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.

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.

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.

Photonic Lab-on-a-Chip: Integration of Optical Spectroscopy in Microfluidic Systems.

The integration of micro-optical elements with microfluidics leads to the highly promising photonic lab-on-a-chip analytical systems (PhLoCs). In this work, we re-examine the main principles which are underneath the on-chip spectrophotometric detection, approaching the PhLoC concept to a nonexpert audience.

Self-oriented Ag-based polycrystalline cubic nanostructures through polymer stabilization.

This paper presents the study of the dynamics of the formation of polymer-assisted highly-orientated polycrystalline cubic structures (CS) by a fractal-mediated mechanism. This mechanism involves the formation of seed Ag@Co nanoparticles by InterMatrix Synthesis and subsequent overgrowth after incubation at a low temperature in chloride and phosphate solutions. These ions promote the dissolution and recrystallization in an ordered configuration of pre-synthetized nanoparticles initially embedded in negatively-charged polymeric matrices. During recrystallization, silver ions aggregate in AgCl@Co fractal-like structures, then evolve into regular polycrystalline solid nanostructures (e.g. CS) in a single crystallization step on specific regions of the ion exchange resin (IER) which maintain the integrity of polycrystalline nanocubes. Here, we study the essential role of the IER in the formation of these CS for the maintenance of their integrity and stability. Thus, this synthesis protocol may be easily expanded to the composition of other nanoparticles providing an interesting, cheap and simple alternative for cubic structure formation and isolation.

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.

Dual photonic-electrochemical lab on a chip for online simultaneous absorbance and amperometric measurements.

A dual lab on a chip (DLOC) approach that enables simultaneous optical and electrochemical detection working in a continuous flow regime is presented. Both detection modes are integrated for the first time into a single detection volume and operate simultaneously with no evidence of cross-talk. The electrochemical cell was characterized amperometrically by measuring the current in ferrocyanide solutions at +0.4 V vs gold pseudoreference electrode, at a flow rate of 200 µL min(-1). The experimental results for ferrocyanide concentrations ranging from 0.005 to 2 mM were in good agreement with the values predicted by the Levich equation for a microelectrode inside a rectangular channel, with a sensitivity of 2.059 ± 0.004 µA mM(-1) and a limit of detection (LoD) of (2.303 ± 0.004) × 10(-3) mM. Besides, optical detection was evaluated by measuring the absorbance of ferricyanide solutions at 420 nm. The results obtained therein coincide with those predicted by the Beer-Lambert law for a range of ferricyanide concentrations from 0.005 to 0.3 mM and showed an estimated LoD of (0.553 ± 0.001) × 10(-3) mM. The DLOC was finally applied to the analysis of L-lactate via a bienzymatic reaction involving lactate oxidase (LOX) and horseradish peroxidase (HRP). Here, the consumption of the reagent of the reaction (ferrocyanide) was continuously monitored by amperometry whereas the product of the reaction (ferricyanide) was recorded by absorbance. The DLOC presented good performance in terms of sensitivity and limit of detection, comparable to other fluidic systems found in the literature. Additionally, the ability to simultaneously quantify enzymatic reagent consumption and product generation confers the DLOC a self-verifying capability which in turn enhances its robustness and reliability.

Characterization of fibrous polymer silver/cobalt nanocomposite with enhanced bactericide activity.

This manuscript describes the synthesis (based on the intermatrix synthesis (IMS) method), optimization, and application to bacterial disinfection of Ag@Co polymer-metal nanocomposite materials with magnetic and bactericidal properties. This material showed ideal bactericide features for being applied to bacterial disinfection of water, particularly (1) an enhanced bactericidal activity (when compared with other nanocomposites only containing Ag or Co nanoparticles), with a cell viability close to 0% for bacterial suspensions with an initial concentration below 10(5) colony forming units per milliliter (CFU/mL) after a single pass through the material, (2) capacity of killing a wide range of bacterial types (from coliforms to gram-positive bacteria), and (3) a long performance-time, with an efficiency of 100% (0% viability) up to 1 h of operation and higher than 90% during the first 24 h of continuous operation. The nanocomposite also showed a good performance when applied to water samples from natural sources with more complex matrices with efficiencies always higher than 80%.

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.

Influence of the Photorhabdus luminescens phosphomannose isomerase gene, manA, on mannose utilization, exopolysaccharide structure, and biofilm formation.

Extracellular polysaccharide (EPS) is produced by diverse bacterial pathogens and fulfills assorted roles, including providing a structural matrix for biofilm formation and more specific functions in virulence, such as protection against immune defenses. We report here the first investigation of some of the genes important for biofilm formation in Photorhabdus luminescens and demonstrate the key role of the phosphomannose isomerase gene, manA, in the structure of functional EPS. Phenotypic analyses of a manA-deficient mutant showed the importance of EPS in motility, insect virulence, and biofilm formation on abiotic surfaces as well as the requirement of this gene for the use of mannose as the sole carbon source. Conversely, this defect had no apparent impact on symbiosis with the heterorhabditid nematode vector. A more detailed analysis of biofilm formation revealed that the manA mutant was able to attach to surfaces with the same efficiency as that of the wild-type strain but could not develop the more extended biofilm matrix structures. A compositional analysis of P. luminescens EPS reveals how the manA mutation has a major effect on the formation of a complete, branched EPS.

Transient storage of electrical charge in biofilms of Shewanella oneidensis MR-1 growing in a microbial fuel cell.

Current output of microbial fuel cells (MFCs) depends on a number of engineering variables mainly related to the design of the fuel cell reactor and the materials used. In most cases the engineering of MFCs relies on the premise that for a constant biomass, current output correlates well with the metabolic activity of the cells. In this study we analyze to what extent, MFC output is also affected by the mode of operation, emphasizing how discontinuous operation can affect temporal patterns of current output. The experimental work has been carried out with Shewanella oneidensis MR-1, grown in conventional two-chamber MFCs subject to periodic interruptions of the external circuit. Our results indicate that after closure of the external circuit, current intensity shows a peak that decays back to basal values. The result suggests that the MFC has the ability to store charge during open circuit situations. Further studies using chronoamperometric analyses were carried out using isolated biofilms of Shewanella oneidensis MR-1 developed in a MFC and placed in an electrochemistry chamber in the presence of an electron donor. The results of these studies indicate that the amount of excess current over the basal level released by the biofilm after periods of circuit disconnection is proportional to the duration of the disconnection period up to a maximum of approximately 60 min. The results indicate that biofilms of Shewanella oneidensis MR-1 have the ability to store charge when oxidizing organic substrates in the absence of an external acceptor.

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.

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.

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.

Photorhabdus adhesion modification protein (Pam) binds extracellular polysaccharide and alters bacterial attachment.

BACKGROUND: Photorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts. RESULTS: A comparison of the proteins secreted by a clinical isolate of P. asymbiotica at simulated insect (28 degrees C) and human (37 degrees C) temperatures led to the identification of a small and highly abundant protein, designated Pam, that is only secreted at the lower temperature. The pam gene is present in all Photorhabdus strains tested and shows a high level of conservation across the whole genus, suggesting it is both ancestral to the genus and probably important to the biology of the bacterium. The Pam protein shows limited sequence similarity to the 13.6 kDa component of a binary toxin of Bacillus thuringiensis. Nevertheless, injection or feeding of heterologously produced Pam showed no insecticidal activity to either Galleria mellonella or Manduca sexta larvae. In bacterial colonies, Pam is associated with an extracellular polysaccharide (EPS)-like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. Interestingly, Surface Plasmon Resonance (SPR) binding studies revealed that the Pam protein itself has adhesive properties. Although Pam is produced throughout insect infection, genetic knockout does not affect either insect virulence or the ability of P. luminescens to form a symbiotic association with its host nematode, Heterorhabditis bacteriophora. CONCLUSIONS: We studied a highly abundant protein, Pam, which is secreted in a temperature-dependent manner in P. asymbiotica. Our findings indicate that Pam plays an important role in enhancing surface attachment in insect blood. Its association with exopolysaccharide suggests it may exert its effect through mediation of EPS properties. Despite its abundance and conservation in the genus, we find no evidence for a role of Pam in either virulence or symbiosis.

The interaction of serum albumin with cholesterol containing lipid vesicles.

In this paper, the interaction of both human blood serum (the primary fraction of which is serum albumin) and pure human serum albumin (HSA) with surface immobilised lipid vesicles was measured by combined Surface Plasmon Resonance (SPR) and Surface Plasmon enhanced Fluorescence (SPEFS), and fluorescence microscopy. It was found that both blood serum and HSA showed specific binding to vesicles which contained cholesterol, resulting in increased membrane permeability and release of encapsulated fluorescent dye. This effect was not seen with heat inactivated blood serum, heat inactivated HSA or in vesicles not containing cholesterol. These results suggest that HSA may have a physiological role over and beyond that of fatty acid carrier, possibly acting to regulate vascular endothelial cell cholesterol concentration.