Microtiter Plate Reader Research Articles

PhagoScreener: A novel phagogram platform based on a capillary-wave microbioreactor

Due to the overuse of antibiotics, the number of multidrug-resistant pathogen bacteria is rising in recent years posing a serious threat to human health. One promising alternative for treatment is the application of phage therapy using highly selective bacteriophages. Because of their selectivity, individual screens called phagograms for each patient are required to select phages from a phage library. Phagograms are mostly performed via bacterial cultivation on double layer agar plates and phage addition causing bacterial lysis. However, these assays are work-intensive and have a low ability for parallelization and automation. Hence, highly parallelizable and automatable microbioreactors in the lowest microliter scale could offer an economic solution increasing the throughput of phagograms. This paper demonstrates the applicability of a novel capillary-wave microbioreactor (cwMBR) to perform phagograms. Due to its small volume of only 7 µL and the open-droplet design, it can be easily automated and parallelized in future. Furthermore, the ability of online biomass measurement makes the cwMBR a perfect phagogram platform in the future. Herein, phagograms with E. coli and different concentrations of the phages MM02 and EASG3 were performed as proof of concept for phagograms in the cwMBR. Thereby, the cwMBR was able to measure differences in lysis kinetics of different phages. Furthermore, the phagograms were compared to those in conventional microtiter plate readers revealing the cwMBR as ideal alternative for phagograms as it combines favorable mixing conditions and a phage repellent hydrophilic glass surface with online biomass measurement in an open-droplet design for future parallelization and automation.

Unraveling the influence of CRISPR/Cas13a reaction components on enhancing trans-cleavage activity for ultrasensitive on-chip RNA detection.

The CRISPR/Cas13 nucleases have been widely documented for nucleic acid detection. Understanding the intricacies of CRISPR/Cas13's reaction components is pivotal for harnessing its full potential for biosensing applications. Herein, we report onthe influence of CRISPR/Cas13a reaction components on its trans-cleavage activity and the development of an on-chip total internal reflection fluorescence microscopy (TIRFM)-powered RNA sensing system. We used SARS-CoV-2 synthetic RNA and pseudovirus as a model system. Our results show that optimizing Mg2+ concentration, reporter length, and crRNA combination significantly improves the detection sensitivity. Under optimized conditions, we detected 100 fM unamplified SARS-CoV-2 synthetic RNA using a microtiter plate reader. To furtherimprove sensitivity and provide a new amplification-free RNA sensing toolbox, we developed a TIRFM-based amplification-free RNA sensing system. We were able to detect RNA down to 100 aM. Furthermore, the TIRM-based detection system developed in this study is 1000-fold more sensitive than the off-coverslip assay. The possible clinical applicability of the system was demonstrated by detecting SARS-CoV-2 pseudovirus RNA. Our proposed sensing system has the potential to detect any target RNA with slight modifications to the existing setup, providing a universal RNA detection platform.

Analysis of long-lasting tumor-specific antibodies from murine tumors treated with Aliya pulsed electric fields.

e14543 Background: Pulsed electric field (PEF) tissue destruction has demonstrated meaningful systemic immune responses in mice that are superior to thermal ablation, and synergize effectively with systemic chemotherapy and checkpoint blockade (CPB) immunotherapy. Immune cell populations are increased by PEF, including tumor specific activated CD8 T-cells and plasma B-cells. Antitumor immunity can include the presence of circulating anti-cancer antibodies. However, the presence and role of circulating tumor specific antibodies induced by PEF have not been explored. Serological antibodies are often detected with the ELISA assay, where tumor-associated antigens are coated to a microtiter plate to attract the corresponding serum antibodies. Here, we quantify IgG tumor specific antibodies after PEF using an in-house ELISA. Methods: Mice bearing immune-warm (EMT6) and immune-cold (4T1) orthotopic murine breast tumors were treated with a biphasic PEF dose comparable to Aliya titrated for mouse tumors. To detect tumor specific circulating IgG1 immunoglobulin in serum, an ELISA plate was coated with EMT6 and 4T1 protein lysate, as well as with a known 4T1-expressed tumor-specific antigen (gp70). Serum draws from the treated, untreated, and naïve mice were exposed to the coated wells for 1 hour. Bound IgG were detected by anti-mouse IgG (HRP tagged) antibody added to the wells and incubated 1 hour. A TMB substrate was added and color development was allowed for 15 minutes. The optical density was quantified using a microtiter plate reader. Results: ELISA quantification of serum IgG at multiple timepoints show that 4T1 tumor-specific IgG antibodies were increased 14.3- and 2.6-fold by day 20 in the PEF-treated and untreated group survivors relative to Naïve mice, respectively, (OD=0.86 and 0.15 v. 0.06). Furthermore, IgG antibodies specific to the gp70 antigen of the 4T1 cell line were increased by 44.9- and 2.7-fold in the PEF-treated and untreated groups relative to naïve mice (OD=3.14 and 0.19 v. 0.07). In the EMT6 model, at 3 months post-treatment, only the PEF-ablated group mice survived, and showed 15.5-fold higher antibodies against EMT6 tumor antigens compared to naive mice (OD=0.62 v. 0.04). Conclusions: The specific form of PEF in this study generated tumor-specific IgG long-lived antibodies in mice for immune-warm and immune-cold tumor models, corroborating previous findings of increased murine B-cell and plasma cell populations. This effect has not been well-reported for other ablation technologies. Increases were evident by Day 20 post-PEF and persisted at least 3 months. These murine data suggest the PEF used may invoke potent, long-lived immune responses and anti-tumor immune surveillance, offering a potential approach for in situ tumor vaccination to improve metastatic disease outcomes. Future studies should determine response durability and its role in attaining distant tumor clearance.

Enzyme-free and label-free detection of HER2 in human serum based on Ag+-released hybridization chain reaction signal amplification

The overexpression of human epidermal growth factor receptor 2 (HER2) has been linked to aggressive breast cancer and worse clinical outcomes. However, accurately measuring HER2 remains challenging, due to its low serum content. Herein, we developed a novel method for HER2 detection in serum by the combination of aptamer with C-Ag+-C structure, Ag+-released hybridization chain reaction (Ag+-HCR) and coprecipitation of Ag+ and gold nanoparticles (AuNPs) in the solution including Eosin Y (EY) and AgI colloids. Following the aptamer bound to HER2, signal amplification occurred through the release of numerous Ag+ by Ag+-HCR. This process subsequently induced AuNPs aggregation on the surface of AgI colloids, resulting in reduced fluorescence intensity of EY in the supernatant. The reduced value of EY fluorescence intensity in the supernatant was proportional to the level of HER2. Under optimized reaction conditions, a wide detection range was obtained between 0.09 and 9000 pg/mL with a limit of detection of 11.7 fg/mL in diluted human serum, and 96 serum samples in a 96-well plate could be measured simultaneously by a microtiter plate reader within 2 h. Moreover, the quantitative results of HER2 were consistent with those obtained using the enzyme-linked immunosorbent assay kit (r = 0.9995). Therefore, leveraging the delicate design of Ag+-HCR signal amplification, this enzyme-free, label-free, and effective method has demonstrated to be capable of detecting HER2 in serum due to its high sensitivity. Remarkably, this method may be extended to detect other cancer-related proteins and genes by replacing the corresponding target sequences.

Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron.

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target for anti-cancer approaches. The development of novel compounds and the identification of leads for further modification requires that proof of mechanism assays be carried out. There are many assays to evaluate the impact on proliferation; however, the ability to chelate iron is an important and sometimes overlooked end-point measure due to the high costs of equipment and the challenge to quickly and reproducibly quantify the strength of chelation. Here, we describe a quantifiable and inexpensive cell-free fluorescent method to confirm the ability of novel compounds to chelate iron. Our assay relies on the commercially available inexpensive fluorescent dye Calcein, whose fluorescence can be quantified on most fluorescent microtiter plate readers. Calcein is a weak iron chelator, and its fluorescence is quenched when it binds Fe2+/3+; fluorescence is restored when a novel chelator outcompetes Calcein for bound Fe2+/3+. The removal of fluorescent quenching and the resulting increase in fluorescence allows the chelation ability of a novel putative chelator to be determined. Therefore, we offer an inexpensive, high-throughput assay that allows the rapid screening of novel candidate chelator compounds.

Evaluation of in vitro antimalarial activity of <i>Citrus aurantifolia</i> (CHRISTM.) and <i>Ziziphus mauritiana</i> (LAM) using heme polymerization inhibition assay

Citrus aurantifolia and Ziziphus mauritiana (Lam) are plants that have numerous medicinal properties and are used extensively in traditional medicine for the treatment of several diseases. Malaria parasites metabolize haemoglobin and detoxify the resulting haem by polymerizing it to form haemozoin (malaria pigment). This study aimed at evaluating the antiplasmodial activity through in vitro cell-free heme polymerization inhibition assay (HPIA) of C. aurantifolia and Z. mauritiana. The analysis was conducted using a 96-well microtiter plate reader at an absorption of 400 nm, at different concentrations (0-2 mg/ml). The IC50 was determined by non-linear regression analysis using GraphPad Prism. The result indicated that both the root extracts of C. aurantifolia and Z. mauritiana showed significant difference (p˂0.0001) with IC50 of 0.81 mg/ml and 0.77 mg/ml respectively compared to positive control. Phytochemical screening of the ethanol root extracts showed the presence of saponins, alkaloids, terpenoids, glycosides, steroids, and anthraquinone, while tannins and reducing sugars were not detected in both samples used, flavonoid was detected in C. aurantifolia. Both the extracts exhibited potential for inhibiting heme polymerization in vitro.

CRISPR-Cas12-based field-deployable system for rapid detection of synthetic DNA sequence of the monkeypox virus genome.

The global outbreak of the monkeypox virus (MPXV) highlights the need for rapid and cost-effective MPXV detection tools to effectively monitor and control the monkeypox disease. Herein, we demonstrated a portable CRISPR-Cas-based system for naked-eye detection of MPXV. The system harnesses the high selectivity of CRISPR-Cas12 and the isothermal nucleic acid amplification potential of recombinase polymerase amplification. It can detect both the current circulating MPXV clade and the original clades. We reached alimit of detection (LoD) of 22.4 aM (13.5 copies/µl) using a microtiter plate reader, while the visual LoD of the system is 75 aM (45 copies/µl) in a two-step assay, which is further reduced to 25 aM (15 copies/µl) in a one-pot system. We compared our results with quantitative polymerase chain reaction andobtained satisfactory consistency. For clinical application, we demonstrated a sensitive and precise visual detection method with attomolar sensitivity and a sample-to-answer time of 35 min.

BRET measurement on CCD camera-based microtiter plate readers.

NanoBRET assays, which utilize bioluminescence energy resonance transfer (BRET), have been widely adopted in drug discovery for measuring both protein-protein interactions and drug target engagement. While the EnVision and other traditional well-scanning plate readers that measure a single well at a time are satisfactory for signal detection for smaller experiments, it becomes challenging to scale these assays to applications that require higher throughput. To address this, we explored the adaptation of the ViewLux and FLIPR plate readers for measuring NanoBRET signal. These plate readers utilize charge-coupled device (CCD) cameras for detection, which enable imaging of the entire assay plate simultaneously. We used tool compounds to generate data from each plate reader and found that the image-based plate readers can be used to measure NanoBRET signals with high S/B and Z´, resulting in comparable IC50 values to those obtained from the EnVision, while requiring less time to complete reads. Consequently, utilization of image-based plate readers for NanoBRET measurement may enable applications that require faster reads, such as for high-throughput screening or kinetics studies.

Applicability and limitations of high-throughput algal growth rate measurements using invivo fluorescence in microtiter plates

Micro-algae play important roles in primary production, nutrient cycling, and applied biotechnology. Mico-algae are phylogenetically, morphologically and physiologically diverse and often easy to grow in cultures. Growth is the key parameter of viability and crucial for survival insitu and efficiency invitro. Therefore, growth is often used in eco-physiological experiments to describe the fundamental niche and optimum conditions. Invivo chlorophyll-a fluorescence (IVCF) is a well-established proxy for the growth of micro-algae and is based on constant cell properties during exponential growth. High-throughput IVCF measurements have been applied for about 20 years for few, mostly green algal strains. Here, we tested the IVCF using microtiter plates on strains of four different algal phyla (Chlorophyceae, Cyanobacteria, Bacillariophyceae, Rhodophyceae) and compared the data with cell counts and chlorophyll contents. The microtiter plate reader based IVCF (MPR-IVCF) approach worked best for the diatom Cylindrothecaclosterium and the cyanobacterium Synechococcuselongatus. Differences between IVCF and cell count-based growth rates were found for Chlorellavulgaris and Rhodella sp. The IVCF growth rates were lower than the growth rates based on cell counts in both cases, thus underestimating absolute growth rates. The MPR-IVCF method is robust in terms of light scattering and non-photosynthetic contamination but prone to changes in light regimes and also to aggregating algal cells. Thus, we recommend using the MPR-IVCF approach (1) for diatoms and cyanobacteria as an absolute growth rate measure, (2) for relative measures of growth rates, intra and interspecific during the same experiment, also for all algal phyla except Rhodophyta.

Multifunctional 3D-printed platform integrated with a smartphone ambient light sensor for halocarbon contaminants monitoring

3D-printed platforms integrated with smartphone light sensors have become a powerful tool for the development of frugal and facile biosensing systems. Herein, we report a 3D-printed platform for multimodal analysis (3D-PMA) based on a smartphone ambient light sensor (ALS). The proposed approach was first validated using a microtiter plate reader, and then its application potential was demonstrated by measuring halohydrin dehalogenase activities employing three different sample modes, viz . using enzyme-linked immunosorbent assay (ELISA) plate strips, cuvettes, and the glass fiber membrane as sample containers. For real-sample analysis, 1,3-dichloro-2-propanol (1,3-DCP), a toxic halogenated compound, was successfully detected in spiked river water samples with recoveries in the range of 101.95–109.70%. After demonstrating the suitability for liquid-phase assays, the 3D-PMA was also optimized for 1,3-DCP detection using a glass fiber membrane. The detection could be done using a 10 μ L reaction system with an assay time of 2 min. The assay showed two linear ranges. The calibration equation of Y = 81.687x + 140.060 was obtained from the lower part of the calibration curve and was used to calculate the detection limit of the system. Under optimized conditions, a detection limit of 80 μ M was achieved for 1,3-DCP. The small size of the device endows it with great promise for use as a benchtop and an on-demand detection system. The proposed low-cost and portable system has great potential for the (bio) sensing of different biological and environmental samples where biocomponent-analyte interaction leads to a color change of the reaction system, indicating its applicability in resource-constrained settings. • A smartphone assisted portable system was developed for environmental contaminants monitoring. • 3D-printed platform demonstrated potential utility for multimodal analysis. • Smartphone Ambient Light Sensor as a potential transducer for halocarbon detection.

Development of a novel high throughput brain-on-chip with 3D structure and its application in evaluation of pesticide-induced-neurotoxicity

We designed and fabricated a novel high throughput brain-on-chip with three dimensional structure with the aim to simulate the in vivo three-dimensional growth environment for brain tissues. The chip consists of a porous filter and 3D brain cell particles, and is loaded into a conventional 96-well plate for use. The filter and the particle molds were fabricated by using computer modeling, 3D printing of positive mold and agarose-PDMS double reversal mold. The 3D cell particles were made by pouring and solidifying a suspension of mouse embryonic brain cells with sodium alginate into a cell particle mold, and then cutting the resulting hydrogel into pieces. The loaded brain-on-chip was used to determine the neurotoxicity of pesticides. The cell particles were exposed to 0, 10, 30, 50, 100 and 200 µmol/L of chlorpyrifos or imidacloprid, separated conveniently from the medium by removing the porous filter after cultivation. Subsequently, cell proliferation, acetylcholinesterase activity and lactate dehydrogenase release were determined for toxicity evaluation. The embryonic brain cells were able to grow and proliferate normally in the hydrogel particles loaded into the filter in a 96-well plate. Pesticide neurotoxicity test showed that both chlorpyrifos and imidacloprid presented dose-dependent inhibition on cell growth and proliferation. Moreover, the pesticides showed inhibition on acetylcholinesterase activity and increase release of lactate dehydrogenase. However, the effect of imidacloprid was significantly weaker than that of chlorpyrifos. In conclusion, a novel brain-on-chip was developed in this study, which can be used to efficiently assess the drug neurotoxicity, pharmacodynamics, and disease mechanism by combining with a microtiterplate reader.

Platelet-Based Factor VIII Activity Is Inhibited By Anti-C2 Domain Antibodies That Don’t Significantly Inhibit Factor VIII Activity in a Standard Assay

The major complication of hemophilia A treatment is the development of inhibitory antibodies against factor VIII (fVIII), causing severe bleeding in spite of infused fVIII. Most antibodies are targeted to the A2 or the C2 domain of fVIII and the degree of inhibition by these antibodies measured in clinical fVIII assays fails to predict the severity of bleeding risk. We investigated whether the degree of inhibition of platelet dependent fVIII activity by a panel of monoclonal anti-C2 domain mAbs deviated from the degree of inhibition in a commercial one-stage assay.Methods: Platelet dependent fVIII activity was measured in a defined activated platelet time (APT) assay containing fresh, density gradient-purified human platelets and fVIII deficient plasma supplemented with fVIII. Coagulation was triggered by simultaneous addition of calcium, thrombin receptor activation peptides PAR-1 and PAR-4, and factor XIa without other exogenous activators or vesicles. Results were compared to a commercial aPTT assay (Helena Laboratories, TX) and to results of a novel factor Xase assay developed to recapitulate the conditions in an APT. In the novel assay fresh, density-gradient purified platelets were activated by thrombin followed by inactivation with hirudin and incubated with fVIII and VWF, fIXa, and fX. In a second step, phospholipid vesicles, fVa and prothrombin were added to produce thrombin in proportion to the fXa generated in the first step. For comparison to the aPTT this assay was also done using purified sonicated phospholipid vesicles instead of activated platelets in the first step. The reaction was quenched with EDTA and thrombin generation was determined immediately in a thermosttated kinetics microtiter plate reader (Molecular Devices) at 250C using the chromogenic substrate S-2238 (Diapharma). A panel of 10 defined, epitope mapped mAbs against the fVIIIC2 domain was chosen in order to survey the relationship between inhibition and binding of anti-C2 antibodies.Results: The APT exhibited a log-linear relationship between fVIII and time to fibrin strand formation over a 4-log range, encompassing 0.01% - 100% plasma fVIII. Nine of 10 mAbs inhibited fVIII activity 89-96% while mAb F85 did not inhibit. There was excellent correlation for the degree of inhibition in the platelet-based factor Xase assay for 8 of 10 antibodies with F85 inhibiting the platelet Xase but not the APT and I109 inhibiting the platelet Xase assay less than the APT. In contrast, there was no correlation between inhibition of the aPTT-based assay and the platelet assay. Four mAbs did not inhibit the aPTT assay yet inhibited approximately 90% of platelet-based activity. The novel factor Xase assay, using phospholipid vesicles instead of activated platelets, showed inhibitory results that correlated to the degree of inhibition in the aPTT assay (which also utilizes purified phospholipids). Thus, the novel assay indicates that the degree of inhibition in both platelet-based and vesicle-based clotting assays is a consequence of the membrane type rather than unidentified clotting modifiers in plasma.Conclusions: We have developed a novel factor Xase assay to measure the low levels of residual fVIII activity, at physiologic concentrations, in the presence of inhibitory antibodies. Antibody inhibition of fVIII activity correlated with inhibition measured in the aPTT and APT clotting assays,. Our results confirm that the anti-C2 inhibitory antibodies have substantially different inhibition on a platelet membrane than in an aPTT-based assay and that the differing degree of inhibition is primarily due to fVIII susceptibility to inhibition when functioning on platelet membranes vs. phospholipid vesicles. These results suggest that current inhibitor assays might underestimate the frequency of significant inhibitory antibodies. DisclosuresNo relevant conflicts of interest to declare.

Relationship of Salivary Cortisol Level With Severe Depression and Family History.

Salivary cortisol level is considered a prominent depression biomarker, as saliva induces less stress as compared to blood. The study was performed in the physiology department, Sheikh Zayed Medical Institute Lahore from April 2015 to December 2015. Sixty participants, including 14 (46.67%) males and 16 (53.33%) females, in each non-depressive and depressive group of over 17 years of age, were undertaken. The depression diagnostics were performed both outdoors and in clinics and confirmed with the standards of the Diagnostic and Statistical Manual of Mental Disorders and Beck’s Inventory. Saliva specimens were collected and processed for enzyme-linked immunoassay (ELISA), and absorbance was calculated on a microtiter plate reader. The statistics with the Statistical Package for the Social Packages (SPSS) 26.0 (IBM Corp., Armonk, NY) show that patients of the non-depressive category exhibited mean ages 35.73±6.89 years and 39.10±6.89 years in the depressive group (p-value: 0.178). The mean cortisol level was 1.46±0.91 ng/ml among non-depressive and 2.23±1.69 in depressive patients (p-value: 0.031). The mean depression score among non-depressive was 5.73±4.05 and 52.03±5.08 while there was no statistical difference in the mean height and weight of subjects in normal and depressive patients (p-value: 0.0001). Meanwhile, the mean cortisol level was 1.46±0.91 ng/ml among non-depressive patients, while it was 2.23±1.69 in the depressive group, with no statistical difference in mean ages (p-value: 0.031). These findings proved the cortisol level directly linked with severe depression and useful for depression diagnostics and management.

Optimization of the Biocatalysis for D-DIBOA Synthesis Using a Quick and Sensitive New Spectrophotometric Quantification Method.

D-DIBOA (4-hydroxy-(2H)-1,4-benzoxazin-3-(4H)-one) is an allelopathic-derived compound with interesting herbicidal, fungicidal, and insecticide properties whose production has been successfully achieved by biocatalysis using a genetically engineered Escherichia coli strain. However, improvement and scaling-up of this process are hampered by the current methodology for D-DIBOA quantification, which is based on high-performance liquid chromatographic (HPLC), a time-consuming technique that requires expensive equipment and the use of environmentally unsafe solvents. In this work, we established and validated a rapid, simple, and sensitive spectrophotometric method for the quantification of the D-DIBOA produced by whole-cell biocatalysis, with limits of detection and quantification of 0.0165 and 0.0501 µmol·mL−1 respectively. This analysis takes place in only a few seconds and can be carried out using 100 µL of the sample in a microtiter plate reader. We performed several whole-cell biocatalysis strategies to optimize the process by monitoring D-DIBOA production every hour to keep control of both precursor and D-DIBOA concentrations in the bioreactor. These experiments allowed increasing the D-DIBOA production from the previously reported 5.01 mM up to 7.17 mM (43% increase). This methodology will facilitate processes such as the optimization of the biocatalyst, the scaling up, and the downstream purification.

Background-free cell surface glycan analysis using persistent luminescence nanoparticle as an optical probe

In this work, we report a novel cell surface glycan analysis method based on persistent luminescence nanoparticle (PLNP) ZnGa2O4: Cr3+ (ZGC) as an optical probe. ZGC was first silanized by (3-Aminopropyl) triethoxysilane (APTES), followed by PEGylation with NHS-P EG-Biotin, which not only introduces biotin, but significantly improves the dispersibility and stability of the nanoparticles. Neutral-avidin was then coupled on ZGC surface through the specific biotin-avidin interaction, producing a ZGC-PEG-avidin nanoprobe. As for cell surface glycan detection, different surface glycans are recognized with their corresponding biotinylated lectins, which are then traced by ZGC-PEG-avidin. The persistent luminescence signal is recorded by a microtiter plate reader in time-resolved fluorescence mode. Glycans expression profiling on prostate cancer cell DU145 and normal prostate cell RWPE-1 was analyzed by the proposed detection platform. Similar results were observed from the conventional horseradish peroxidase (HRP)-catalyzed absorbent assay and confocal microscope-based fluorescence imaging, demonstrating the applicability of the proposed platform. The approach based on the long afterglow property of ZGC efficiently eliminates the background noise from cells and substrate, resulting in the best signal-to-noise ratio and high detection sensitivity.

Fluorescence calibration standards made from broadband emitters encapsulated in polymer beads for fluorescence microscopy and flow cytometry

We present here the design and characterization of a set of spectral calibration beads. These calibration beads are intended for the determination and regular control of the spectral characteristics of fluorescence microscopes and other fluorescence measuring devices for the readout of bead-based assays. This set consists of micrometer-sized polymer beads loaded with dyes from the liquid Calibration Kit Spectral Fluorescence Standards developed and certified by BAM for the wavelength-dependent determination of the spectral responsivity of fluorescence measuring devices like spectrofluorometers. To cover the wavelength region from 400 to 800 nm, two new near-infrared emissive dyes were included, which were spectroscopically characterized in solution and encapsulated in the beads. The resulting set of beads presents the first step towards a new platform of spectral calibration beads for the determination of the spectral characteristics of fluorescence instruments like fluorescence microscopes, FCM setups, and microtiter plate readers, thereby meeting the increasing demand for reliable and comparable fluorescence data especially in strongly regulated areas, e.g., medical diagnostics. This will eventually provide the basis for standardized calibration procedures for imaging systems as an alternative to microchannel slides containing dye solutions previously reported by us.Graphical abstract

Emerging Approaches for Fluorescence-Based Newborn Screening of Mucopolysaccharidoses.

Interest in newborn screening for mucopolysaccharidoses (MPS) is growing, due in part to ongoing efforts to develop new therapies for these disorders and new screening assays to identify increased risk for the individual MPSs on the basis of deficiency in the cognate enzyme. Existing tests for MPSs utilize either fluorescence or mass spectrometry detection methods to measure biomarkers of disease (e.g., enzyme function or glycosaminoglycans) using either urine or dried blood spot (DBS) samples. There are currently two approaches to fluorescence-based enzyme function assays from DBS: (1) manual reaction mixing, incubation, and termination followed by detection on a microtiter plate reader; and (2) miniaturized automation of these same assay steps using digital microfluidics technology. This article describes the origins of laboratory assays for enzyme activity measurement, the maturation and clinical application of fluorescent enzyme assays for MPS newborn screening, and considerations for future expansion of the technology.

Nonsteroidal anti-inflammatory drugs as potential ecto-nucleotide phosphodiesterase inhibitors

Phosphodiesterases (PDE) are group of enzymes which catalyze the hydrolysis of cAMP and cGMP. Since these cyclic phosphate moieties worked as intracellular second messengers in numerous physiological processes, their inhibition can affect normal physiology of living system. NSAIDs are among the frequently prescribed medications, because of their efficacy as analgesic, antipyretic and anti-inflammatory agents. They are known to block cyclooxygenase pathway. In limited data NSAIDs has been shown anti-tumor potential, and phosphodiesterase inhibition has assumed to be one of the mechanism. To date no further evaluation being done. Further, NSAIDs are classified as cyclooxygenase inhibitors and phosphodiesterase inhibition can imprint its side effects. This study first time investigates the effects of NSAIDs on phosphodiesterase 1 inhibition. The activity against snake venom phosphodiesterase 1 was assayed on a microtitre plate reader spectrophotometer. Selective COX-2 inhibitor, celecoxib, exhibited a potent PDE1 inhibitory activity, at therapeutic doses, with an IC50 value of 29.4 µM. The findings of our study are indicative of new pharmacological actions of cyclooxygenase inhibitors. This article presents the PDE inhibitory properties as a new effects of already existing drugs. These additional effects could be potentially helpful for researchers to assess other physiological and pathological states.

The effects of cell-free extracts derived from probiotic strains Bifidobacterium bifidum and Lactobacillus reuteri on the proliferation and biofilm formation by Lactobacillus reuteri in vitro

The aim of the research was to investigate the ability of cell-free extracts, containing derivatives of probiotic strains Bifidobacterium bifidum and Lactobacillus reuteri , to influence the proliferation and biofilm formation by Lactobacillus reuteri in vitro ; to evaluate the prospects for the creation of new metabiotics and means of increasing the overall productivity of probiotic cell biomass based on them. Materials and methods . Cell-free extracts were obtained from probiotic strains B. bifidum 1 and L. reuteri DSM 17938 by the authors’ method. Probiotic strain L. reuteri DSM 17938 used as a test culture. The investigation of the proliferation and biofilm formation by L. reuteri was carried out by spectrophotometric method using a microtiter-plate reader “Lisa Scan EM” (Erba Lachema s.r.o., Czech Republic). Results . It has been established that the cell-free extract obtained from L. reuteri culture grown in its own disintegrate, supplemented with glycerol and glucose, is the only one of all studied, which stimulates both proliferation and biofilm formation by L. reuteri . The cell-free extracts, obtained from L. reuteri and B. bifidum disintegrates and from L. reuteri culture, grown in its own disintegrate, stimulate proliferation of test-culture to varying degrees (depending on the type of extract and its content in the culture medium), but have a significant inhibitory effect on the biofilm formation by L. reuteri . The extract, obtained from B. bifidum culture, grown in its own disintegrate, does not have a significant effect on proliferation and greatly suppresses the biofilm formation by L. reuteri . Conclusions . The results of the study allow us to positively evaluate the prospects for the creation of new metabiotics based on probiotic derivatives. Cell-free extract, obtained from L. reuteri culture, grown in its own disintegrate supplemented with glycerol and glucose can contribute to the survival and facilitate inoculation of the introduced probiotic in the gastrointestinal tract when used together. Derivative-containing extracts with a pronounced growth-stimulating effect can be the basis for creating the means of increasing the overall productivity of the probiotic cell biomass. The use of such means will increase the economic efficiency of probiotic cultures production. Obtained data induce further study of the biochemical composition, elucidation of the mechanism of cell-free extracts action and confirmation of their efficacy in vivo .

A novel smartphone-based CD-spectrometer for high sensitive and cost-effective colorimetric detection of ascorbic acid

As a powerful tool for medical diagnosis and bioanalysis, conventional optical spectrometers are generally expensive, bulky and always require an accompanying data processing device. In this work, we developed a novel smartphone-based CD-spectrometer (SCDS) for high sensitive and ultra-portable colorimetric analysis, with the advantage of cost-effective and simplicity. The distance between the light source and slit, the structure of SCDS and the parameters of camera in the smartphone were all optimized to ensure the best analytical performance. Besides, the SCDS employed HSV color model and utilized the overall intensity calculated by summing V-value of adjacent position for the absorbance measurement. In this way the errors caused by the low resolution of CD-grating can effectively be eliminated to promote the sensitivity of the SCDS. The performance of the SCDS was first validated for colorimetric detection of BSA with a detection limit of 0.0073 mg/mL, which is superior compared to that of the microtiter plate reader (MTPR). Moreover, by combining with 3,3′,5,5′-tetramethylbenzidine-manganese dioxide (TMB-MnO2) nanosheets reaction, a high sensitive and specific system for ascorbic acid detection was established. The SCDS gives a detection range from 0.6250 μM to 40 μM with a detection limit of 0.4946 μM for AA detection. Compared to other studies, the SCDS features wide detection range and very low detection limit with low cost instrument. Therefore, the SCDS will be an ideal and promising colorimetric system for point-of-care (POC) application in food security, disease diagnosis and environmental monitoring.