Design of "Off-On-Off" fluorescence sensors for Heparin detection by precise modulation of molecular structure.

In this strategy, the fluorescence sensor Nap-Co-T1 employing the fluorescence resonance energy transfer (FRET) mechanism was designed and synthesized to have an efficient response to Heparin, and the FRET mechanism was explored for different excitation-emission wavelengths with different distances between the energy acceptor and the energy donor (comparing with fluorescence sensor Nap-TPA-T2). Upon the addition of Heparin, the fluorescence emission of Nap-Co-T1 was turned on at 565 nm, and the fluorescence color changed of the solution from colorless to bright yellow. The limit of detection (LOD) was as low as 0.04 µg/mL. With the addition of antagonistic protamine (PRTM) to the sensor complex with Heparin, the fluorescence emission was turned off to a certain extent, and the reversibility of the "off-on-off" system was maintained for five cycles or more. In addition, Nap-Co-T1 provides rapid and sensitive detection of Heparin in human serum albumin solution and artificial urine and is highly sensitive to environmental viscosity.

Enhancing a multi-purpose artificial urine for culture and gene expression studies of uropathogenic Escherichia coli strains.

AIMS: The main objective of this study was to modify a recently reported multi-purpose artificial urine (MP-AU) for culture and gene expression studies of uropathogenic Escherichia coli (UPEC) strains. METHODS AND RESULTS: We used liquid chromatography mass spectrometry (LC-MS) to identify and adjust the metabolic profile of MP-AU closer to that of pooled human urine (PHU). Modification in this way facilitated growth of UPEC strains with growth rates similar to those obtained in PHU. Transcriptomic analysis of UPEC strains cultured in enhanced artificial urine (enhanced AU) and PHU showed that the gene expression profiles are similar, with <7% of genes differentially expressed between the two conditions. CONCLUSIONS: Enhancing an MP-AU with metabolites identified in PHU allows the enhanced AU to be used as a substitute for the culture and in vitro gene expression studies of UPEC strains.

Highly sensitive detection of the neurodegenerative biomarker Tau by using the concentration effect of the pyro-electrohydrodynamic jetting.

It is largely documented that neurodegenerative diseases can be effectively treated only if early diagnosed. In this context, the structural changes of some biomolecules such as Tau, seem to play a key role in neurodegeneration mechanism becoming eligible targets for an early diagnosis. Post-translational modifications are responsible to drive the Tau protein towards a transition phase from a native disorder conformation into a preaggregation state, which then straight recruits the final fibrillization process. Here, we show for the first time the detection of pre-aggregated Tau in artificial urine at femto-molar level, through the concentration effect of the pyro-electrohydrodynamic jet (p-jet) technique. An excellent linear calibration curve is demonstrated at the femto-molar level with a limit of detection (LOD) of 130 fM. Moreover, for the first time we show here the structure stability of the protein after p-jet application through a deep spectroscopic investigation. Thanks to the small volumes required and the relatively compact and cost-effective characteristics, this technique represents an innovative breakthrough in monitoring the early stage associated to neurodegeneration syndromes in different scenarios of point of care (POC) and such as for example in long-term human space exploration missions.

Meta-analysis guided development of a standard artificial urine.

In this study, we present the first meta-analysis of human urine reported in the literature, drawing data from a total of 35 articles with a combined participant count of 14,021. Through this analysis, we have developed an artificial urine (AU) composition that can be adjusted within typical physiological parameters for in vitro applications. Our findings demonstrate the utility of this AU in determining the solubility of nitrofurantoin, particularly in the context of crystalluria. Notably, we observe that in saline, nitrofurantoin solubility, within the framework of its urinary pharmacokinetics, suggests a risk of crystalluria. However, in AU, this risk is mitigated due to complexation with urea. More broadly, we anticipate that our developed formulation will serve as a foundation for translational studies across biomedical and pharmaceutical sciences.

Bacteria-Infected Artificial Urine Characterization Based on a Combined Approach Using an Electronic Tongue Complemented with 1H-NMR and Flow Cytometry.

The prevailing form of bacterial infection is within the urinary tract, encompassing a wide array of bacteria that harness the urinary metabolome for their growth. Through their metabolic actions, the chemical composition of the growth medium undergoes modifications as the bacteria metabolize urine compounds, leading to the subsequent release of metabolites. These changes can indirectly indicate the existence and proliferation of bacterial organisms. Here, we investigate the use of an electronic tongue, a powerful analytical instrument based on a combination of non-selective chemical sensors with a partial specificity for data gathering combined with principal component analysis, to distinguish between infected and non-infected artificial urine samples. Three prevalent bacteria found in urinary tract infections were investigated, Escherichia coli, Klebsiella pneumoniae, and Enterococcus faecalis. Furthermore, the electronic tongue analysis was supplemented with 1H NMR spectroscopy and flow cytometry. Bacteria-specific changes in compound consumption allowed for a qualitative differentiation between artificial urine medium and bacterial growth.

Metal-free electrocatalytic nanocomposites of poly azovan blue-decorated graphitic carbon nitride for simultaneously sensing paracetamol and 4-aminophenol.

Excessive consumption of paracetamol (PA) and 4-aminophenol (4-AP) can have harmful effects on the human body. This study developed a novel electroanalytical technique that utilizes the nanocomposites of poly azovan blue (PAB)-decorated graphitic carbon nitride (g-C3N4), deposited onto a screen-printed carbon electrode (SPCE), for the concurrent sensing of PA and 4-AP. The fabricated g-C3N4@PAB/SPCE exhibited exceptional synergistic effects, such as a high active electrochemical surface area and excellent electron transfer properties. The electrochemical behavior of g-C3N4@PAB/SPCE for simultaneous PA and 4-AP sensing was evaluated in the linear dynamic ranges of 0.08-75 and 0.05-90 µM, with the detection limits (S/N = 3) of 0.011 and 0.016 µM and sensitivities of 2.974 and 2.857 µA/µM/cm-2 for PA and 4-AP, respectively. Additionally, g-C3N4@PAB/SPCE showed long-term stability, high reproducibility (RSD = 2.17%, n = 4), and superior anti-interference capabilities. Finally, when g-C3N4@PAB/SPCE was tested for simultaneously sensing both PA and 4-AP in tap water and artificial urine models, it exhibited satisfactory recoveries, demonstrating its potential use for various industrial and clinical applications.

Novel sensitive immunosensor for the selective detection of Engrailed 2 urinary prostate cancer biomarker.

Engrailed 2 (EN2) is a homeodomain-containing transcription factor expressed in prostate cancer (PCa) cell lines and is secreted into the urines. It is nowadays considered as a promising non-invasive biomarker for PCa early diagnosis. Herein, we report the design of an electrochemical immunosensor for EN2 detection. The biosensor fabrication involved a covalent immobilization of anti-EN2 antibodies onto a poly para amino benzoic acid (PABA) film electropolymerized on a gold electrode. Square wave voltammetry was investigated for EN2 detection in a phosphate buffer solution in a concentration range of 10-5 ng/mL to 1 µg/mL. The limit of detection of the designed sensor was equal to 10-5 ng/mL and the sensitivity was of order of (29 ± 2) µL/ng. The dissociation constant Kd of the "complex" EN2/anti-EN2, estimated from a Hill model, was of order of (0.9 ± 0.2) fM. Experimental results revealed that the immunosensor enabled selective detection of EN2 in a mixture of three proteins which can be found in men' urine: human serum albumin (HSA), prostate-specific antigen (PSA) and immunoglobulin G (IgG). Tests in artificial urine, with an ionic strength of 0.18 M, have been done and results were found comparable to those obtained in PBS (0.16 M). These encouraging results show a potentially promising future for the development of an electrochemical biosensor for robust and accurate urinary biomarkers detection.

Microbial fuel cell scale-up options: Performance evaluation of membrane (c-MFC) and membrane-less (s-MFC) systems under different feeding regimes.

In recent years, bioelectrochemical systems have advanced towards upscaling applications and tested during field trials, primarily for wastewater treatment. Amongst reported trials, two designs of urine-fed microbial fuel cells (MFCs) were tested successfully on a pilot scale as autonomous sanitation systems for decentralised area. These designs, known as ceramic MFCs ( c -MFCs) and self-stratifying MFCs ( s -MFC), have never been calibrated under similar conditions. Here, the most advanced versions of both designs were assembled and tested under similar feeding conditions. The performance and efficiency were evaluated under different hydraulic retention times (HRT), through chemical oxygen demand measures and polarisation experiments. Results show that c -MFCs displayed constant performance independently from the HRT (32.2 ± 3.9 W m-3) whilst displaying high energy conversion efficiency at longer HRT (NER COD  = 2.092 ± 0.119 KWh.Kg COD -1, at 24h HRT). The s -MFC showed a correlation between performance and HRT. The highest performance was reached under short HRT (69.7 ± 0.4 W m-3 at 3h HRT), but the energy conversion efficiency was constant independently from the HRT (0.338 ± 0.029 KWh.Kg COD -1). The c -MFCs and s -MFCs similarly showed the highest volumetric efficiency under long HRT (65h) with NER V of 0.747 ± 0.010 KWh.m-3 and 0.825 ± 0.086 KWh.m-3, respectively. Overall, c -MFCs seems more appropriate for longer HRT and s -MFCs for shorter HRT.

Colorimetric paper-based sarcosine assay with improved sensitivity.

This manuscript reports on a simple paper-based bienzymatic colorimetric assay for sarcosine as an important urinary biomarker of prostate cancer. All required assay reagents are pre-deposited on hydrophilic filter paper spots surrounded by a hydrophobic barrier. Sarcosine in the sample solution is selectively oxidized in the presence of sarcosine oxidase (SOx), resulting in the formation of hydrogen peroxide, which is subsequently detected through the horseradish peroxidase (HRP)-catalyzed conversion of the colorless indicator 3,3',5,5'-tetramethylbenzidine (TMB) into its blue-colored oxidation product. By the modification of the paper with positively charged poly(allylamine hydrochloride) (PAH), a linear response to sarcosine between 0 and 10 µM and a significant lowering of the limit of detection (LOD) (0.6 µM) compared to the unmodified paper substrate (12.6 µM) has been achieved. The improvement of the LOD was attributed to the fact that the presence of the polymer limits the enzyme-driven colorimetric reaction to the surface of the paper substrate, resulting in stronger color development. In experiments in artificial urine matrix, the bicarbonate anion was identified as an inhibitor of the colorimetric reaction. This inhibition was successfully eliminated through on-device sample pH adjustments with pH-buffer components pre-deposited onto assay devices. The LOD for sarcosine achieved in artificial urine matrix (2.5 µM) is below the 5 µM threshold value for this urinary biomarker required for diagnostic purposes. Finally, good selectivity over all 20 natural amino acids and satisfactory long-term storage stability of reagent-modified paper substrates at - 20 °C for a period of 50 days were confirmed.

Assessment of encrustation and physicochemical properties of poly(lactide-glycolide) - Papaverine hydrochloride coating on ureteral double-J stents after long-term flow of artificial urine.

Implantation of ureteral stents is associated with inconvenience for the patient, which is related to the natural ability of the ureter to contract. The most frequently used solution is the systemic administration of a diastolic drug, which has a relaxing effect on smooth muscle cells and decreases inconvenience. Current interdisciplinary research aimed at reducing the complications after the implantation of ureteral stents used in the treatment of upper urinary tracts with regard to infection, initiation of encrustation, and fragmentation of stents, and patient pain has not been resolved. This study presents the results of research regarding the impact of a biodegradable coating with the active substance on the physical and chemical properties of ureteral stents used in the treatment of the upper urinary tract. The surface of polyurethane double-J stents was coated with poly(lactide-glycolide) (PLGA) 85/15 loaded with papaverine hydrochloride (PAP) with diastolic properties. The coating for ureteral stents has been designed for short-term implantation. The effect of the coating on the process of encrustation and PAP release by the dynamic in vitro model with artificial urine (AU) up to 30 days was evaluated. The influence of AU on the physical and chemical properties of ureteral stents was determined. As part of the study, surface structure and topography researches; chemical composition analyses using X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, and wetting; and surface roughness studies of both PUR stents and coated stents were carried out. The proposed biodegradable PLGA+PAP coating is characterized by controlled drug release, while optimal physicochemical properties does not increase the encrustation process.

Physical Properties of Electropolished CoCrMo Alloy Coated with Biodegradable Polymeric Coatings Releasing Heparin after Prolonged Exposure to Artificial Urine.

In this study, we aimed to determine the effect of long-term exposure to artificial urine on the physical properties of CoCrMo alloy with biodegradable heparin-releasing polymeric coatings. Variants of polymer coatings of poly(L,L-lactide-ɛ-caprolactone) (P(L,L-L/CL)) and poly(D,L-lactide-ɛ-caprolactone) (P(D,L-L/CL)) constituting the base for heparin-releasing (HEP) polyvinyl alcohol (PVA) coatings were analyzed. The coatings were applied by the dip-coating method. Heparin was used to counteract the incrustation process in the artificial urine. The study included tests of wettability, resistance to pitting and crevice corrosion, determination of the mass density of metal ions penetrating into the artificial urine, and the kinetics of heparin release. In addition, microscopic observations of surface roughness and adhesion to the metal substrate were performed. Electrolytically polished CoCrMo samples (as a reference level) and samples with polymer coatings were used for the tests. The tests were conducted on samples in the initial state and after 30, 60, and 90 days of exposure to artificial urine. The analysis of the test results shows that the polymer coatings contribute by improving the resistance of the metal substrate to pitting and crevice corrosion in the initial state and reducing (as compared with the metal substrate) the mass density of metal ion release into the artificial urine. Moreover, the PVA + HEP coating, regardless of the base polymer coatings used, contributes to a reduction in the incrustation process in the first 30 days of exposure to the artificial urine.

Label-free silver triangular nanoplates for spectrophotometric determination of catecholamines and their metabolites.

A novel method towards spectrophotometric determination of catecholamines and their metabolites differing in their functional groups has been developed. This method is based on a change in morphology of silver triangular nanoplates upon the action of cateсholamines and their metabolites, which is manifested by the decrease of the nanoparticle local surface plasmon resonance (LSPR) band intensity or its shift to the short-wavelength region of the spectrum. The shift value of the LSPR band or the change of its intensity increases with increasing concentration of catecholamines or their metabolites, which is proposed for their spectrophotometric determination. The limits of detection of catecholamines and their metabolites under selected conditions increase in the series homovanillic acid < vanillylmandelic acid < L-epinephrine < L-norepinephrine < dopamine and are 0.25, 1.2, 3.0, 64, and 130 µmol L-1, respectively. The selectivity of the proposed method was assessed using vanillylmandelic acid as example. It was found that the determination of vanillylmandelic acid does is not interfered in the presence of 4000-fold excess of Na+, K+, CH3COO-, and 1000-fold excess of Mg2+, Ca2+, Al3+, NO3-. The method also allows for the selective determination of vanillylmandelic acid in the presence of a 1000-fold excess of structurally related substances that do not contain either a catechol fragment or an electron donor substituent. The proposed approach was successfully applied to the determination of catecholamines in pharmaceuticals and artificial urine. Graphical abstract.

Selective reaction of homocysteine with o-phthalaldehyde under flow conditions in highly alkaline medium: fluorimetric determination using zone fluidics.

In the present study we report the reaction between homocysteine and o-phthalaldehyde under flow conditions. Homocysteine reacts on-line with the derivatization reagent in a strong alkaline medium and in the absence of nucleophilic reagents to yield a fluorescent derivative (λex /λem = 370/480 nm). The reaction variables were investigated using the concept of zone fluidics. Selectivity factors against other compounds were calculated at 10-fold excess. The findings formed the basis of an automated proposed method that was found to be linear in the range 0.1-1.5 µmol L-1 , with a limit of detection of 20 nmol L-1 and relative standard deviation < 0.5% (within-day) and 3.2% (between-day). The method proved to be rapid, offering a practical sampling rate of 24 h-1 and accurate following application to an artificial urine matrix with minimum dilution.

Proteus mirabilis outcompetes Klebsiella pneumoniae in artificial urine medium through secretion of ammonia and other volatile compounds.

Klebsiella pneumoniae and Proteus mirabilis form mixed biofilms in catheter-associated urinary tract infections. However, co-inoculation of P. mirabilis with K. pneumoniae in artificial urine medium (AUM) resulted in a drastic reduction of K. pneumoniae cells in both biofilm and planktonic growth. Here, the mechanism behind this competitive interaction was studied. Both pH and aqueous ammonia (NH3aq) increased in mixed cultures (to 9.3 and 150 mM, respectively), while K. pneumoniae viable cells dramatically diminished over time (>6-log reduction, p < 0.05). Mixed cultures developed in either 2-(N-morpholino) ethanesulfonic acid (MES)-buffered AUM (pH 6.5) or AUM without urea did not show bacterial competition, evidencing that the increase in pH and/or NH3aq concentration play a role in the competitive interaction. Viability of K. pneumoniae single-species cultures decreased 1.5-log in alkaline AUM containing 150 mM NH3aq after 24 h inoculation, suggesting that ammonia is involved in this inter-species competition. Besides NH3aq, additional antimicrobials should be present to get the whole competitive effect. Supernatants from P. mirabilis-containing cultures significantly diminished K. pneumoniae viability in planktonic cultures and affected biofilm biomass (p < 0.05). When subjected to evaporation, these supernatants lost their antimicrobial activity suggesting the volatile nature of the antimicrobial compounds. Exposure of K. pneumoniae to volatile compounds released by P. mirabilis significantly decreased cell viability in both planktonic and biofilm cultures (p < 0.05). The current investigation also evidenced a similar bactericidal effect of P. mirabilis volatiles over Escherichia coli and Morganella morganii. Altogether, these results evidence the secretion of ammonia and other volatile compounds by P. mirabilis, with antimicrobial activity against gram-negative uropathogens including K. pneumoniae. This investigation provides novel insight into competitive inter-species interactions that are mediated by production of volatile molecules.

Smartphone-integrated urinary CTX-II immunosensor based on wavelength filtering from chromogenic reaction.

The integration of smart IT devices and biochemical assays with optical biosensing technology facilitates the development of efficacious optical biosensors for many practical diagnostic fields, owing to their minimized use of high-technical electronic components and simple operation. Herein, we introduced a simple optical biosensing system based on the specific wavelength filtering principle and count-based analysis method. The developed system uses a smartphone with a paper-based signal guide and a biosensing channel. The paper-based signal guide was prepared by printing red patterns of various brightness on a black background. Given that a blue product is generated as a result of horseradish peroxidase (HRP)-based enzymatic reaction in the biosensing channel, the channel could be used as a blue filter that absorbs red light. When red light reflected from the red pattern is absorbed by the channel, the pattern appears black. As such, the color of the patterns is assimilated with the black background, so it seems to disappear. Consequently, the amount of blue product relative to the concentration of the target analyte can be measured by counting the number of observed patterns on the paper-based signal guide. In this study, the concentration of urinary C-telopeptide fragment of type II collagen (uCTX-II, 0-10 ng/mL) was measured using the developed system without complicated equipment. In addition, the quantitative analysis of uCTX-II in the real urine sample was successfully performed. Therefore, we expect that the developed optical transducing system could be practically used for point-of-care testing (POCT) diagnosis under resource-limited environmental conditions.

Culture Methods for Proteus mirabilis.

Proteus mirabilis is generally easy to culture, but its tendency to swarm on a wide variety of media can interfere with isolation of single colonies or identification of other species in a sample. Therefore, specialized media may be needed to control swarming or to study the bacteria under chemically defined conditions. Here, methods are described for routine culture of P. mirabilis, isolation of P. mirabilis from mixed cultures, and culture of P. mirabilis on physiologically relevant media.

Biodegradable ciprofloxacin-incorporated waterborne polyurethane polymers prevent bacterial biofilm formation in vitro.

The aim of the present study was to explore whether ciprofloxacin-incorporated waterborne polyurethane (WBPU) polymers have the capacity to inhibit bacterial biofilm formation in vitro. WBPU polymers were incorporated with ciprofloxacin and were cultured with Escherichia coli (E. coli) or Staphylococcus aureus (S. aureus) in media for 2, 4 or 7 days. In another experiment, the WBPU membranes were cultured with Proteus mirabilis (P. mirabilis) in artificial urine for 2, 4 or 7 days. Colony counting, scanning electron microscopy and fluorescence confocal microscopy were utilized to examine bacterial biofilms on the surfaces of membranes. The membranes were further co-cultured with P. mirabilis in a simple model of an artificial catheterized bladder in order to evaluate their ability to control encrustation. The WBPU films with ciprofloxacin effectively inhibited bacterial biofilm formation in the culture medium and in artificial urine. In addition, in artificial urine, the films with ciprofloxacin reduced catheter obstruction. In conclusion, ciprofloxacin-incorporated WBPU polymers are able to effectively inhibit bacterial biofilm formation in vitro.

Simultaneous voltammetric detection of 5-hydroxyindole-3-acetic acid and 5-hydroxytryptamine using a glassy carbon electrode modified with conducting polymer and platinised carbon nanofibers.

The authors describe a method for simultaneous voltammetric determination of 5-hydroxytryptamine (serotonin; 5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA). A glassy carbon electrode was modified with poly(pyrrole-3-carboxylic acid) and with platinised carbon nanofibers to obtain a sensor that can quantify 5-HT and 5-HIAA with detection limits of 10 nM and 20 nM, respectively. The peak currents, best measured at voltages of 170 mV and 500 mV (vs. Ag/AgCl) for 5-HT and 5-HIAA, increase linearly in the 0.01-100 µM concentration range for both analytes. The method was successfully applied to the quantitation of 5-HT and 5-HIAA in spiked artificial urine samples, and the sensor can be used up to 10 days. Graphical abstract A new electroanalytical device was developed for separation and quantitation of 5-hydroxytryptamine (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA), based on stripping square wave voltammetry, exploiting conducting polymer surfaces on platinised carbon nanofiber supports.

Luminescent Device for the Detection of Oxidative Stress Biomarkers in Artificial Urine.

A luminescent paper-based device for the visual detection of oxidative stress biomarkers is reported. The device consists of a polyvinylidene fluoride membrane impregnated with poly(3-alkoxy-4-methylthiophene) (PT) for colorimetric detection. 8-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker associated with oxidative stress, is used as a model system for validating the proposed methodology. The detection strategy is based on monitoring the changes in optical properties of PT associated with its conformational changes upon interaction with an aptamer in the presence and in the absence of 8-OHdG. Fluorometric and colorimetric monitoring revealed linear responses for 8-OHdG concentrations between 50 pM and 500 nM (∼14 pg/mL to 140 ng/mL), with limits of detection of ∼300 pM and  ∼350 pM, respectively for ( n = 3). Colorimetric responses in artificial urine ascertained rapid, sensitive, and selective detection of 8-OHdG at clinically relevant (pM to nM) concentration levels. Furthermore, the proposed methodology enables point-of-care diagnostics for oxidative stress without requiring sophisticated instrumentation.

Urinary p-cresol diagnosis using nanocomposite of ZnO/MoS2 and molecular imprinted polymer on optical fiber based lossy mode resonance sensor.

A lossy mode resonance (LMR) based sensor for urinary p-cresol testing on optical fiber substrate is developed. The sensor probe fabrication includes dip coating of nanocomposite layer of zinc oxide and molybdenum sulphide (ZnO/MoS2) over unclad core of optical fiber as the transducer layer followed by the layer of molecular imprinted polymer (MIP) as the recognition medium. The addition of molybdenum sulphide in the transducer layer increases the absorption of light in the medium which enhances the LMR properties of zinc oxide thereby increasing the conductivity and hence the sensitivity of the sensor. The sensor probe is characterized for p-cresol concentration range from 0µM (reference sample) to 1000µM in artificially prepared urine. Optimizations of various probe fabrication parameters are carried to bring out the sensor's optimal performance with a sensitivity of 11.86nm/µM and 28nM as the limit of detection (LOD). A two-order improvement in LOD is obtained as compared to the recently reported p-cresol sensor. The proposed sensor possesses a response time of 15s which is 8 times better than that reported in the literature utilizing electrochemical method. Its response time is also better than the p-cresol sensor currently available in the market for the medical field. Thus, with a fast response, significant stability and repeatability, the proposed sensor holds practical implementation possibilities in the medical field. Further, the realization of sensor probe over optical fiber substrate adds remote sensing and online monitoring feasibilities.