosity and enhanced biosensing sensitivity. The bulk functionalization of SF with the enzymes glucose oxidase and peroxidase and the mediator ABTS produces colourless and transparent SF films that respond to blood glucose increasing 2.5 times the sensitivity of conventional ABTS-based assays. This enhanced sensitivity results from the formation of SF-ABTS complexes, where SF becomes part of the bioassay. Additionally, SF films triple the durability of most stable cellulose-based sensors. Although demonstrated for glucose, SF microfluidic test strips may incorporate other optical bioassays, e.g. immunoassays, with the aim of transferring them from central laboratories to the place of patient's care.

Sonochemical coating of Prussian Blue for the production of smart bacterial-sensing hospital textiles.

In healthcare facilities, environmental microbes are responsible for numerous infections leading to patient's health complications and even death. The detection of the pathogens present on contaminated surfaces is crucial, although not always possible with current microbial detection technologies requiring sample collection and transfer to the laboratory. Based on a simple sonochemical coating process, smart hospital fabrics with the capacity to detect live bacteria by a simple change of colour are presented here. Prussian Blue nanoparticles (PB-NPs) are sonochemically coated on polyester-cotton textiles in a single-step requiring 15 min. The presence of PB-NPs confers the textile with an intensive blue colour and with bacterial-sensing capacity. Live bacteria in the textile metabolize PB-NPs and reduce them to colourless Prussian White (PW), enabling in situ detection of bacterial presence in less than 6 h with the bare eye (complete colour change requires 40 h). The smart textile is sensitive to both Gram-positive and Gram-negative bacteria, responsible for most nosocomial infections. The redox reaction is completely reversible and the textile recovers its initial blue colour by re-oxidation with environmental oxygen, enabling its re-use. Due to its simplicity and versatility, the current technology can be employed in different types of materials for control and prevention of microbial infections in hospitals, industries, schools and at home.

Integrated Photonic System for Early Warning of Cyanobacterial Blooms in Aquaponics.

Cyanobacterial blooms produce hazardous toxins, deplete oxygen, and secrete compounds that confer undesirable organoleptic properties to water. To prevent bloom appearance, the World Health Organization has established an alert level between 500 and 2000 cells·mL-1, beyond the capabilities of most optical sensors detecting the cyanobacteria fluorescent pigments. Flow cytometry, cell culturing, and microscopy may reach these detection limits, but they involve both bulky and expensive laboratory equipment or long and tedious protocols. Thus, no current technology allows fast, sensitive, and in situ detection of cyanobacteria. Here, we present a simple, user-friendly, low-cost, and portable photonic system for in situ detection of low cyanobacterial concentrations in water samples. The system integrates high-performance preconcentration elements and optical components for fluorescence measurement of specific cyanobacterial pigments, that is, phycocyanin. Phycocyanin has demonstrated to be more selective to cyanobacteria than other pigments, such as chlorophyll-a, and to present an excellent linear correlation with bacterial concentration from 102 to 104 cell·mL-1 (R2 = 0.99). Additionally, the high performance of the preconcentration system leads to detection limits below 435 cells·mL-1 after 10 min in aquaponic water samples. Due to its simplicity, compactness, and sensitivity, we envision the current technology as a powerful tool for early warning and detection of low pathogen concentrations in water samples.

Nanoplasmonic Paper-Based Platform for General Screening of Biomacromolecules.

Hygiene assessment in industrial and clinical environments is crucial in the prevention of health risks. Current technologies for routine cleanliness evaluation rely on the detection of specific biomolecules, thus requiring more than one test for broad-range screening. Herein, the modulation of the catalytic activity of gold nanoparticles (AuNPs) by biomacromolecules was employed to develop a nanoplasmonic platform for general hygiene screening. AuNPs were immobilized on cellulose paper by simple adsorption. When ferricyanide was dispensed onto the paper, the AuNPs catalysed the ferricyanide's dissociation, releasing free cyanide ions that dissolved them. The AuNP dissolution produced an intense colour shift detectable with the naked eye. When biomacromolecules (e.g., proteins and polysaccharides) were present, they spontaneously attached to AuNPs, forming a biomolecular corona (biocorona), reducing their catalytic activity until complete suppression when the NPs were fully covered by molecules. The concentration-dependent decrease in the catalytic activity was here used to quantify biomacromolecules and complex samples such as milk, eggs, soy sauce and yeast extract (in 20 min), with detection limits comparable to those of standard methods, i.e., 0.25 µg mL-1 for albumin. This nano-enabled technology may be applied as a broad-range (unspecific) alert system for inexpensive cleanliness evaluation, with potential applications in sensitive sectors including productive industries and hospitals.

Impedance model for voltage optimization of parabens extraction in an electromembrane millifluidic device.

In sample pre-treatment, millifluidic electromembrane platforms have been developed to extract and pre-concentrate target molecules with good clean-up that minimize matrix effects. Optimal operation conditions are normally determined experimentally, repeating the extractions at different conditions and determining the efficiencies by an analytical technique. To shorten and simplify the optimization protocol, millifluidic platforms have been electrically characterized by impedance spectroscopy. The magnitude of the resistance of the electromembrane has been found very predictive of the migration capacity and extraction efficiency of three different parabens on real time. The optimal conditions (4 V of applied potential) (Electromembrane extraction low voltage) have been successfully applied in the extraction of parabens from urine samples, that not only improves the extraction efficiency (100% for all compounds) but also provides a very low current intensity (7 µA), which is very important in electromembrane to minimize electrolysis phenomena. The possibility to optimize one of the most critical and important parameters such as the voltage with a simple electrical model may accelerate the production of application-specific millifluidic electromembrane platforms in a short development time. The results showed that millifluidic electromembrane extraction based low voltage has a future potential as a simple, selective, and time-efficient sample preparation technique allowing a simple battery as power supply.

Photoelectro-Enzymatic Glucose Reusable Biosensor by Using Dithienylethene Mediators.

In the development of colorimetric biosensors, the use of electrochromic mediators has been accepted and widely used during decades. The main drawback of these types of enzymatic substrates is the difficult recovery of the initial redox state of the molecule, which can be done electrochemically or by antioxidants addition, complicating the initially simple structure of the biosensor. those strategies are rarely followed Actually, being the disposable biosensor configuration the most extended for this detection mechanisms. Alternatively, we propose the first reported use of a diacid dithienylethene 1,2-bis(5-carboxy-2-methylthien-3-yl)cyclopentene (DTE) photoelectrochromic compound as a substrate of the horseradish peroxidase (HRP). The photoisomerization between the open (DTEo) and closed (DTEc) forms of the molecule and the respective shift in the redox potential allowed the light-induced enzymatic detection of glucose in the glucose oxidase [(GOx)]-HRP cascade system. This fast and easy control over the enzymatic substrate availability by light pulses permits a gradually consumption and the light-regeneration of the biosensor for a number of cycles. We consider the presented results transcendent in the development of reusable and light-controlled photonic biosensing systems.

A self-calibrating and multiplexed electrochemical lab-on-a-chip for cell culture analysis and high-resolution imaging.

In vitro analysis requires cell proliferation in conditions close to physiological ones. Lab-on-a-chip (LoC) devices simplify, miniaturize and automate traditional protocols, with the advantages of being less expensive and faster due to their shorter diffusion distances. The main limitation of current LoCs is still the control of the culture conditions. Most LoCs employ off-chip equipment to determine cell culture activity, which confers limited monitoring capacity. The few systems integrating transducers on-chip present important functional problems mostly associated with the attachment of biomolecules to the transducer surface (i.e., biofouling) and the impossibility of re-calibrating the sensors during cell culturing. This limitation is addressed in the present LoC containing a network of micro-channels and micro-chambers, which allows (i) cell seeding and cultivation, avoiding biofouling risk, (ii) multiplexed analysis of cell culture, reactivation and recalibration of the (bio)sensors without compromising cell viability, (iii) cell imaging and (iv) reference electrode compartmentalization to guarantee stability. The activity of the culture is monitored with four independent electrochemical micro-electrodes for glucose, hydrogen peroxide, conductivity and oxidation reduction potential. Electrochemical analysis is complemented with high-resolution confocal microscopy analysis. This paper demonstrates the suitability of the current configuration for cell culture monitoring and future applications in drug screening or organ-on-a-chip development.

Ultrasensitive Photonic Microsystem Enabling Sub-micrometric Monitoring of Arterial Oscillations for Advanced Cardiovascular Studies.

Cardiovascular diseases are the first cause of death globally. Their early diagnosis requires ultrasensitive tools enabling the detection of minor structural and functional alterations in small arteries. Such analyses have been traditionally performed with video imaging-based myographs, which helped to investigate the pathophysiology of the microvessels. Since new vascular questions have emerged, substantial modifications are necessary to improve the performance of imaging and tracking software, reducing the cost and minimizing the microvessel cleaning and manipulation. To address these limitations, we present a photonic microsystem fabricated in polydimethylsiloxane and integrating micro-optical elements and a lightguide-cantilever for sub-micrometric analysis of small arteries (between 125 and 400 µm of basal diameter). This technology enables simultaneous measurement of arterial distension, stiffness, vasomotion, and heartbeat and without the need for advanced imaging system. The microsystem has a limit of detection of 2 µm, five times lower than video imaging-based myographs, is two times more sensitive than them (0.5 µm/mmHg), reduces variability to half and doubles the linear range reported in these myographs. More importantly, it allows the analysis of intact arteries preserving the integrity and function of surrounding tissues. Assays can be conducted in three configurations according to the surrounding tissue: (i) isolated arteries (in vitro) where the surrounding tissue is partially removed, (ii) non-isolated arteries (in vivo) with surrounding tissue partially removed, and (iii) intact arteries in vivo preserving surrounding tissue as well as function and integrity. This technology represents a step forward in the prediction of cardiovascular risk.

Microfluidic-controlled optical router for lab on a chip.

In multiplexed analysis, lab on a chip (LoC) devices are advantageous due to the low sample and reagent volumes required. Although optical detection is preferred for providing high sensitivity in a contactless configuration, multiplexed optical LoCs are limited by the technological complexity for integrating multiple light sources and detectors in a single device. To address this issue, we present a microfluidic-controlled optical router that enables measurement in four individual optical channels using a single light source and detector, and without movable parts. The optofluidic device is entirely fabricated in polydimethylsiloxane (PDMS) by soft-lithography, compatible with standard microfabrication technologies, enabling monolithic integration in LoCs. In the device, in-coupled light from an optical fiber is collimated by a polymeric micro-lens and guided through a set of four sequentially connected micro-chambers. When a micro-chamber is filled with water, light is transmitted to the next one. If it is empty of liquid, however, total internal reflection (TIR) occurs at the PDMS-air interface, re-directing the light to the output optical fiber. The router presents high performance, with low cross-talk (<2%) and high switching frequencies (up to 0.343 ± 0.006 Hz), and provides a stable signal for up to 91% of the switching time. With this miniaturized, low-cost, simple and robust design, we expect the current technology to be integrated in the new generation of multiplexed photonic LoCs for biomarker analysis, even at the point of care.

Fast phage detection and quantification: An optical density-based approach.

Since 1959 with the proposal of Double Agar Layer (DAL) method for phage detection and quantification, many sophisticated methods have emerged meanwhile. However, many of them are either too complex/expensive or insensitive to replace routine utilization of DAL method in clinical, environmental and industrial environments. For that purpose, we have explored an alternative method for the detection and quantification of bacteriophages that fulfills the criteria of being rapid, simple and inexpensive. In this paper we have developed a method based on the analysis of optical density kinetics in bacterial cultures exposed to phage-containing samples. Although the decrease in optical density caused by cell lysis was one of the first observable consequences of the effect of viral infection in bacterial cultures, the potential of the method for the assessment of phage abundance has never been fully exploited. In this work we carry out a detailed study of optical density kinetics in phage-infected bacterial cultures, as a function of both, phage abundance and initial concentration of the host organisms. In total, 90 different combinations of bacteria/phage concentrations have been used. The data obtained provide valuable information about sensitivity ranges, duration of the assay, percentages of inhibition and type of lysing behavior for each phage concentration. The method described can detect, as few as 10 phage particles per assay volume after a phage incubation period of 3.5h. The duration of the assay can be shortened to 45min at the expense of losing sensitivity and increasing the limit of detection to 108 pfu/ml. Despite using non-sophisticated technology, the method described has shown sensitivity and response time comparable to other high-end methods. The simplicity of the technology and of the analytical steps involved, make the system susceptible of miniaturization and automation for high-throughput applications which can be implemented in routine analysis in many environments.

Reconfigurable multiplexed point of Care System for monitoring type 1 diabetes patients.

At the point of care (POC), on-side clinical testing allows fast biomarkers determination even in resource-limited environments. Current POC systems rely on tests selective to a single analyte or complex multiplexed systems with important portability and performance limitations. Hence, there is a need for handheld POC devices enabling the detection of multiple analytes with accuracy and simplicity. Here we present a reconfigurable smartphone-interfaced electrochemical Lab-on-a-Chip (LoC) with two working electrodes for dual analyte determination enabling biomarkers' selection in situ and on-demand. Biomarkers selection was achieved by the use of electrodepositable alginate hydrogels. Alginate membranes containing either glucose oxidase (GOx) or lactate oxidase (LOx) were selectively electrodeposited on the surface of each working electrode in around 4 min, completing sample measurement in less than 1 min. Glucose and lactate determination was performed simultaneously and without cross-talk in buffer, fetal bovine serum (FBS) and whole blood samples, the latter being possible by the size-exclusion filtration capacity of the hydrogels. At optimal conditions, glucose and lactate were determined in a wide linear range (0-12 mM and 0-5 mM, respectively) and with high sensitivities (0.24 and 0.54 µA cm-2 mM-1, respectively), which allowed monitoring of Type-1 diabetic patients with a simple dual analysis system. After the measurement, membranes were removed by disaggregation with the calcium-chelator phosphate buffer. At this point, new membranes could be electrodeposited, this time being selective to the same or another analyte. This conferred the system with on-demand biomarkers' selection capacity. The versatility and flexibility of the current architecture is expected to impact in POC analysis in applications ranging from homecare to sanitary emergencies.

Cost-effective smartphone-based reconfigurable electrochemical instrument for alcohol determination in whole blood samples.

The determination of ethanol intoxication in whole blood samples may open the opportunity for a precise and quick point-of-measurement in the ambit of medical emergency or law enforcement. In contrast with traditional techniques based on breath sampling, direct blood measurements present greater immunity to errors specially in case of unconscious or non-collaborative patients. In this context, a portable, sensitive and easy-to-use instrument is highly desirable. In the current work we present a smartphone-based µPotentiostat which combines a novel circuital technique for sensor readout digitalization with a reusable lab-on-a-chip (LoC) concept. Such system allows both chronoamperometric and cyclic voltammetry measurements with a reduced number of electronic components on a very compact PCB (38.5â€¯× 22.5 mm2). Power, data-link and user interface are provided in combination with a standard smartphone, enabling cost-effectiveness and reconfigurability without sacrificing precision. The readout platform discussed in this work has been coupled to a LoC for point-of-care combining Pt electrodes microfabricated on silicon substrate for electrochemical measurement and a microfluidic structure of methacrylate for fluid management. Biosensing is enabled by in situ electrodeposition of a calcium alginate hydrogel containing horseradish peroxidase (HPR) and alcohol oxidase (AOx) for selective ethanol detection. Alginate membrane electrodeposition has been here optimized for rapid generation (2 min) and to retain the cellular fraction, thus allowing the measurement in whole blood samples. The µPotentiostat features a sensitivity of 36 nA/g L-1 to ethanol concentration in blood in the 0-1.25 g;L-1 range, with a limit of quantification (LoQ) of 4.5 nA, which is a suitable response for discerning the legal, illegal, severely illegal thresholds in a 40 µL sample of blood.