Detection of Cannabinoids in Oral Fluid Specimens as the Preferred Biological Matrix for a Point-of-Care Biosensor Diagnostic Device.

An increasing number of countries have started to decriminalize or legalize the consumption of cannabis for recreational and medical purposes. The active ingredients in cannabis, termed cannabinoids, affect multiple functions in the human body, including coordination, motor skills, memory, response time to external stimuli, and even judgment. Cannabinoids are a unique class of terpeno-phenolic compounds, with 120 molecules discovered so far. There are certain situations when people under the influence of cannabis may be a risk to themselves or the public safety. Over the past two decades, there has been a growing research interest in detecting cannabinoids from various biological matrices. There is a need to develop a rapid, accurate, and reliable method of detecting cannabinoids in oral fluid as it can reveal the recent intake in comparison with urine specimens, which only show a history of consumption. Significant improvements are continuously made in the analytical formats of various technologies, mainly concerning improving their sensitivity, miniaturization, and making them more user-friendly. Additionally, sample collection and pretreatment have been extensively studied, and specific devices for collecting oral fluid specimens have been perfected to allow rapid and effective sample collection. This review presents the recent findings regarding the use of oral fluid specimens as the preferred biological matrix for cannabinoid detection in a point-of-care biosensor diagnostic device. A critical review is presented, discussing the findings from a collection of review and research articles, as well as publicly available data from companies that manufacture oral fluid screening devices. Firstly, the various conventional methods used to detect cannabinoids in biological matrices are presented. Secondly, the detection of cannabinoids using point-of-care biosensors is discussed, emphasizing oral fluid specimens. This review presents the current pressing technological challenges and highlights the gaps where new technological solutions can be implemented.

Characterization and optimization of bioluminescent bacterial cells immobilization process in calcium alginate hydrogel tablets.

AIMS: Whole-cell biosensors are increasingly utilized in various applications. These platforms integrate cells with a signal measurement device. One of the main challenges in the development of such platforms is the immobilization matrix that is used to keep the cells stable, which also affects the portability of the device. In this study, a portable and simple immobilization of bioluminescent bacterial cells in calcium alginate hydrogel was examined. METHODS AND RESULTS: The effects of several physical parameters were investigated (e.g. calcium alginate solution volume, drying, incubation time, mixing procedure, bacterial concentration, and tablet location within the cylinder). An alginate solution volume of 3 ml was preferred as well as the addition of 400 µl solution after the 15 min of compressing step and before the polymerization step. Also, a stirring mixing mode is favored over vortexing due to the creation of better homogenized tablets, as well as a bacterial concentration of 0.15 OD600nm that produced a high light response while maintaining a lower variance. Lastly, the findings showed a significantly higher response [induction factor (IF)] in the tablets using the optimized immobilization protocol (IF = 8.814) than the old one (IF = 1.979). CONCLUSIONS: To conclude, bacterial cells immobilization in calcium alginate tablets provides improved sensitivity and storability.

Biocompatibility characterization of vaterite with a bacterial whole-cell biosensor.

The growing biomedical challenges impose the continuous development of novel platforms. Ensuring the biocompatibility of drug delivery and implantable biomedical devices is an essential requirement. Calcium carbonate (CaCO3) in the form of vaterite nanoparticles is a promising platform, which has demonstrated distinctive optical and biochemical properties, including high porosity and metastability. In this study, the biocompatibility of differently shaped CaCO3 vaterite particles (toroids, ellipsoids, and spheroids) are evaluated by bacterial toxicity mode-of-action with a whole-cell biosensor. Different Escherichia coli (E. coli) strains were used in the bioluminescent assay, including cytotoxicity, genotoxicity and quorum-sensing. Firstly, both scanning electron microscopy (SEM) and fluorescence microscopy characterizations were conducted. Bacterial cell death and aggregates were observed only in the highest tested concentration of the vaterite particles, especially in toroids 15-25 µm. After, the bioluminescent bacterial panel was exposed to the vaterite particles, and their bioluminescent signal reflected their toxicity mode-of-action. The vaterite particles resulted in an induction factor (IF > 1) on the bacterial panel, which was higher after exposure to the toroids (1.557 ≤ IF ≤ 2.271) and ellipsoids particles (1.712 ≤ IF ≤ 2.018), as compared to the spheroids particles (1.134 ≤ IF ≤ 1.494), in all the tested bacterial strains. Furthermore, the vaterite particles did not affect the viability of the bacterial cells. The bacterial monitoring demonstrated the biofriendly nature of especially spheroids vaterite nanoparticles.

Portable biosensors for rapid on-site determination of cannabinoids in cannabis, a review.

Recent studies highlight the therapeutic virtues of cannabidiol (CBD). Furthermore, due to their molecular enriched profiles, cannabis inflorescences are biologically superior to a single cannabinoid for the treatment of various health conditions. Thus, there is flourishing demand for Cannabis sativa varieties containing high levels of CBD. Additionally, legal regulations around the world restrict the cultivation and consumption of tetrahydrocannabinol (THC)-rich cannabis plants for their psychotropic effects. Therefore, the use of cannabis varieties that are high in CBD is permitted as long as their THC content does not exceed a low threshold of 0.3%-0.5%, depending on the jurisdiction. These chemovars are legally termed 'hemp'. This controlled cannabinoid requirement highlights the need to detect low levels of THC, already in the field. In this review, cannabis profiling and the existing methods used for the detection of cannabinoids are firstly evaluated. Then, selected valuable biosensor technologies are discussed, which suggest portable, rapid, sensitive, reproducible, and reliable methods for on-site identification of cannabinoids levels, mainly THC. Recent cutting-edge techniques of promising potential usage for both cannabis and hemp analysis are identified, as part of the future cultivation and agricultural improvement of this crop.

Whole-cell bacterial biosensor with the capability to detect red palm weevil, Rhynchophorus ferrugineus, in date palm trees, Phoenix dactylifera: a proof of concept study.

The red palm weevil (RPW), Rhynchophorus ferrugineus, is considered a severe pest of palms. Usually, the early stages of infection are without visible signs. An attractive early sensing approach of non-visible infections is based on volatile organic compounds (VOCs). In this study, a whole-cell bacterial biosensor was used for the identification of RPW in date palm (Phoenix dactylifera). The cells are genetically modified to produce light in the presence of general stresses. The bioluminescent bacterial panel is based on three genetically engineered Escherichia coli strains that are sensitive to cytotoxicity (TV1061), genotoxicity (DPD2794), or quorum-sensing (K802NR). The bioluminescent bacterial panel detects the presence of VOCs and a change in the light signal is then generated, reflecting the health status of the date palm tree. The bioreporter bacteria cells are immobilized in calcium alginate tablets and placed in a sealed jar without direct contact with the tested sample, thereby exposing them only to the VOCs in the surrounding air. The immobilized bacteria cells were exposed to the air near infected by RPW or uninfected sugar canes, date palm tree pieces, and on date palm trees. Commercial plate reader was used for signal measurement. The findings show that quorum-sensing was induced by all the tested samples of infected sugar canes, date palm tree pieces, and date palm trees. While, cytotoxicity was induced only by infected date palm tree pieces, and genotoxicity was induced only by infected date palm trees. The bacterial monitoring results enable the identification of specific signatures that will allow a quick and accurate diagnosis.

Whole-cell bacterial biosensor for volatile detection from Pectobacterium-infected potatoes enables early identification of potato tuber soft rot disease.

Half of the harvested food is lost due to rots caused by microorganisms. Plants emit various volatile organic compounds (VOCs) into their surrounding environment, and the VOC profiles of healthy crops are altered upon infection. In this study, a whole-cell bacterial biosensor was used for the early identification of potato tuber soft rot disease caused by the pectinolytic bacteria Pectobacterium in potato tubers. The detection is based on monitoring the luminescent responses of the bacteria panel to changes in the VOC profile following inoculation. First, gas chromatography-mass spectrometry (GC-MS) was used to specify the differences between the VOC patterns of the inoculated and non-inoculated potato tubers during early infection. Five VOCs were identified, 1-octanol, phenylethyl alcohol, 2-ethyl hexanol, nonanal, and 1-octen-3-ol. Then, the infection was detected by the bioreporter bacterial panel, firstly measured in a 96-well plate in solution, and then also tested in potato plugs and validated in whole tubers. Examination of the bacterial panel responses showed an extensive cytotoxic effect over the testing period, as seen by the elevated induction factor (IF) values in the bacterial strain TV1061 after exposure to both potato plugs and whole tubers. Moreover, quorum sensing influences were also observed by the elevated IF values in the bacterial strain K802NR. The developed whole-cell biosensor system based on bacterial detection will allow more efficient crop management during postharvest, storage, and transport of crops, to reduce food losses.

Characterization of the selective binding of modified chitosan nanoparticles to Gram-negative bacteria strains.

Chitosan is a nature-sourced polysaccharide widely used in numerous applications. The antibacterial potential of chitosan has attracted researchers to further develop and utilize this polymer for the formation of biocompatible antibacterial agents for both the food and healthcare industries. The tested hypothesis in this study is that modified N-alkylaminated chitosan nanoparticles (CNPs) have selective binding properties to Gram-negative bacteria strains that result in bacterial aggregation. Various bacterial strains were tested of five Gram-negative bacteria including Erwinia carotovora, Escherichia coli, Pseudomonas aeruginosa, Salmonella, and Serratia marcescens, as well as three Gram-positive bacteria strains including Bacillus licheniformis, Bacillus megaterium, and Bacillus subtilis. The fluorescence microscopy characterization showed that the presence of CNPs caused the aggregation of Escherichia coli bacteria cells, where modified CNPs with a shorter chain length of the substituent caused a higher aggregation effect. Moreover, it was found that the CNPs exhibited a selective binding behavior to Gram-negative as compared to Gram-positive bacteria strains, mainly to Escherichia coli and Salmonella. Also, the scanning electron microscopy characterization showed that CNPs exhibited selective binding to Gram-negative bacteria, which was especially understood when both Gram-negative and Gram-positive bacteria strains were within the same sample. In addition, the bacterial viability assay suggests that CNPs with a lower degree of substitution have a higher inhibitory effect on bacterial growth. CNPs with longer side chains had a less inhibitory effect on the bacterial growth of Gram-negative strains, where a concentration-dependent response pattern was only seen for the cases of Gram-negative strains, and not for the case of Gram-positive strain. To conclude, the further understanding of the selective binding of CNPs to Gram-negative bacteria strains may produce new opportunities for the discovery and characterization of effective antibacterial agents.

Cigarette smoke toxicity modes of action estimated by a bioluminescent bioreporter bacterial panel.

Cigarette smoking is considered to be a risk factor for several chronic diseases and even premature death. However, despite the importance of this detrimental habit, little seems known in terms of the overall toxicity potential of its ingredients in humans. In this study, a panel of genetically modified bioluminescent bioreporter bacteria was used to evaluate its usefulness in estimating the cigarette smoke's complex molecular mixture on a bacterial toxicity-bioreporter panel, both filtered or unfiltered. This work enabled to confirm the usefulness of cigarette filters, with better protection found in higher priced brands despite both having genotoxic and cytotoxic attributes. Quorum sensing interference was also shown, which may explain why cigarette smokers are at greater risk for pulmonary infections. Moreover, the findings of this study support the fact that the filter is a dominating contributor to reducing the harm caused by cigarette smoke. Increased efforts should be conducted to reduce the harmful effects of cigarette smoke, via increasingly effective filters. To conclude, the panel of bioreporter bacteria was found to be useful in the evaluation of the general effect of the toxic mixture found in cigarette smoke and therefore has the potential to be used in cigarette research, helping researchers pinpoint the reduction of toxicity when working with filter improvement.

Blood biomarkers to detect new-onset atrial fibrillation and cardioembolism in ischemic stroke patients.

BACKGROUND: Accumulating data suggest blood biomarkers could inform stroke etiology. OBJECTIVE: The purpose of this study was to investigate the performance of multiple blood biomarkers in elucidating stroke etiology with a focus on new-onset atrial fibrillation (AF) and cardioembolism. METHODS: Between January and December 2017, information on clinical and laboratory parameters and stroke characteristics was prospectively collected from ischemic stroke patients recruited from the National University Hospital, Singapore. Multiple blood biomarkers (N-terminal pro-brain natriuretic peptide [NT-proBNP], d-dimer, S100ß, neuron-specific enolase, vitamin D, cortisol, interleukin-6, insulin, uric acid, and albumin) were measured in plasma. These variables were compared with stroke etiology and the risk of new-onset AF and cardioembolism using multivariable regression methods. RESULTS: Of the 515 ischemic stroke patients (mean age 61 years; 71% men), 44 (8.5%) were diagnosed with new-onset AF, and 75 (14.5%) had cardioembolism. The combination of 2 laboratory parameters (total cholesterol ≤169 mg/dL; triglycerides ≤44.5 mg/dL) and 3 biomarkers (NT-proBNP ≥294 pg/mL; S100ß ≥64 pg/mL; cortisol ≥471 nmol/l) identified patients with new-onset AF (negative predictive value [NPV] 90%; positive predictive value [PPV] 73%; area under curve [AUC] 85%). The combination of 2 laboratory parameters (total cholesterol ≤169 mg/dL; triglycerides ≤44.5 mg/dL) and 2 biomarkers (NT-proBNP ≥507 pg/mL; S100ß ≥65 pg/mL) identified those with cardioembolism (NPV 86%; PPV 78%; AUC 87%). Adding clinical predictors did not improve the performance of these models. CONCLUSION: Blood biomarkers could identify patients with increased likelihood of cardioembolism and direct the search for occult AF.

The effect of cannabis toxicity on a model microbiome bacterium epitomized by a panel of bioluminescent E. coli.

The world acceptance of medical cannabis slowly widens. Cannabinoids are known as the main therapeutic active compounds in the cannabis plant, yet their bioactive physiological effects are still unknown. In this study, the mode of action of nine selected cannabinoids was examined using a bioluminescent bacterial panel, as well as the extracts of six different cannabis varieties and cannabinoids standards artificial mixtures. The bacterial panel was composed of genetically modified E. coli bacteria that is commonly found in the gut microbiome, to which a lux operon was added to various stress promoters. The panel was exposed to the cannabinoids in order to identify bacterial defense mechanism, via the aforementioned specific stress types response. This enables the understanding of the toxicity mode of action of cannabinoids. From all the tested cannabinoids, only delta-9-tetrahydrocannabinol (THC) and delta-9-tetrahydrocannabinolic acid A (THCA) produced a genotoxic effect, while the other tested cannabinoids, demonstrated cytotoxic or oxidative damages. Unlike pure cannabinoids, cannabis plant extracts exhibited mostly genotoxicity, with minor cytotoxicity or oxidative stress responses. Moreover, cannabinoids standards artificial mixtures produced a different response patterns compared to their individual effects, which may be due to additional synergistic or antagonistic reactions between the mixed chemicals on the bacterial panel. The results showed that despite the lack of cannabigerol (CBG), cannabidivarin (CBDV), cannabinol (CBN), and cannabichromene (CBC) in the artificial solution mimicking the CN6 cannabis variety, a similar response pattern to the cannabinoids standards mixture was obtained. This work contributes to the understanding of such correlations and may provide a realistic view of cannabinoid effects on the human microbiome.

The Incorporation of Amplified Metal-Enhanced Fluorescence in a CMOS-Based Biosensor Increased the Detection Sensitivity of a DNA Marker of the Pathogenic Fungus Colletotrichum gloeosporioides.

Half of the global agricultural fresh produce is lost, mainly because of rots that are caused by various pathogenic fungi. In this study, a complementary metal-oxide-semiconductor (CMOS)-based biosensor was developed, which integrates specific DNA strands that allow the detection of enoyl-CoA-hydratase/isomerase, which is a quiescent marker of Colletotrichum gloeosporioides fungi. The developed biosensor mechanism is based on the metal-enhanced fluorescence (MEF) phenomenon, which is amplified by depositing silver onto a glass surface. A surface DNA strand is then immobilized on the surface, and in the presence of the target mRNA within the sample, the reporter DNA strand that is linked to horseradish peroxidase (HRP) enzyme will also bind to it. The light signal that is later produced from the HRP enzyme and its substrate is enhanced and detected by the coupled CMOS sensor. Several parameters that affect the silver-deposition procedure were examined, including silver solution temperature and volume, heating mode, and the tank material. Moreover, the effect of blocking treatment (skim milk or bovine serum albumin (BSA)) on the silver-layer stability and nonspecific DNA absorption was tested. Most importantly, the effect of the deposition reaction duration on the silver-layer formation and the MEF amplification was also investigated. In the study findings a preferred silver-deposition reaction duration was identified as 5-8 min, which increased the deposition of silver on the glass surface up to 13-times, and also resulted in the amplification of the MEF phenomenon with a maximum light signal of 50 relative light units (RLU). It was found that MEF can be amplified by a customized silver-deposition procedure that results in increased detection sensitivity. The implementation of the improved conditions increased the biosensor sensitivity to 3.3 nM (4500 RLU) with a higher detected light signal as compared to the initial protocol (400 RLU). Moreover, the light signal was amplified 18.75-, 11.11-, 5.5-, 11.25-, and 3.75-times in the improved protocol for all the tested concentrations of the target DNA strand of 1000, 100, 10, 3.3, and 2 nM, respectively. The developed biosensor system may allow the detection of the pathogenic fungus in postharvest produce and determine its pathogenicity state.

Environmental pollutants induce noninherited antibiotic resistance to polymyxin B in Escherichia coli.

Aim: The mechanisms behind antibiotic resistance by bacteria are important to create alternative molecules. Objective: This study focuses on the impact of environmental pollutants on bacterial resistance to antibiotics. Materials & methods: The effect of various environmental pollutants on noninherited bacterial resistance to antibiotics was examined. Results: The tolerance to the polymyxin-B antibiotic was shown to be conferred to Escherichia coli, by pretreatment with subinhibitory concentrations of environmental toxicants. The cell survival to a sublethal dosage of antibiotics was tested. Exposure to low concentrations of toxic compounds (500 ppb copper, 2% [v/v] ethanol or 0.5 µg/ml trimethoprim) stimulated the bacterial heat shock systems and led to increased tolerance to polymyxin B. Conclusion: Environmental pollutants induce a temporary bacterial noninheritable resistance to antibiotic.

Blood-Based Biomarkers Are Associated with Different Ischemic Stroke Mechanisms and Enable Rapid Classification between Cardioembolic and Atherosclerosis Etiologies.

Stroke is a top leading cause of death, which occurs due to interference in the blood flow of the brain. Ischemic stroke (blockage) accounts for most cases (87%) and is further subtyped into cardioembolic, atherosclerosis, lacunar, other causes, and cryptogenic strokes. The main value of subtyping ischemic stroke patients is for a better therapeutic decision-making process. The current classification methods are complex and time-consuming (hours to days). Specific blood-based biomarker measurements have promising potential to improve ischemic stroke mechanism classification. Over the past decades, the hypothesis that different blood-based biomarkers are associated with different ischemic stroke mechanisms is increasingly investigated. This review presents the recent studies that investigated blood-based biomarker characteristics differentiation between ischemic stroke mechanisms. Different blood-based biomarkers are specifically discussed (b-type natriuretic peptide, d-dimer, c-reactive protein, tumor necrosis factor-α, interleukin-6, interleukin-1ß, neutrophil-lymphocyte ratio, total cholesterol, triglycerides, low-density lipoprotein, high-density lipoprotein and apolipoprotein A), as well as the different cut-off values that may be useful in specific classifications for cardioembolic and atherosclerosis etiologies. Lastly, the structure of a point-of-care biosensor device is presented, as a measuring tool on-site. The information presented in this review will hopefully contribute to the major efforts to improve the care for stroke patients.

Smartphone-Based Whole-Cell Biosensor Platform Utilizing an Immobilization Approach on a Filter Membrane Disk for the Monitoring of Water Toxicants.

Bioluminescent bacteria whole-cell biosensors (WCBs) have been widely used in a range of sensing applications in environmental monitoring and medical diagnostics. However, most of them use planktonic bacteria cells that require complicated signal measurement processes and therefore limit the portability of the biosensor device. In this study, a simple and low-cost immobilization method was examined. The bioluminescent bioreporter bacteria was absorbed on a filter membrane disk. Further optimization of the immobilization process was conducted by comparing different surface materials (polyester and parafilm) or by adding glucose and ampicillin. The filter membrane disks with immobilized bacteria cells were stored at -20 °C for three weeks without a compromise in the stability of its biosensing functionality for water toxicants monitoring. Also, the bacterial immobilized disks were integrated with smartphones-based signal detection. Then, they were exposed to water samples with ethanol, chloroform, and H2O2, as common toxicants. The sensitivity of the smartphone-based WCB for the detection of ethanol, chloroform, and H2O2 was 1% (v/v), 0.02% (v/v), and 0.0006% (v/v), respectively. To conclude, this bacterial immobilization approach demonstrated higher sensitivity, portability, and improved storability than the planktonic counterpart. The developed smartphone-based WCB establishes a model for future applications in the detection of environmental water toxicants.

B-Type Natriuretic Peptide as a Significant Brain Biomarker for Stroke Triaging Using a Bedside Point-of-Care Monitoring Biosensor.

Stroke is a widespread condition that causes 7 million deaths globally. Survivors suffer from a range of disabilities that affect their everyday life. It is a complex condition and there is a need to monitor the different signals that are associated with it. Stroke patients need to be rapidly diagnosed in the emergency department in order to allow the admission of the time-limited treatment of tissue plasminogen activator (tPA). Stroke diagnostics show the use of sophisticated technologies; however, they still contain limitations. The hidden information and technological advancements behind the utilization of biomarkers for stroke triaging are significant. Stroke biomarkers can revolutionize the way stroke patients are diagnosed, monitored, and how they recover. Different biomarkers indicate different cascades and exhibit unique expression patterns which are connected to certain pathologies in the human body. Over the past decades, B-type natriuretic peptide (BNP) and its derivative N-terminal fragment (NT-proBNP) have been increasingly investigated and highlighted as significant cardiovascular biomarkers. This work reviews the recent studies that have reported on the usefulness of BNP and NT-proBNP for stroke triaging. Their classification association is also presented, with increased mortality in stroke, correlation with cardioembolic stroke, and an indication of a second stroke recurrence. Moreover, recent scientific efforts conducted for the technological advancement of a bedside point-of-care (POC) device for BNP and NT-proBNP measurements are discussed. The conclusions presented in this review may hopefully assist in the major efforts that are currently being conducted in order to improve the care of stroke patients.

Functionalized silicon dioxide self-referenced plasmonic chip as point-of-care biosensor for stroke biomarkers NT-proBNP and S100ß.

Surface plasmon resonance (SPR) biosensors are often used in the detection of solid, liquid or gaseous samples in diagnostics, pharmaceutics and military defense. Plasmon waveguide resonance (PWR) mode is obtained when a dielectric waveguide layer is added to the metal film. In this study, a self-referenced PWR (SRPWR) silicon dioxide (SiO2) chip was examined. The self-referenced measurement is important to compensate for temperature fluctuations, other instabilities and allows RI signal measurement without an additional reference sample, thus minimising the sample volume needed. The chip was fabricated with a multi-layer of metals and dielectrics, consisting of a 420 nm SiO2 layer, a 40 nm Ag layer and another 480 nm SiO2 layer. This chip was shown to give one internal plasmon excited on the bottom interface SiO2/Ag, which is used as self-reference in the detection. The top layer acts as a waveguide layer and can be designed to give modes with ultrahigh penetration depth. A direct assay was developed, where the recognition molecule (specific antibody) was immobilized onto the SiO2 plasmonic chip surface, via a covalent coupling protocol based on 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde. The SRPWR biosensor was developed for the sensing of two chosen stroke biomarkers: NT-proBNP and S100ß, which are sensitive and specific for stroke diagnostics. For both biomarkers, a linear decreasing pattern in the RI signal was recognized with the increasing biomarkers concentrations. Biomarkers detection was conducted in deionized water and validation was done in spiked porcine plasma. The SiO2 based plasmonic chip demonstrates a limit-of-detection of less than 1 ng/mL that is clinically relevant for both stroke biomarkers.

Enhanced Colorimetric Signal for Accurate Signal Detection in Paper-Based Biosensors.

Abstract: Paper-based colorimetric biosensors combine the use of paper with colorimetric signal detection. However, they usually demonstrate lower sensitivities because a signal amplification procedure has not been used. Stopping the reaction of colorimetric signal generation is often used in lab-based assays in order to amplify and stabilize the colorimetric signal for detection. In this study, the generation of a stopped colorimetric signal was examined for accurate and enhanced signal detection in paper-based biosensors. The colorimetric reaction in biosensors is usually based on the interaction between the enzyme horseradish peroxidase (HRP) and a selected chromogenic substrate. The two most commonly used HRP substrates, 3, 3', 5, 5'-tetramethylbenzidine (TMB) and 2'-azinobis (3-ethylbenzothiazoline-6-sulfonic-acid) (ABTS), were compared in terms of their ability to generate a stopped colorimetric signal on membrane. The stopped colorimetric signal was visible for TMB but not for ABTS. Moreover, the generation of stopped colorimetric signal was dependent on the presence of polyvinylidene-difluoride (PVDF) membrane as the separation layer. With PVDF the colorimetric signal (color intensity) was higher (TMB: 126 ± 6 and ABTS: 121 ± 9) in comparison to without PVDF (TMB: 110 ± 2 and ABTS: 102 ± 4). The TMB stopped colorimetric signal demonstrated a more stable signal detection with lower standard deviation values. To conclude, a stopped colorimetric signal can be generated in paper-based biosensors for enhanced and accurate signal detection.

Point-of-Care Surface Plasmon Resonance Biosensor for Stroke Biomarkers NT-proBNP and S100ß Using a Functionalized Gold Chip with Specific Antibody.

Surface-plasmon-resonance (SPR) is a quantum-electromagnetic phenomenon arising from the interaction of light with free electrons at a metal-dielectric interface. At a specific angle/wavelength of light, the photon's energy is transferred to excite the oscillation of the free electrons on the surface. A change in the refractive-index (RI) may occur, which is influenced by the analyte concentration in the medium in close contact with the metal surface. SPR has been widely used for the detection of gaseous, liquid, or solid samples. In this study, a functionalized specific SPR chip was designed and used in a novel point-of-care SPR module (PhotonicSys SPR H5) for the detection of the stroke biomarkers NT-proBNP and S100ß. These biomarkers have proven to be good for stroke diagnosis, with sensitivity and specificity of >85%. Specific detection was done by binding a biomolecular-recognizing antibody onto the Au SPR-chip. Detection was tested in water and plasma samples. NT-proBNP and S100ß were detected in a range of concentrations for stroke, from 0.1 ng/mL to 10 ng/mL. The RI of the blank plasma samples was 1.362412, and the lowest concentration tested for both biomarkers showed a prominent shift in the RI signal (0.25 ng/mL NT-proBNP (1.364215) and S100ß (1.364024)). The sensor demonstrated a clinically relevant limit-of-detection of less than ng/mL.

Dissolvable Polyvinyl-Alcohol Film, a Time-Barrier to Modulate Sample Flow in a 3D-Printed Holder for Capillary Flow Paper Diagnostics.

Integrating a dissolvable membrane into a sensor allows the control of sample flow, location and duration in critical areas. These time-barrier films stop the flow of samples until the membrane has dissolved, thus, for example, allowing increased exposure time between immunoreagents for the formation of greater numbers of immuno-complexes, ensuring higher sensitivity, reactivity, and helping to reduce false-positive signals. In this study, dissolvable polyvinyl alcohol (PVA) films are used in a 3D-printed sensor holder, which enables film integration without the use of glue. PVA is a synthetic hydrophilic linear polymer, its solubility is dependent on its molecular weight and degree of hydrolysis. Three types of PVAs films were tested herein: (1) PVA 1-Mw: 30⁻70 K, 87⁻90% hydrolyzed; (2) PVA 2-Mw: 31⁻50 K, 98⁻99% hydrolyzed and (3) PVA 3-Mw: 89⁻98 K, >99% hydrolyzed. The films were exposed to water in (1) the novel 3D-printed holder and (2) directly immersed into a water droplet. After comparing the time taken to dissolve PVA 1⁻3 films, PVA 1 films of 5⁻20% (w/v) are found to be most suitable as time barrier films, due to their optimal dissolution times and physical properties for integration into the customized 3D-printed holder.

Measuring Artificial Sweeteners Toxicity Using a Bioluminescent Bacterial Panel.

Artificial sweeteners have become increasingly controversial due to their questionable influence on consumers' health. They are introduced in most foods and many consume this added ingredient without their knowledge. Currently, there is still no consensus regarding the health consequences of artificial sweeteners intake as they have not been fully investigated. Consumption of artificial sweeteners has been linked with adverse effects such as cancer, weight gain, metabolic disorders, type-2 diabetes and alteration of gut microbiota activity. Moreover, artificial sweeteners have been identified as emerging environmental pollutants, and can be found in receiving waters, i.e., surface waters, groundwater aquifers and drinking waters. In this study, the relative toxicity of six FDA-approved artificial sweeteners (aspartame, sucralose, saccharine, neotame, advantame and acesulfame potassium-k (ace-k)) and that of ten sport supplements containing these artificial sweeteners, were tested using genetically modified bioluminescent bacteria from E. coli. The bioluminescent bacteria, which luminesce when they detect toxicants, act as a sensing model representative of the complex microbial system. Both induced luminescent signals and bacterial growth were measured. Toxic effects were found when the bacteria were exposed to certain concentrations of the artificial sweeteners. In the bioluminescence activity assay, two toxicity response patterns were observed, namely, the induction and inhibition of the bioluminescent signal. An inhibition response pattern may be observed in the response of sucralose in all the tested strains: TV1061 (MLIC = 1 mg/mL), DPD2544 (MLIC = 50 mg/mL) and DPD2794 (MLIC = 100 mg/mL). It is also observed in neotame in the DPD2544 (MLIC = 2 mg/mL) strain. On the other hand, the induction response pattern may be observed in its response in saccharin in TV1061 (MLIndC = 5 mg/mL) and DPD2794 (MLIndC = 5 mg/mL) strains, aspartame in DPD2794 (MLIndC = 4 mg/mL) strain, and ace-k in DPD2794 (MLIndC = 10 mg/mL) strain. The results of this study may help in understanding the relative toxicity of artificial sweeteners on E. coli, a sensing model representative of the gut bacteria. Furthermore, the tested bioluminescent bacterial panel can potentially be used for detecting artificial sweeteners in the environment, using a specific mode-of-action pattern.