Search Results (7)

Limulus amoebocyte lysate (LAL) assays have emerged as among the most effective approaches for detecting endotoxins and fungi in vitro since they were first tested 50 years ago. Although detailed protocols are publicly available, conventional LAL collection methods (3% sodium chloride) waste as much as 80% of the total LAL during blood accumulation, confirming the incompatibility of these methods with the lasting survival of the American horseshoe crab. For this reason, new implementations of blood collection–suspension buffer combinations are critical. Here, we evaluated the ability of different blood collection solutions to inhibit exocytosis and subsequently treated the cells with CaCl₂ to stimulate exocytosis and improve the yield of LAL. Two test methods, chromogenic and turbidimetric tests for LAL activity, were evaluated. Crabs were bled during the bleeding season. The crab blood samples were collected with the following blood collection solutions: citric acid buffer, malic acid buffer, PBS buffer, and PBS–caffeine buffer. The cell pellets were washed with 3% NaCl and subsequently resuspended in LRW or CaCl₂ to facilitate degranulation. Both the chromogenic test and the turbidimetric assay were used to evaluate the LAL enzyme activity. Citric acid buffer, malic acid buffer, PBS buffer, and PBS–caffeine buffer blocked exocytosis, resulting in the high yields of LAL. There was no observable effect on the activity output of crab size via a chromogenic test with PBS–caffeine buffer during the bleeding season. This protocol substantially benefited prior processes, as the PBS–caffeine collection mixture decreased amoebocyte aggregation/clot formation during processing. Furthermore, we evaluated the specific biochemical parameters of PBS–caffeine-derived LAL. We developed an accessible, promising phosphate–caffeine-based blood collection buffer that prevents amoebocyte degranulation during blood collection, maximizing the LAL yield. Moreover, our analysis revealed that phosphate–caffeine-derived LAL is uniquely adaptable to compatibility with chromogenic and turbidimetric assay techniques. By employing this method for LAL blood extraction, our same-cost approach fostered significantly greater LAL yields, simultaneously ensuring a healthy limulus polyphemus population. Full article

This study aimed to test the effectiveness of Raman spectroscopy in the characterization of the degrees of physiological stress and virulence in clinical swab samples collected from patients affected by oral candidiasis. Raman experiments were conducted on a series of eight isolates, both in an as-collected state and after biofilm purification followed by 3 days of culture. The outputs were matched to optical microscopy observations and the results of conventional chromogenic medium assays. A statistically significant series of ten Raman spectra were collected for each clinical sample, and their averages were examined and interpreted as multiomic snapshots for albicans and non-albicans species. Spectroscopic analyses based on selected Raman parameters previously developed for standard Candida samples revealed an extreme structural complexity for all of the clinical samples, which arose from the concurrent presence of a variety of biofilms and commensal bacteria in the samples, as well as a number of other biochemical circumstances affecting the cells in their physiological stress state. However, three Raman algorithms survived such complexity, which enabled insightful classifications of Candida cells from clinical samples, in terms of their physiological stress and morphogenic state, membrane permeability, and virulence. These three characteristics, in turn, converged into a seemingly “fight or flight” response of the Candida cells. Although yet preliminary, the present study points out criticalities and proposes solutions regarding the potential utility of Raman spectroscopy in fast bedside analyses of surveillance samples. Full article

Antibiotic-resistant bacteria (ARB) adhesion onto plastic substrates is a potential threat to environmental and human health. This current research investigates the prevalence of two relevant human pathogens, Staphylococcus spp. and Klebsiella spp., and their sophisticated equipment of antibiotic-resistant genes (ARGs), retrieved from plastic substrates submerged into an inland water body. The results of microbiological analysis on selective and chromogenic media revealed the presence of colonies with distinctive phenotypes, which were identified using biochemical and molecular methods. 16S rDNA sequencing and BLAST analysis confirmed the presence of Klebsiella spp., while in the case of Staphylococcus spp., 63.6% of strains were found to be members of Lysinibacillus spp., and the remaining 36.3% were identified as Exiguobacterium acetylicum. The Kirby–Bauer disc diffusion assay was performed to test the susceptibility of the isolates to nine commercially available antibiotics, while the genotypic resistant profile was determined for two genes of class 1 integrons and eighteen ARGs belonging to different classes of antibiotics. All isolated bacteria displayed a high prevalence of resistance against all tested antibiotics. These findings provide insights into the emerging risks linked to colonization by potential human opportunistic pathogens on plastic waste commonly found in aquatic ecosystems. Full article

Factor VIII (FVIII) is an important component of blood coagulation as its congenital deficiency results in life-threatening bleeding. Current prophylactic therapy of the disease (hemophilia A) is based on 3–4 intravenous infusions of therapeutic FVIII per week. This poses a burden on patients, demanding reduction of infusion frequency by using FVIII with extended plasma half-life (EHL). Development of these products requires understanding FVIII plasma clearance mechanisms. This paper overviews (i) an up-to-date state of the research in this field and (ii) current EHL FVIII products, including recently approved efanesoctocog alfa, for which the plasma half-life exceeds a biochemical barrier posed by von Willebrand factor, complexed with FVIII in plasma, which results in ~1 per week infusion frequency. We focus on the EHL FVIII products’ structure and function, in particular related to the known discrepancy in results of one-stage clotting (OC) and chromogenic substrate (CS) assays used to assign the products’ potency, dosing, and for clinical monitoring in plasma. We suggest a possible root cause of these assays’ discrepancy that is also pertinent to EHL factor IX variants used to treat hemophilia B. Finally, we discuss approaches in designing future EHL FVIII variants, including those to be used for hemophilia A gene therapy. Full article

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can serve as biochemical markers of various pathologies like liver disfunction and poisonings by nerve agents. Ellman’s assay is the standard spectrophotometric method to measure cholinesterase activity in clinical laboratories. The authors present a new colorimetric test to assess AChE and BChE activity in biological samples using chromogenic reagents, treated 3D-printed measuring pads and a smartphone camera as a signal detector. Multiwell pads treated with reagent substrates 2,6-dichlorophenolindophenyl acetate, indoxylacetate, ethoxyresorufin and methoxyresorufin were prepared and tested for AChE and BChE. In the experiments, 3D-printed pads containing indoxylacetate as a chromogenic substrate were optimal for analytical purposes. The best results were achieved using the red (R) channel, where the limit of detection was 4.05 µkat/mL for BChE and 4.38 µkat/mL for AChE using a 40 µL sample and a 60 min assay. The major advantage of this method is its overall simplicity, as samples are applied directly without any specific treatment or added reagents. The assay was also validated to the standard Ellman’s assay using human plasma samples. In conclusion, this smartphone camera-based colorimetric assay appears to have practical applicability and to be a suitable method for point-of-care testing because it does not require specific manipulation, additional education of staff or use of sophisticated analytical instruments. Full article

Recently, smartphone-based chromogenic sensing with paper-based microfluidic technology has played an increasingly important role in biochemical assays. However, generally there were three defects: (i) the paper-based chips still required complicated fabrication, and the hydrophobic boundaries on the chips were not clear enough; (ii) the chromogenic signals could not be steadily captured; (iii) the smartphone apps were restricted to the detection of specific target analytes and could not be extended for different assays unless reprogrammed. To solve these problems, in this study, a portable smartphone-based sensing system with a 3D-printed chip was developed. A 3D-printed imaging platform was designed to significantly reduce sensing errors generated during signal capture, and a brand-new strategy for signal processing in downloadable apps was established. As a proof-of-concept, the system was applied for detection of organophosphorus pesticides and multi-assay of fruit juice, showing excellent sensing performance. For different target analytes, the most efficient color channel could be selected for signal analysis, and the calibration equation could be directly set in user interface rather than programming environment, thus the developed system could be flexibly extended for other biochemical assays. Consequently, this study provides a novel methodology for smartphone-based biochemical sensing. Full article

To overcome the time and space constraints in disease diagnosis via the biosensing approach, we developed a new signal-transducing strategy that can be applied to colorimetric optical biosensors. Our study is focused on implementation of a signal transduction technology that can directly translate the color intensity signals—that require complicated optical equipment for the analysis—into signals that can be easily counted with the naked eye. Based on the selective light absorption and wavelength-filtering principles, our new optical signaling transducer was built from a common computer monitor and a smartphone. In this signal transducer, the liquid crystal display (LCD) panel of the computer monitor served as a light source and a signal guide generator. In addition, the smartphone was used as an optical receiver and signal display. As a biorecognition layer, a transparent and soft material-based biosensing channel was employed generating blue output via a target-specific bienzymatic chromogenic reaction. Using graphics editor software, we displayed the optical signal guide patterns containing multiple polygons (a triangle, circle, pentagon, heptagon, and 3/4 circle, each associated with a specified color ratio) on the LCD monitor panel. During observation of signal guide patterns displayed on the LCD monitor panel using a smartphone camera via the target analyte-loaded biosensing channel as a color-filtering layer, the number of observed polygons changed according to the concentration of the target analyte via the spectral correlation between absorbance changes in a solution of the biosensing channel and color emission properties of each type of polygon. By simple counting of the changes in the number of polygons registered by the smartphone camera, we could efficiently measure the concentration of a target analyte in a sample without complicated and expensive optical instruments. In a demonstration test on glucose as a model analyte, we could easily measure the concentration of glucose in the range from 0 to 10 mM. Full article