Fluorescence Microplate Assay Research Articles

Fluorescent quantum dot-based nanotool for targeted identification and evaluation of the schistosomiasis circulating cathodic antigen in tissue samples

Schistosomiasis represents a serious public health problem, a disease for which the circulating cathodic antigen (CCA) is a relevant biomarker. Quantum dots (QDs) are advantageous fluorescent nanoparticles that can be used as specific nanoprobes. In this study, a nanotool based on QDs and anti-CCA antibodies was developed, which, in association with fluorescence microscopy, was applied to trace and evaluate the CCA profile in schistosomiasis-infected tissue samples. Kidney and liver tissues from mice at different disease phases were used as models. QDs and the conjugates were characterized by absorption and emission spectroscopies. Microscopy analyses were used to map and assess CCA accumulation in infected tissue slices in respect to non-infected control samples. The fluorescent microplate assay (FMA) and Zeta potential (ζ) analyses indicated an effective conjugation, which was corroborated by the absence of labeling in non-infected tissue slices (which lack CCA) after incubation with the nanoprobe. Infected liver and kidney tissues exhibited notable staining by the QDs-anti-CCA conjugate. The CCA accumulation increased as follows: 30 < 60 = 120 days post-infection, with 30, 60, and 120 days corresponding to the pre-patent, acute, and beginning of chronic disease phases, respectively. Therefore, this innovative approach, combining imaging acquisition with the sensitivity and specificity of the QDs-anti-CCA conjugate, demonstrated efficiency in locating and comparatively evaluating CCA deposition in biological samples, thereby opening new possibilities for schistosomiasis research.

Neonatal stress disrupts the glymphatic system development and increases the susceptibility to Parkinson's disease in later life.

Neonatal stress disrupts brain development and increases the risk of neurological disorders later in life. However, the impact of neonatal stress on the development of the glymphatic system and susceptibility to Parkinson's disease (PD) remains largely unknown. Neonatal maternal deprivation (NMD) was performed on mice for 14 consecutive days to model chronic neonatal stress. Adeno-associated virus expressing A53T-α-synuclein (α-syn) was injected into the substantia nigra to establish PD model mice. Glymphatic activity was determined using invivo magnetic resonance imaging, exvivo fluorescence imaging and microplate assay. The transcription and expression of aquaporin-4 (AQP4) and other molecules were evaluated by qPCR, western blotting, and immunofluorescence. Animal's responses to NMD and α-syn overexpression were observed using behavioral tests. Glymphatic activity was impaired in adult NMD mice. AQP4 polarization and platelet-derived growth factor B (PDGF-B) signaling were reduced in the frontal cortex and hippocampus of both young and adult NMD mice. Furthermore, exogenous α-syn accumulation was increased and PD-like symptoms were aggravated in adult NMD mice. The results demonstrated that NMD could disrupt the development of the glymphatic system through PDGF-B signaling and increase the risk of PD later in life, indicating that alleviating neonatal stress could be beneficial in protecting the glymphatic system and reducing susceptibility to neurodegeneration.

Antioxidative effects of molybdenum and its association with reduced prevalence of hyperuricemia in the adult population.

The relationship between molybdenum and kidney-related disease outcomes, including hyperuricemia, is not well investigated. This study aims to determine whether molybdenum and its antioxidative property are associated with systemic inflammation and kidney-related disease parameters including hyperuricemia. Urinary molybdenum's epidemiological relationship to hyperuricemia and kidney-disease related outcomes was evaluated in 15,370 adult participants in the National Health and Nutrition Examination Survey (NHANES) collected between 1999 and 2016. Individuals' urinary molybdenum levels were corrected to their urinary creatinine concentrations. The association between urinary molybdenum-to-creatinine ratio and kidney-disease related outcomes were assessed by multivariable linear and logistic regression analyses, adjusting for covariates including age, sex, ethnicity, diabetes mellitus, hypertension, body mass index, and estimated glomerular filtration rate. Antimony and tungsten were used as control trace metals. Experimentally, HK-2 cell was used to assess molybdenum's antioxidative properties. HK-2 cells were challenged with H2O2-induced oxidative stress. Oxidative stress was measured using a fluorescent microplate assay for reactive oxygen species (ROS) and antioxidation levels were assessed by measuring the expression of manganese superoxide dismutase. In the adult NHANES population, urinary molybdenum-to-creatinine ratio was significantly associated with decreased serum uric acid (β, -0.119; 95% CI, -0.148 to -0.090) concentrations, and decreased prevalence of hyperuricemia (OR, 0.73; 95% CI, 0.64-0.83) and gout (OR, 0.71; 95% CI, 0.52-0.94). Higher urinary molybdenum levels were associated with lower levels of systemic oxidative stress (gamma-glutamyltransferase levels; β, -0.052; 95% CI, -0.067 to -0.037) and inflammation (C-reactive protein levels; β, -0.184; 95% CI, -0.220 to -0.148). In HK-2 cells under H2O2-induced oxidative stress, molybdenum upregulated manganese superoxide dismutase expression and decreased oxidative stress. Urinary molybdenum levels are associated with decreased prevalence of hyperuricemia and gout in adult population. Molybdenum's antioxidative properties might have acted as an important mechanism for the reduction of systemic inflammation, ROS, and uric acid levels.

The effect of factor XIa on thrombin and plasmin generation, clot formation, lysis and density in coagulation factors deficiencies

IntroductionGrowing evidence supports the importance of factor (F) XI activation for thrombosis and hemostasis as well as inflammation and complement systems. In this study, we evaluated the effect of activated FXI (FXIa) on the detection of factor deficiencies by global hemostasis assays of thrombin generation (TG), plasmin generation (PG), and clot formation and lysis (CFL). Materials and methodsAn absorbance and fluorescence microplate assay was used to simultaneously observe TG, PG, and CFL in FV-, FVII-, FVIII-, and FIX-deficient plasmas supplemented with purified factors. Coagulation was initiated with tissue factor with or without FXIa in the presence of tissue plasminogen activator. Thrombin and plasmin peak heights (TPH and PPH), maximal clot density (MCD), times to clotting (CT), thrombin and plasmin peaks (TPT and PPT) and clot lysis (LyT) and a new parameter, clot lifetime (LiT), were evaluated. ResultsTG/CFL were elevated by the FXIa at low FV (below 0.1 IU/mL), and at FVIII and FIX above 0.01 IU/mL. FXIa affected PG only at low FV and FVII. At high factor concentrations, FXIa reduced MCD. Thrombin and plasmin substrates had effect on CT, LyT, LiT and MCD parameters. ConclusionsFXIa reveals new relationships between TG, PG and CFL parameters in factor deficiencies suggesting potential benefits for discrimination of bleeding phenotypes.

A fluorescent quantum dot conjugate to probe the interaction of Enterolobium contortisiliquum trypsin inhibitor with cancer cells

Serine proteases play crucial biological roles and have their activity controlled by inhibitors, such as the EcTI, a serine protease inhibitor purified from Enterolobium contortisiliquum seeds, which has anticancer activity. This study aimed to conjugate EcTI with quantum dots (QDs), fluorophores with outstanding optical properties, and investigate the interaction of QDs-EcTI nanoprobe with cancer cells. The conjugation was evaluated by fluorescence correlation spectroscopy (FCS) and fluorescence microplate assay (FMA). EcTI inhibitory activity after interaction with QDs was also analyzed. From FCS, the conjugate presented a hydrodynamic diameter about 4× greater than bare QDs, suggesting a successful conjugation. This was supported by FMA, which showed a relative fluorescence intensity of ca. 3815% for the nanosystem, concerning bare QDs or EcTI alone. The EcTI inhibitory activity remained intact after its interaction with QDs. From flow cytometry analyses, approximately 62% of MDA-MB-231 and 90% of HeLa cells were labeled with the QD-EcTI conjugate, suggesting that their membranes have different protease levels to which EcTI exhibits an affinity. Concluding, the QD-EcTI represents a valuable nanotool to study the interaction of this inhibitor with cancer cells using fluorescence-based techniques with the potential to unravel the intricate dynamics of interplays between proteases and inhibitors in cancer biology.

Exploring the supplementary potential of all-trans retinoic acid with methotrexate in rheumatoid arthritis: modulation of synovial cell apoptosis and autophagy.

The imbalance between apoptosis and proliferation in fibroblast-like synoviocytes (FLSs) plays a key role in the pathogenesis of rheumatoid arthritis (RA). This study aims to investigate the potential of all-trans retinoic acid (ATRA) as a supplementary therapeutic agent alongside methotrexate (MTX) for RA, by examining its ability to inhibit synovial cell proliferation and enhance apoptosis through the ROS-JNK signalling pathway. The viability, apoptosis, and autophagy levels of human rheumatoid arthritis fibroblast-like synovial cells (HFLS-RA) were evaluated, while ROS generation was measured through the DCFH-DA fluorescence microplate assay. Western blotting was used to analyse the expression levels of JNK signalling pathway-related proteins. To assess therapeutic potential in vivo, a collagen-induced arthritis (CIA) model was established in Wistar rats. Small doses of MTX did not significantly affect the viability of HFLS-RAs or induce apoptosis. However, when ATRA was added to the treatment, the therapy markedly inhibited cell proliferation and induced apoptosis and excessive autophagy. Mechanistically, ATRA activated the ROS/JNK signalling pathway in HFLS-RAs. ROS scavengers and JNK inhibitors significantly attenuated ATRA-induced apoptosis and autophagy. In vivo, the combination therapy demonstrated a remarkable enhancement of the anti-arthritic efficacy in CIA rats. The ability of ATRA to inhibit proliferation in RA FLSs through autophagy and apoptosis underscores its potential as a supplementary therapeutic agent alongside MTX for RA, particularly when compared to the limited impact of MTX on these processes. This combined strategy holds promise for enhancing therapeutic outcomes and warrants further investigation in the management of RA.

A bioorthogonal chemistry approach to detect the K1 polysialic acid capsule in Escherichia coli.

Most Escherichia coli strains associated with neonatal meningitis express the K1 capsule, a sialic acid polysaccharide that is directly related to their pathogenicity. Metabolic oligosaccharide engineering (MOE) has mostly been developed in eukaryotes, but has also been successfully applied to the study of several oligosaccharides or polysaccharides constitutive of the bacterial cell wall. However, bacterial capsules are seldom targeted despite their important role as virulence factors, and the K1 polysialic acid (PSA) antigen that shields bacteria from the immune system still remains untackled. Herein, we report a fluorescence microplate assay that allows the fast and facile detection of K1 capsules with an approach that combines MOE and bioorthogonal chemistry. We exploit the incorporation of synthetic analogues of N-acetylmannosamine or N-acetylneuraminic acid, metabolic precursors of PSA, and copper-catalysed azide-alkyne cycloaddition (CuAAC) as the click chemistry reaction to specifically label the modified K1 antigen with a fluorophore. The method was optimized, validated by capsule purification and fluorescence microscopy, and applied to the detection of whole encapsulated bacteria in a miniaturized assay. We observe that analogues of ManNAc are readily incorporated into the capsule while those of Neu5Ac are less efficiently metabolized, which provides useful information regarding the capsule biosynthetic pathways and the promiscuity of the enzymes involved. Moreover, this microplate assay is transferable to screening approaches and may provide a platform to identify novel capsule-targeted antibiotics that would circumvent resistance issues.

Anaerobic Bacterial Biofilms Inhibit Neutrophil Responses via the Release of Stable Small‐Molecule Effectors

Background and ObjectiveFusobacterium and Porphyromonasare two genera of opportunistic pathogens that often aggregate in complex communities known as biofilms. Host innate immune cells are frequently frustrated when they encounter biofilm bacteria, resulting in ineffectual clearance and persistent infection. Anaerobic pathogens generate or accumulate organic molecules and use them as defense against environmental pressures such as oxidative stress or attack from host immune cells. Porphyromonasspecies utilize protoporphyrin IX (PPIX) to protect against neutrophil oxidative‐killing. The objective was to develop a model of mixed‐species biofilm generation and to evaluate bovine neutrophil responses to bioactive molecules released from either biofilm or planktonic bacteria. We hypothesized that biofilm‐conditioned growth medium would contain more PPIX than planktonic medium and therefore inhibit the functional responses of peripheral neutrophils.MethodsNeutrophils were isolated from whole blood via hypotonic lysis and bacterial biofilms were generated using previously validated methods for the cultivation of mixed‐species biofilms. Spent media from biofilm and overnight planktonic cultures were collected, centrifuged, and filtered to remove viable bacteria. Supernatants were fractionated via &lt;3kDa‐cutoff filtration and exposed directly to neutrophils. Neutrophil responses to the supernatants were assessed with a fluorescent microplate assay for reactive oxygen species (ROS) and chemotaxis was measured with a Transwell® transmigration assay. The specific mechanism of neutrophil activation was investigated by employing NF‐κB and NADPH oxidase inhibitors.ResultsNeutrophils exposed to planktonic supernatant showed significantly elevated oxidative and chemotactic responses compared to biofilm products from identical bacteria. Ultra‐filtration revealed that a &lt;3kDa molecule is differentially expressed in the supernatants and allows biofilm‐bacteria to inhibit neutrophil responses. Intensive heat, nuclease, lipase, and protease treatment of the &lt;3kDa fractions did not alter neutrophil functional responses. Preliminary experiments suggest that the accumulation of PPIX in the biofilm supernatant attenuates neutrophil chemotaxis and ROS production. In addition, chemical inhibition experiments indicated that neutrophils respond to planktonic supernatants independently of NF‐κB signaling.ConclusionsExperiments reveal a minimal neutrophil response to molecules released from biofilm bacteria and may explain the inadequate host response to biofilm‐mediated infections. The phenotypic changes in the biofilm mode of growth allow for the generation and accumulation of PPIX, providing protection from host immune clearance mechanisms. Understanding the mechanism of immune avoidance is a crucial step to reduce the persistent inflammation and negative clinical outcomes associated with chronic biofilm‐mediated disease.

Determination of dissolved nickel in natural waters using a rapid microplate fluorescence assay method

A new microplate analytical procedure is described for the determination of nickel (Ni2+) ions in natural water samples. A lophine analogue fluorescent sensor was synthesized and a spectral study showed a selective fluorescence quenching effect of chemical sensor by Ni2+ under optimized conditions. Density functional theory (DFT) calculations confirmed the formation of a Ni(II)L3 complex obtained by the Job plot. The calculations showed that the fluorescence emission peak of L collapses due to the distortion of L in the complex. The simple and fast microplate procedure allowed us to quantify Ni2+ with a linear response from 1.6 to 40 µg L-1 and a quantification limit of 5.4 µg L-1 without the need of a preconcentration step. The optimized procedure using high-throughput microplate assay has been applied for the determination of Ni2+ in natural water samples with good analytical performances.

Membrane perturbation, altered morphology and killing of Staphylococcus epidermidis upon contact with a cytocompatible peptide-based antibacterial surface

One possibility to prevent prosthetic infections is to produce biomaterials resistant to bacterial colonization by anchoring membrane active antimicrobial peptides (AMPs) onto the implant surface. In this perspective, a deeper understanding of the mode of action of the immobilized peptides should improve the development of AMP-inspired infection-resistant biomaterials. The aim of the present study was to characterize the bactericidal mechanism against Staphylococcus epidermidis of the AMP BMAP27(1–18), immobilized on titanium disks and on a model resin support, by applying viability counts, Field Emission Scanning Electron Microscopy (FE-SEM), and a fluorescence microplate assay with a membrane potential-sensitive dye. The cytocompatibility to osteoblast-like MG-63 cells was investigated in monoculture and in co-culture with bacteria. The impact of peptide orientation was explored by using N- and C- anchored analogues. On titanium, the ∼50 % drop in bacteria viability and dramatically affected morphology indicate a contact-killing action exerted by the N- and C-immobilized peptides to the same extent. As further shown by the fluorescence assay with the resin-anchored peptides, the bactericidal effect was mediated by rapid membrane perturbation, similar to free peptides. However, at peptide MBC resin equivalents the C-oriented analogue proved more effective with more than 99 % killing and maximum fluorescence increase, compared to half-maximum fluorescence with more than 90 % killing produced by the N-orientation. Confocal microscopy analyses revealed 4–5 times better MG-63 cell adhesion on peptide-functionalized titanium both in monoculture and in co-culture with bacteria, regardless of peptide orientation, thus stimulating further studies on the effects of the immobilized BMAP27(1–18) on osteoblast cells.

Simple Fluorescence Assay for Triethylamine Based on the Palladium Catalytic Dimerization of Benzofuran-2-boronic Acid.

Although benzofuran-2-boronic acid hardly emits fluorescence, it can be rapidly converted to a highly fluorescent benzofuran dimer after mixing with a palladium catalyst and amine. We found that a fluorescence enhancement accompanying dimerization was quantitatively promoted upon increasing the concentration of amine. In the present study, we developed a simple fluorescence assay for amines based on the promotive effect. As the result of a fluorescence measurement of the reaction mixture of 19 kinds of typical amines, it was found that tertiary amines including triethylamine (TEA) provided a significant fluorescence enhancement. Finally, the fluorogenic reaction could be applied to develop a high-throughput fluorescent microplate assay for TEA with the limit of detection (blank + 3SD) of 0.091 μM.

Total synthesis of lindbladione, a Hes1 dimerization inhibitor and neural stem cell activator isolated from Lindbladia tubulina

Lindbladione (1) is a neural stem cell differentiation activator isolated from Lindbladia tubulina by our group. Hes1 dimerization inhibitory activity of lindbladione (1) was discovered using our original fluorescent Hes1 dimer microplate assay. We also found that lindbladione (1) accelerates the differentiation of neural stem cells. We conducted the first total synthesis of lindbladione (1) via Heck reaction of 1-hexene-3-one 7 with iodinated naphthoquinone 12, which was provided by Friedel–Crafts acylation followed by Claisen condensation, in the presence of Pd (II) acetate. Careful deprotection of the benzyl groups of 13 successively provided lindbladione (1). Synthesized lindbladione (1) exhibited potent Hes1 dimer inhibition (IC50 of 2.7 μM) in our previously developed fluorescent Hes1 dimer microplate assay. Synthesized lindbladione (1) also accelerated the differentiation of C17.2 mouse neural stem cells into neurons dose dependently, increasing the number of neurons by 59% (2.5 μM) and 112% (10 μM) compared to the control. These activities are comparable to those of naturally occurring lindbladione (1) isolated from L. tublina.

Biofilm and Planktonic Bacteria Differentially Express a Small Molecule (&lt;3kD), Heat‐Stable, Protease‐Resistant Factor that Affects Bovine Neutrophil Function in a NF‐Kappa β‐Independent Mechanism

IntroductionChronic bacterial infections and the associated inflammation have a significant impact in both veterinary and human medicine. Bacterial biofilms are a major cause of the persistent inflammation observed in chronic infections and nearly 80% of all infections involve biofilms. Biofilms are typically polymicrobial in composition and many of these mixed‐infections involve anaerobic bacteria. Recent evidence demonstrates that immune cells often disregard the signals released from biofilm bacteria, and ineffectual clearance of biofilms may explain the chronicity of this sort of infection. A mixed‐species model of anaerobic biofilm growth was developed to evaluate medically relevant biofilms and their interactions with bovine neutrophils.AimsTo generate mixed‐species bacterial biofilms composed of the two opportunistic pathogens Fusobacterium necrophorum and Porphyromonas levii, and to employ the in vitro system to investigate factors associated with biofilms that may limit neutrophil functional ability.MethodsMixed‐species biofilm formation was verified with SEM, CLSM and viable cell counts. Neutrophil oxidative burst was assessed with a fluorescent microplate assay for hydrogen peroxide. Chemotaxis was measured with a Transwell transmigration assay. Extracellular factors released from planktonic cultures and biofilms were assessed by size‐exclusion filtration, lipopolysaccharide (LPS) removal, protease and heat treatment. Mechanistic experiments to study neutrophil responses to these supernatants were conducted with specific inhibitors of function, such as NF‐κβ signaling (BAY‐11) and inflammasome activation (glyburide).ResultsNeutrophils exposed to planktonic bacterial supernatants showed significantly elevated oxidative (4‐fold increase in fluorescence) and chemotactic responses compared to neutrophils exposed to biofilm products from identical bacteria. LPS plays a significant role in the stimulation of neutrophils; in this experiment biofilms produced substantially more LPS than planktonic bacteria despite inducing less neutrophil response. Removal of LPS resulted in similar neutrophil responses to planktonic and biofilm supernatants.Size‐exclusion filtration of bacterial supernatants and subsequent exposure to neutrophils revealed that the active stimulatory molecule is below 3 kD and is released strictly from planktonic cultures. Intensive heat and protease treatment of the &lt;3kD fractions did not alter neutrophil functional responses. Treatment with a NF‐κβ inhibitor demonstrated that neutrophils respond specifically to planktonic supernatants independently of NF‐κβ signaling.ConclusionsWe describe a heat‐stable, protease‐resistant, non‐LPS, small (&lt;3kD) factor produced by in vitro planktonic cultures of F. necrophorum and P. levii that activates bovine neutrophils. Neutrophils did not exhibit the same oxidative and chemotactic responses upon exposure to in vitro generated biofilm supernatants and these differential responses are the result of a NF‐κβ independent mechanism, perhaps through an inflammasome‐mediated process.Support or Funding InformationFunding provided by the University of Calgary Grant 10002461 to DW Morck.

A fluorescence microplate assay based on molecularly imprinted silica coated quantum dot optosensing materials for the separation and detection of okadaic acid in shellfish.

Molecularly imprinted polymers (MIPs) are attracting substantial interest as artificial plastic antibodies because of their biometric capability for targeting small molecules. In this study, molecularly imprinted silica material-coated quantum dots (MIS-QDs) with selective recognition capability to okadaic acid (OA) were developed and characterized. The synthesized MIS-QDs with specific imprinting cavities exhibited excellent recognition capability similar to those of biological antibodies and high fluorescence (FL) quenching selectivity for OA. Furthermore, the MIS-QDs with unsaturated bonds were immobilized onto the surface of 96-well microplates by cold plasma-induced grafting. A novel direct competitive microplate assay strategy was then proposed. The FL quenching properties of the developed microplate assay showed an excellent linear relationship with OA in the range of 10.0-100.0μg/kg with a correlation coefficient of 0.9961. The limit of detection for OA was 0.25μg/kg in the shellfish samples. The mean quantitative recoveries were 92.5%-101.0% and 92.9%-101.3%, with relative standard deviations of <7.7% and 7.6% for pure solvents and purified shellfish samples, respectively. The established microplate assay strategy can be used as a rapid and high-throughput method for analyzing OA marine toxins in biological samples.

Optimization of a Microplate Assay for Generating Listeria Monocytogenes, E. Coli O157:H7, and Salmonella Biofilms and Enzymatic Recovery for Enumeration.

Biofilms enable the persistence of pathogens in food processing environments. Sanitizing agents are needed that are effective against pathogens entrapped in biofilms that are more difficult to inactivate than planktonic cells that are displaced and found on equipment surfaces. We examined conditions to develop, analyze, and enumerate the enhanced biofilms of three different foodborne pathogens assisted by fluorescence adherence assay and enzymatic detachment. We compared three different isomeric forms of fluorescent substrates that are readily taken up by bacterial cells based on carboxy-fluorescein diacetate (5-CFDA, 5,6-CFDA, 5,6-CFDA, SE). Biofilm-forming strains of Escherichia coli O157:H7 F4546 and Salmonella Montevideo FSIS 051 were identified using a microplate fluorescence assay defined previously for L. monocytogenes. Adherence levels were determined by differences in relative fluorescence units (RFU) as well as recovered bacterial cells. Multiple hydrolytic enzymes were examined for each representative pathogen for the most suitable enzyme for detachment and enumeration to confirm adherence data obtained by fluorescence assay. Cultures were grown overnight in microplates, incubated, washed and replenished with fresh sterile growth medium; this cycle was repeated for seven consecutive days to enrich for robust biofilms. Treatments were performed in triplicate and compared by one-way analysis of variance (ANOVA) to determine significant differences (p < 0.05).

Evaluating the glycophenotype on breast cancer tissues with quantum dots-Cramoll lectin conjugates

During carcinogenesis, changes in the glycosylation can modulate many biological processes. Thus, the interest in exploring and understanding the roles of carbohydrates as cancer biomarkers has been increasing. Lectins have been applied as useful tools in glycobiology, especially when associated with fluorescent reporters. Therefore, to take advantage of the physicochemical properties of quantum dots (QDs), herein, we conjugated Cramoll, a lectin that recognizes glucose/mannose residues, with those nanoparticles. We applied the conjugates to investigate the glycocode of normal, fibroadenoma (FB), and invasive ductal carcinoma (IDC) human breast tissues. Additionally, we proposed a method to quantitatively evaluate the tissue labeling intensity by a fluorescence microplate assay (FMA). Conjugates showed intense fluorescence and specificity. The lectin activity and secondary structure were also preserved after the conjugation with QDs. Moreover, fluorescence images showed that ductal cells of normal and FB tissues were preferentially labeled by conjugates, whereas both cells and stroma were strongly labeled in IDC. FMA showed in a quantitative, practical, and sensitive way that the level of exposed glucose/mannose residues increased accordingly to the sample malignancy degree. In conclusion, QDs-Cramoll conjugates can be considered effective, specific, and versatile probes to evaluate glycan profiles in normal and transformed tissues, by fluorescence microscopy as well as FMA quantification. Furthermore, FMA showed to be a potential method that can be applied with other fluorescent conjugates.

Detection of hydrogen sulfide in water samples with 2-(4-hydroxyphenyl)-4,5-di(2-pyridyl)imidazole-copper(II) complex using environmentally green microplate fluorescence assay method

Hydrogen sulfide (H2S) is a colorless toxic gas which can be found as HS− in rivers and waste waters especially in the occupational susceptible environment. Herein we synthesized a lophine analogue, 2-(4-hydroxyphenyl)-4,5-di(2-pyridyl)imidazole (HPI), which fluoresces at 410 nm after excitation at 280 nm. HPI has an imidazole ring and a pyridine ring which are capable of forming coordinate bonds with copper (Cu(II)) that cause quenching of HPI fluorescence. We found that HS− can selectively liberate HPI from the complex via formation of CuS, thus, HPI regains its fluorescence properties. Interestingly, the probe was proved to be regenerable. This reaction was used for the development of a fluorescence microplate assay for the determination of HS− in environmental samples. The method was applied to river water samples and was able to detect HS− in concentrations down to 5 ppb with acceptable accuracy (90.3–103.0%) and good precision (%RSD ≤ 4.1). The method showed many advantages over the previously reported ones including instantaneous reaction, simple probe synthesis, high-throughput, high selectivity toward hydrogen sulfide over other ions and sulfur or thiol containing compounds and at last, it complies with the green chemistry rules through using a regenerable probe, aqueous solvents, and miniaturized measurement system.

Multimodal highly fluorescent-magnetic nanoplatform to target transferrin receptors in cancer cells

BackgroundSite-specific multimodal nanoplatforms with fluorescent-magnetic properties have great potential for biological sciences. For this reason, we developed a multimodal nanoprobe (BNPs-Tf), by covalently conjugating an optical-magnetically active bimodal nanosystem, based on quantum dots and iron oxide nanoparticles, with the human holo-transferrin (Tf). MethodsThe Tf bioconjugation efficiency was evaluated by the fluorescence microplate assay (FMA) and the amount of Tf immobilized on BNPs was quantified by fluorescence spectroscopy. Moreover, relaxometric and fluorescent properties of the BNPs-Tf were evaluated, as well as its ability to label specifically HeLa cells. Cytotoxicity was also performed by Alamar Blue assay. ResultsThe FMA confirmed an efficient bioconjugation and the fluorescence spectroscopy analysis indicated that 98% of Tf was immobilized on BNPs. BNPs-Tf also presented a bright fluorescence and a transversal/longitudinal relaxivities ratio (r2/r1) of 65. Importantly, the developed BNPs-Tf were able to label, efficiently and specifically, the Tf receptors in HeLa cells, as shown by fluorescence and magnetic resonance imaging assays. Moreover, this multimodal system did not cause noteworthy cytotoxicity. ConclusionsThe prepared BNPs-Tf hold great promise as an effective and specific multimodal, highly fluorescent-magnetic, nanoplatform for fluorescence analyses and T2-weighted images. General significanceThis study developed an attractive and versatile multimodal nanoplatform that has potential to be applied in a variety of in vitro and in vivo studies, addressing biological processes, diagnostic, and therapeutics. Moreover, this work opens new possibilities for designing other efficient multimodal nanosystems, considering other biomolecules in their composition able to provide them important functional properties.

Fewer Defects in the Surface Slows the Hydrolysis Rate, Decreases the ROS Generation Potential, and Improves the Non‐ROS Antimicrobial Activity of MgO

Magnesium oxide (MgO) is recognised as exhibiting a contact-based antibacterial activity. However, a comprehensive study of the impact of atomic-scale surface features on MgO's antibacterial activity is lacking. In this study, the nature and abundance of the native surface defects on different MgO powders are thoroughly investigated. Their impacts on the hydrolysis kinetics, antibacterial activity against Escherichia coli (ATCC 47076), Staphylococcus epidermidis and Pseudomonas aeruginosa and the reactive oxygen species (ROS) generation potential are determined and explained. It is shown that a reduction in the abundance of low-coordinated oxygen atoms on the surface of the MgO improves its resistance to both hydrolysis and antibacterial activity. The ROS generation potential, determined in-situ using a fluorescence microplate assay and electron paramagnetic resonance spectroscopy, is not an inherent property of the studied MgO, rather it is a side product of hydrolysis (only for the most highly defected MgO particles) and/or a consequence of the MgO/bacteria interaction. The evaluation of the mutual correlations of the hydrolysis, the antibacterial activity and the ROS generation, with their origin in the surface defects' peculiarities, led to the conclusion that the acid/base reaction between the MgO surface and the bacterial wall contributes considerably to the MgO's antibacterial activity.

Fluorescent microplate assay method for high-throughput detection of lipase transesterification activity

This study describes a sensitive and fluorescent microplate assay method to detect lipase transesterification activity. Lipase-catalyzed transesterification between butyryl 4-methyl umbelliferone (Bu-4-Mu) and methanol in tert-butanol was selected as the model reaction. The release of 4-methylumbelliferone (4-Mu) in the reaction was determined by detecting the fluorescence intensity at λex 330 nm and λem 390 nm. Several lipases were used to investigate the accuracy and efficiency of the proposed method. Apparent Michaelis constant (Km) was calculated for transesterification between Bu-4-Mu and methanol by the lipases. The main advantages of the assay method include high sensitivity, inexpensive reagents, and simple detection process.