Fluorescein Isothiocyanate Fluorescence Research Articles

Screening high affinity monoclonal antibody producing hybridomas using a graphene oxide-based fluorescence biosensor

Screening monoclonal antibody (mAb) producing hybridomas by ELISA using microplates is time-consuming and expensive. The combination of biosensing and flow cytometry represents a promising rapid, high-throughput method for screening mAb producing clones. Based on the assembly of graphene oxide (GO) and fluorescein isothiocyanate (FITC)-labeled peptide that included the N-terminal portion of selenoprotein K (SELENOK) antigen epitope, we designed a novel GO fluorescence biosensor for screening high affinity mAb producing hybridomas. The FITC-labeled peptide was strongly adsorbed on the surface of GO, which effectively quenched the FITC fluorescence. The specific SELENOK mAb competitively bound to FITC-Peptide, resulting in FITC-Peptide release from the GO surface and fluorescence recovery. This sensor was successfully utilized to sensitively detect SELENOK mAb with low detection limit of 0.25 U. In addition, we employed this FITC-Peptide/GO biosensor to in situ visualizing and high-throughput screening SELENOK mAb hybridomas (SMAHs) at the single-cell level. The proposed sensor is simple, relatively inexpensive, highly specific, and applicable for cell sort assay, demonstrating the effective use for peptide-antibody interactions in the specific screening for other mAb hybridomas libraries.

Intelligent detection of tetracycline by a rare earth multicolor fluorescent probe based on guanosine-5′-monophosphate

Long-term discharge of tetracycline (TC) in the environment exerts selective pressure on the bacteria, increasing resistance in sensitive bacteria and posing a serious threat to public health. Therefore, it is crucial to develop real-time detection of tetracycline. In this study, we reported a novel rare-earth fluorescent nanosensor FITC-GMP-Eu, based on the formation of guanosine-5′-monophosphate disodium salt (GMP) and fluorescein isothiocyanate (FITC), which enabled the real-time identification of tetracycline in the environment. When there was no TC in the environmental sample, the FITC-GMP-Eu nanosensor exhibited the green fluorescence of FITC due to the fluorescence quenching effect of water molecules on Eu3+. When the environmental samples contained TC, the red fluorescence of Eu3+ could be sensitized due to the diketone structure in the tetracycline molecule, which enabled the naked-eye visualized identification of TC. The nanosensor had high sensitivity (detection limit of 19 nM) and a wide detection range (0–30 μM) for TC. At the same time, the sensor was characterized by high visualization and anti-interference ability due to the strong fluorescence of both FITC and Eu complexes. In order to improve the practicality of the detection, this study also prepared a paper-based and sodium alginate gelated fluorescence detection platform with low background fluorescence. Combined with the common color analysis and RGB extraction procedures in mobile phones, real-time and on-site identification of TC on the surface of various fruit samples can be achieved.

Evaluation of Autoantibody Binding to Cardiac Tissue in Multisystem Inflammatory Syndrome in Children and COVID-19 Vaccination–Induced Myocarditis

Cardiac dysfunction and myocarditis have emerged as serious complications of multisystem inflammatory syndrome in children (MIS-C) and vaccines against SARS-CoV-2. Understanding the role of autoantibodies in these conditions is essential for guiding MIS-C management and vaccination strategies in children. To investigate the presence of anticardiac autoantibodies in MIS-C or COVID-19 vaccine-induced myocarditis. This diagnostic study included children with acute MIS-C or acute vaccine myocarditis, adults with myocarditis or inflammatory cardiomyopathy, healthy children prior to the COVID-19 pandemic, and healthy COVID-19 vaccinated adults. Participants were recruited into research studies in the US, United Kingdom, and Austria starting January 2021. Immunoglobulin G (IgG), IgM, and IgA anticardiac autoantibodies were identified with immunofluorescence staining of left ventricular myocardial tissue from 2 human donors treated with sera from patients and controls. Secondary antibodies were fluorescein isothiocyanate-conjugated antihuman IgG, IgM, and IgA. Images were taken for detection of specific IgG, IgM, and IgA deposits and measurement of fluorescein isothiocyanate fluorescence intensity. Data were analyzed through March 10, 2023. IgG, IgM and IgA antibody binding to cardiac tissue. By cohort, there were a total of 10 children with MIS-C (median [IQR] age, 10 [13-14] years; 6 male), 10 with vaccine myocarditis (median age, 15 [14-16] years; 10 male), 8 adults with myocarditis or inflammatory cardiomyopathy (median age, 55 [46-63] years; 6 male), 10 healthy pediatric controls (median age, 8 [13-14] years; 5 male), and 10 healthy vaccinated adults (all older than 21 years, 5 male). No antibody binding above background was observed in human cardiac tissue treated with sera from pediatric patients with MIS-C or vaccine myocarditis. One of the 8 adult patients with myocarditis or cardiomyopathy had positive IgG staining with raised fluorescence intensity (median [IQR] intensity, 11 060 [10 223-11 858] AU). There were no significant differences in median fluorescence intensity in all other patient cohorts compared with controls for IgG (MIS-C, 6033 [5834-6756] AU; vaccine myocarditis, 6392 [5710-6836] AU; adult myocarditis or inflammatory cardiomyopathy, 5688 [5277-5990] AU; healthy pediatric controls, 6235 [5924-6708] AU; healthy vaccinated adults, 7000 [6423-7739] AU), IgM (MIS-C, 3354 [3110-4043] AU; vaccine myocarditis, 3843 [3288-4748] AU; healthy pediatric controls, 3436 [3313-4237] AU; healthy vaccinated adults, 3543 [2997-4607] AU) and IgA (MIS-C, 3559 [2788-4466] AU; vaccine myocarditis, 4389 [2393-4780] AU; healthy pediatric controls, 3436 [2425-4077] AU; healthy vaccinated adults, 4561 [3164-6309] AU). This etiological diagnostic study found no evidence of antibodies from MIS-C and COVID-19 vaccine myocarditis serum binding cardiac tissue, suggesting that the cardiac pathology in both conditions is unlikely to be driven by direct anticardiac antibody-mediated mechanisms.

Local pH mapping in the cell adhesion nano-interfaces on a pH-responsive fluorescence-dye-immobilized substrate.

Cell-substrate adhesion nano-interfaces can, in principle, exhibit a spatial distribution of local pH values under the influence of the weakly acidic microenvironment of glycocalyx grafted on lipid bilayer cell membrane which is compressed and closely attached to culture substrate in the vicinity of integrin-adhesion complexes. However, a simple local pH distribution imaging methodology has not been developed. In this study, to visualize the local pH distribution at the cell adhesion interface, we prepared glass substrates chemically modified with a pH-responsive fluorescent dye fluorescein isothiocyanate (FITC), observed the distribution of FITC fluorescence intensity at the adhesion interface of fibroblast (NIH/3T3) and cancer cells (HeLa), and compared the FITC images with the observed distribution of focal adhesions. FITC images were converted to pH mapping based on the pH-fluorescence calibration data of surface-immobilized FITC pre-measured in different pH media, which showed significantly larger regions with lowered pH level (6.8-7.0) from outside the cell (pH 7.4) were observed at the thick inner periphery of HeLa cells while 3T3 cells exhibited smaller lowered pH regions at the thin periphery. The lowered pH regions overlapped with many focal adhesions, and image analysis showed that larger focal adhesions tend to possess more lowered pH sites inside, reflecting enhanced glycocalyx compression due to accumulated integrin-adhesion ligand binding. This tendency was stronger for HeLa than for 3T3 cells. The role of glycocalyx compression and the pH reduction at the cell adhesive interface is discussed.

Photo-pH dual stimuli-responsive multicolor fluorescent polymeric nanoparticles for multicolor security ink, optical data encryption and zebrafish imaging

Stimuli-responsive fluorescent polymeric nanoparticles are widely used in bioimaging, anti-counterfeiting and data encryption due to their advantages of simple preparation, good water-dispersibility, high brightness and photostability. In this paper, novel photo-pH dual stimuli-responsive multicolor fluorescent polymeric nanoparticles (SMFPNs) were fabricated via combination of one-pot miniemulsion polymerization and surface modification. Based on fluorescence resonance energy transfer (FRET) principle, 4-ethoxy-9-allyl-1,8-naphthalimide (EANI), fluorescein isothiocyanate (FITC) and spiropyran-linked methacrylate (SPMA) can be used to constitute three pairs of FRET systems. In the nanoparticles, the blue fluorescence of EANI (energy donor) decreases and the green fluorescence of FITC (energy receptor) increases via increasing pH. While pH decreases, it can return to blue fluorescence. Meanwhile, after UV light irradiation, SPMA converts from non-fluorescent spiropyrans (SP) state to red fluorescent merocyanine (MC) state, the transformation from blue or green emission to red emission can be realized. When irradiated with visible light, it reverts to its original blue or green fluorescence. Therefore, the system can achieve the multi-color transformation of blue, green and red under changing pH and illumination conditions. In addition, the as-prepared nanoparticles also exhibited high fluorescence contrast, rapid photo-responsiveness and excellent photoreversibility. Therefore, SMFPNs displayed great potential in the fields of multicolor security ink, advanced anti-counterfeiting, multilevel data encryption and in vivo bioimaging.

A ratiometric fluorescent probe based on peptide modified MnFe2O4 nanoparticles for matrix metalloproteinase-7 activity detection in vitro and in vivo.

Abnormal expression of matrix metalloproteinases plays an important role in tumor invasion and metastasis. In this report, a peptide modified MnFe2O4 ratiometric fluorescent nanoprobe is developed for noninvasively visualizing the distribution of matrix metalloproteinase-7 (MMP-7) in vitro and in vivo. A fluorescein isothiocyanate (FITC) modified peptide containing the specific motif VPLSLTMG for MMP-7 cleavage was conjugated with MnFe2O4 nanoparticles (NPs) to establish a Förster resonance energy transfer (FRET) system for sensing the protease. The rhodamine B (RhB) modified targeting peptide immobilized on the nanoparticle surface was not only used as an internal reference for forming a ratiometric fluorescence system together with the FITC dye, but also used for enhancing the tumor targeting ability. The tumor accumulation amount of the as-developed ratiometric fluorescent probe (termed MnFe2O4-pep-dyes) can be measured by T2-weighted magnetic resonance (T2-weighted MR) imaging because MnFe2O4 NPs display an excellent T2-weighted MR contrast ability. In the presence of MMP-7, FITC detached from the MnFe2O4 surface resulting in the recovery of FITC fluorescence, while no obvious change of the RhB fluorescence was observed. The recovery ratio of FITC to RhB fluorescence intensity is linearly dependent on the MMP-7 concentration within the range of 0.1 to 15 nM in buffer and 5 × 102 to 1 × 104 cells in cell lysates with a limit of detection of 0.1 nM and 436 cells, respectively. MnFe2O4-pep-dyes was further applied to spatially observe MMP-7 expression in a tumor-bearing mouse by in vivo fluorescence imaging with external magnetic field assistance for demonstrating its practicability.

Design of smartphone platform by ratiometric fluorescent for visual detection of silver ions

In this work, a smartphone platform based on ratiometric fluorescence signals was reported, which can achieve fast, sensitive, and on-site visual detection of silver ions. The ratiometric fluorescent probe was constructed with fluorescein isothiocyanate (FITC) and telluride quantum dots (CdTe QDs) through electrostatic interaction. Silver ions will interact with -SH on the surface of CdTe QDs, which can cause the decrease of the fluorescence intensity at 680 nm, while the fluorescence of FITC at 522 nm remains unchanged. It displays the obvious color changes from orange-red to light yellow to green. Based on the changes, the portable smartphone platform was used to identify the RGB values of the system. Then, an analysis model was established according to the variation of RGB values for the visual analysis of Ag+. Under the best experimental conditions, the detection limit of Ag+ by fluorescence spectrometer analysis can be as low as 3.65 nM, the linear range was 12.50 nM to 500 nM. Additionally, the linear range of smartphone platform was 0.125–12.5 μM, and the detection limit was 40 nM. Therefore, visual on-site analysis of Ag+ in environmental water samples can be achieved by capturing the obvious color changes of the system on a smartphone platform.

Fluorescein isothiocyanate, a platform for the selective and sensitive detection of S-Nitrosothiols and hydrogen sulfide

Here we show that the fluorescence of fluorescein isothiocyanate (FITC) is not altered by its reaction with primary amines. However, the fluorescence is rapidly quenched upon reaction with small molecular weight thiols including cysteine, glutathione, homocysteine, dithiothreitol, and sulfide. We have taken advantage of the thiol-dependent quenching of FITC to devise a sulfide specific assay by utilizing polydimethylsiloxane (PDMS) membranes that are permeable to hydrogen sulfide but not to larger charged thiols. In addition, we have discovered that the fluorescein dithiocarbamate (FDTC) formed by the reaction with sulfide can specifically react with S-nitrosothiols (RSNO) to regenerate FITC, thus serving as a specific, fluorogenic reagent to detect picomol levels of RSNO. FDTC was tested as an intracellular RSNO-sensor in germinating tomato seedlings (Solanum lycopersicum) via epifluorescence microscopy. Control plant roots exposed to FDTC showed low intracellular fluorescence which increased ∼3-fold upon exposure to extracellular S-nitrosoglutathione and ∼4-fold in the presence of N6022, a S-nitrosoglutathione reductase (GSNOR) inhibitor, demonstrating that FDTC can be used to visualize intracellular RSNO levels.

Fluorescein isothiocyanate-doped conjugated polymer nanoparticles for two-photon ratiometric fluorescent imaging of intracellular pH fluctuations

Herein, we report a two-photon ratiometric fluorescent pH nanosensor based on conjugated polymer poly(9,9-di-n-octylfluorenyl-2,7-diyl) (PFO) nanoparticles loaded with pH-sensitive fluorescein isothiocyanate (FITC) for intracellular pH monitoring. The obtained nanosensor (FITC-PFO NPs) possesses high sensitivity, excellent stability, good reversibility, favorable two-photon excitability and low cytotoxicity. The ratiometric fluorescence of FITC and PFO (F517/F417) in FITC-PFO NPs solution shows an efficient pH-sensitive response over the pH range from 3 to 10 (pKa = 6.43) under two-photon excitation. Additionally, the FITC-PFO NPs is successfully applied for ratiometric imaging of intracellular pH and its fluctuation in both one-photon and two-photon excitation modes. Overall, the two-photon pH nanosensor based on FITC-PFO NPs exhibits great potential in crucial physiological and biological processes related to intracellular pH fluctuations.

Self-Propelled Asymmetrical Nanomotor for Self-Reported Gas Therapy.

Gas therapy has emerged as a new therapeutic strategy in combating cancer owing to its high therapeutic efficacy and biosafety. However, the clinical translation of gas therapy remains challenging due to the rapid diffusion and limited tissue penetration of therapeutic gases. Herein, a self-propelled, asymmetrical Au@MnO2 nanomotor for efficient delivery of therapeutic gas to deep-seated cancer tissue for enhanced efficacy of gas therapy, is reported. The Au@MnO2 nanoparticles (NPs) catalyze endogenous H2 O2 into O2 that propels NPs into deep solid tumors, where SO2 prodrug is released from the hollow NPs owing to the degradation of MnO2 shells. Fluorescein isothiocyanate (FITC) is conjugated onto the surface of Au via caspase-3 responsive peptide (DEVD) and the therapeutic process of gas therapy can be optically self-reported by the fluorescence of FITC that is turned on in the presence of overexpressed caspase-3 as an apoptosis indicator. Au@MnO2 nanomotors show self-reported therapeutic efficacy and high biocompatibility both in vitro and in vivo, offering important new insights to the design and development of novel nanomotors for efficient payload delivery into deep tumor tissue and in situ monitoring of the therapeutic process.

Plasma extracellular vesicle delivery of miR-210-3p by targeting ATG7 to promote sepsis-induced acute lung injury by regulating autophagy and activating inflammation

Extracellular vesicles (EVs) can be used for intercellular communication by facilitating the transfer of miRNAs from one cell to a recipient cell. MicroRNA (miR)-210-3p is released into the blood during sepsis, inducing cytokine production and promoting leukocyte migration. Thus, the current study aimed to elucidate the role of plasma EVs in delivering miR-210-3p in sepsis-induced acute lung injury (ALI). Plasma EVs were isolated from septic patients, after which the expression of various inflammatory factors was measured using enzyme-linked immunosorbent assay. Cell viability and apoptosis were measured via cell counting kit-8 and flow cytometry. Transendothelial resistance and fluorescein isothiocyanate fluorescence were used to measure endothelial cell permeability. Matrigel was used to examine the tubulogenesis of endothelial cells. The targeting relationship between miR-210-3p and ATG7 was assessed by dual-luciferase reporter assays. The expression of ATG7 and autophagy-related genes was determined to examine autophagic activation. A sepsis mouse model was established by cecal ligation and puncture (CLP)-induced surgery. The level of miR-210-3p was highly enriched in septic EVs. MiR-210-3p enhanced THP-1 macrophage inflammation, BEAS-2B cell apoptosis, and HLMVEC permeability while inhibiting angiogenesis and cellular activity. MiR-210-3p overexpression reduced ATG7 and LC3II/LC3I expression and increased P62 expression. Improvements in vascular density and autophagosome formation, increased ATG7 expression, and changes in the ratio of LC3II/LC3I were detected, as well as reduced P62 expression, in adenovirus-anti-miR-210-3p treated mice after CLP injury. Taken together, the key findings of the current study demonstrate that plasma EVs carrying miR-210-3p target ATG7 to regulate autophagy and inflammatory activation in a sepsis-induced ALI model.

Polyacrylic acid@zeolitic imidazolate framework-8 nanoparticles for detection and absorptive removal of cyanide from aqueous media with high efficiency

A strategy for sensing of cyanide anion by analyte-induced displacement of a preloaded fluorescent molecule from a cage-like structure is developed. For this purpose, the polyacrylic acid@zeolitic imidazolate framework-8 nanoparticles (PAA@ZIF-8 NPs) were synthesised and a fluorescent molecule, fluorescein isothiocyanate (FITC), was loaded in its cavity. On loading, the fluorescence of FITC was quenched, then upon addition of CN− into the PAA@ZIF-8-FITC, the CN− replaced the doped FTIC, the fluorescence of which was then regenerated and it was then used as a highly sensitive signal transduction for detection of CN- in aqueous media. The process was optimized with parameters such as pH, incubation time etc. for best sensitivity and quantification. The kinetics and mechanism of cyanide adsorption have been studied in detail. The PAA@ZIF-8 NPs has been tested for removal of CN- from aqueous media by adsorption and it exhibited remarkable uptake efficiency of 61 and 85 mg g-1 for NaCN and CuCN, respectively. The kinetic adsorption data exhibits good agreement with the pseudo-second order kinetic fit, while the equilibrium data fitted perfectly with the Langmuir adsorption equation. The PAA@ZIF-8 can be used as a packing material of a column for removing cyanide anion. This study demonstrated a viable and sustainable solution for detection and removal of cyanide from contaminated water.

Grafting of Fluorescence-labeled ssDNA onto Inorganic Nanosheets and Detection of a Target DNA

Abstract A single stranded DNA (ssDNA) labeled with fluorescein isothiocyanate (FITC) was grafted on hexaniobate nanosheets as confirmed by infrared spectra, X-ray diffraction, and confocal laser scanning microscopy; this is the first case that a ssDNA was grafted on nanosheets. The grafted ssDNA retained the capability of hybridization with complementary ssDNA to form double stranded DNA so that the fluorescence of FITC was effectively quenched by hybridization with the target ssDNA having a quencher moiety.

Ratiometric fluorescent nanosensor for dosage-sensitive visual discrimination of glucose based on electron transfer mechanism

Owing to the great role of glucose in biological systems and food industry, it is significant to maintain the dynamic balance of organisms. Various methods widely reported are focused on the glucose detection with high sensitivity and low detection limit. However, the sensor with ultra-low concentration sensitivity is not suitable for the direct detection of high concentration of glucose in human serum and food samples. Additionally, the application of ratiometric fluorescence probe is usually limited by the narrow range of color changes and insensitivity color change of the target. Here, a novel dual-emission ratiometric fluorescence probe with green to red fluorescence intensity of 6:1 was first constructed by fluorescein isothiocyanate (FITC) with green fluorescence as signal unit and silica coated quantum dot (QD@SiO2) with red fluorescence as the stable internal standard. The glucose was oxidized to generate H2O2 by the catalysis of glucose oxidase, which can oxidize Fe2+ to form Fe3+ ion. The formation of Fe3+ ion will cause the quenching of FITC fluorescence, which can realize the continuous fluorescence color change from green to yellow to orange to red for the qualitative and quantitative detection of blood glucose and fruit juice. The proposed method is cheap, simple, intuitive and has a wide detection range for glucose detection, which is of significance in designing the dual-emission ratiometric fluorescence sensor with wide range of color change.

Effect of lansoprazole on DNA integrity of human spermatozoa and activity of seminal creatine kinase.

Although lansoprazole (brand name Prevacid) is a commonly used dug to manage various acid-related gastrointestinal diseases, little is known about its effects on human semen quality and sperm parameters. Here, we aimed to investigate the effect of lansoprazole on DNA integrity of human spermatozoa and activity of seminal creatine kinase. DNA integrity of human spermatozoa was assessed by the Apo-Direct™ kit followed by flow cytometry. The activity of creatine kinase was measured by kinetic spectrophotometric method using commercially available kits following the International Federation of Clinical Chemistry recommendations. Lansoprazole at 3µg/ml, after 1-hr incubation period, did not show any significant increase in fluorescein isothiocyanate fluorescence (p>.05) and hence on the content of DNA breaks of human spermatozoa. In addition, there was no significant change (p=.8113) in the activity of seminal creatine kinase by the effect of lansoprazole. In conclusion, lansoprazole at 3µg/ml did not alter DNA integrity of human spermatozoa or activity of seminal creatine kinase after 1-hr incubation period.

Mechanistic insights into the inhibition of NTCP by myrcludex B.

Background & aimsThe sodium taurocholate co-transporting polypeptide (NTCP) is the entry receptor for the hepatitis B and delta virus (HBV/HDV) and the main hepatic uptake transporter of conjugated bile acids. Myrcludex B, a synthetic peptide mimicking the NTCP-binding domain of HBV, blocks HBV/HDV infection and inhibits NTCP-mediated bile acid uptake. In humans this increases systemic bile acid levels, which remain elevated for hours even after Myrcludex B is cleared from the circulation. Here, we investigated the dynamics of Myrcludex B-induced NTCP-mediated bile acid transport inhibition in mice and if/how the duration of this effect relates to NTCP protein turnover.MethodsPlasma bile acids were determined in Myrcludex B-treated OATP1a/1b-deficient mice. In vitro, plasma membrane-resident NTCP was labeled with biotin or fluorescein isothiocyanate (FITC)-labeled Myrcludex B and traced in time using hNTCP-overexpressing U2OS cells. Förster resonance energy transfer by fluorescent lifetime imaging microscopy was used to investigate whether Myrcludex B can transfer to newly synthesized NTCP.ResultsConjugated bile salt levels in plasma peaked 4 h after subcutaneous Myrcludex B administration. After 24 h, plasma bile salt levels were completely normalized, in line with restored NTCP-mediated bile acid transport in vitro. Biotin-labeled NTCP disappeared faster than Myrcludex B-FITC, with almost 40% of FITC signal remaining after 24 h. FITC fluorescence lifetime was strongly decreased upon expression of DY547-labeled acyl carrier protein-tagged NTCP, demonstrating transfer of pre-bound Myrcludex B-FITC to newly formed NTCP.ConclusionsThe dynamics of NTCP protein turnover and Myrcludex B-induced plasma bile salt elevations are similar, suggesting that the Myrcludex B:NTCP interaction is very long-lived. Nevertheless, Myrcludex B is not completely degraded together with NTCP and can transfer to newly synthesized NTCP.Lay summaryThe experimental drug Myrcludex B binds the sodium taurocholate co-transporting polypeptide (NTCP), the viral entry receptor for the hepatitis B and D virus (HBV/HDV), and thereby prevents infection, but also inhibits hepatic bile salt uptake leading to transiently elevated bile salt levels. This study describes that while the normalization of plasma bile salt levels likely depends on the protein turnover rate of NTCP, Myrcludex B partly escapes co-degradation with NTCP by transferring from one NTCP molecule to another. This is of importance to the HBV/HDV research field as it provides a potential explanation for the distinct kinetics and dose-dependence of Myrcludex B’s effects on viral infection versus bile salt transport.

Ultrasensitive fluorescence detection of Fe3+ ions using fluorescein isothiocyanate functionalized Ag/SiO2/SiO2 core–shell nanocomposites

Fluorescein isothiocyanate (FITC) functionalized Ag/SiO2/SiO2 core–shell (CS) nanoparticles (NPs) and nanorods (NRs) are synthesized by chemical method to perform the fluorescence detection of Fe3+ in aqueous medium. The Fe3+ ion sensing is performed on the basis of metal-enhanced fluorescence and subsequent fluorescence quenching of fluorophore (fluorescein isothiocyanate) in aqueous medium. The intensity of fluorescence of FITC is found to get enhanced with increasing the silica shell thickness. The fluorescence intensity of FITC of spherical Ag/SiO2/SiO2 (FITC) CS NPs (AgSiF50) is amplified up to 2.25-folds whereas, in case of Ag/SiO2/SiO2 (FITC) CS NRs (AgSiFNR), the fluorescence intensity of FITC is amplified up to 3.63-folds compared to that of pure FITC. Owing to the amplified fluorescence of FITC, AgSiF50 and AgSiFNR samples are evaluated for detection of Fe3+ in an aqueous medium and the proposed nanoprobes exhibit good sensitivity towards Fe3+ detection because of strong binding affinity of Fe3+ with isothiocyanate group of FITC. The limit of detections for AgSiF50 and AgSiFNR are found to be 19.4 nM and 0.8 nM, respectively. The BET surface areas of AgSiF50 and AgSiFNR are calculated to be 157.64 m2 g−1 and 193.42 m2 g−1, respectively. The higher sensitivity of AgSiFNR compared to AgSiF50 for Fe3+ ion sensing is accredited to the higher adsorption capacity and higher fluorescence of FITC. This Ag/SiO2/SiO2 (FITC) CS NRs probe is a very simple and efficient sensor for Fe3+ ion detection and thus, using this sample, a very simple Arduino based electronic device is fabricated which can give real-time application of the idea.

An efficient method for FITC labelling of proteins using tandem affinity purification.

Fluorescence-based assays are extremely diverse, sensitive and robust experimental methods for investigating the conformational changes, enzyme kinetics, dynamics and molecular interactions. A prerequisite for most of these experimental approaches is to label the protein of interest with one or more extrinsic fluorophores with desired photophysical properties. Fluorescein isothiocyanate (FITC), due to its high quantum efficiency and conjugate stability, is most widely used fluorescence labelling reagent for such experimental approaches. However, the bottlenecks in this labelling reaction is requirement of high protein concentration, maintenance of protein stability during the labelling process as well as high background fluorescence due to ineffective removal of unreacted FITC, prior to fluorescence studies. Therefore, to overcome these inadequacies or limitations, we have modified the existing protocol by introducing tandem affinity purification tags at the N- and C-terminus of target protein. Using this modified method, we have efficiently labelled target protein with significant decrease in precipitation, degradation and background fluorescence of unreacted FITC. This facile and rapid technique may also be used as a basis for labelling procedures with other fluorophores and hence has a broad application in spectroscopic studies.

Aspartate Beta-Hydroxylase (ASPH) As a Novel Therapeutic Target in Acute Myeloid Leukemia

IntroductionDespite recent advances in treatment, acute myeloid leukemia (AML) remains difficult to cure with high rates of relapse. Relapsed/refractory AML patients who are elderly or unfit for cytotoxic chemotherapy and whose disease fails to respond to hypomethylating agents represent an unmet need, and new safe and effective treatment options are needed for this patient population. Aspartate β-hydroxylase (ASPH) is a transmembrane protein that hydroxylates aspartyl and asparaginyl residues of epidermal growth factor (EGF)-like protein domains, and promotes cellular motility, migration, and adhesion. ASPH is highly expressed during fetal development and in placental trophoblasts, but not in any other healthy adult human tissue. ASPH is uniquely upregulated in cancer cells and is reported to be overexpressed in over 20 different solid neoplasms, in which it propagates a malignant phenotype, and is associated with increased cell proliferation, invasiveness, and poor prognosis. ASPH is therapeutically targetable via a fully human monoclonal antibody against ASPH, SNS-622. Subsequently, SNS-622 antibody-drug conjugates (ADCs) and a vaccine have been developed and are under current preclinical and clinical testing, respectively. We have previously demonstrated ASPH overexpression on the MOLM-14 AML cell line and effective in vitro killing of these cells using SNS-622 ADCs. In an effort to further characterize ASPH as a therapeutic target in AML, we report here the first study of ASPH expression patterns in AML patient samples.MethodsBone marrow (BM) aspirate and peripheral blood (PB) samples were collected from AML patients during 2014-2018 on a University of Maryland Greenebaum Comprehensive Cancer Center (UMGCCC) tissue banking protocol. Mononuclear cells were isolated by density centrifugation and were viably cryopreserved at -80ᵒC. Samples were analyzed using 8-color multiparameter flow cytometry to assess cell surface expression of ASPH, using fluorescein isothiocyanate (FITC)-conjugated SNS-622. The remainder of the fluorophores were labeled with standard lineage-specific markers. Expression of ASPH was measured via FITC fluorescence for each cell population and analyzed by two blinded, independent reviewers using FlowLogicTM software. Statistical analysis of ASPH expression was conducted using IBM® SPSS® Statistics for Windows, release 25.0.0 (IBM Corp., Armonk, N.Y., USA). Cohen's kappa (κ) was used to quantify the inter-observer agreement between two independent observers. Visual inspection of the expression data for the whole patient population showed a bimodal distribution, which was used to identify a robust cut-point separating high (i.e. positive) from low (i.e. negative) ASPH expression.ResultsForty-two AML patient samples were evaluated (32 BM, 10 PB). Median patient age was 66 years, 48% (n=20) were female, 69% Caucasian and 21% African American. Disease status was 52% untreated de novo, 19% untreated secondary from antecedent MDS or MPN, and 29% relapsed/refractory. Samples were cytogenetically and molecularly diverse - with 50% exhibiting normal and 14% complex karyotype; 38% FLT3-ITD or FLT3-TKD, 29% NPM1, 19% IDH1/2, and 10% TP53 mutations. Full patient characteristics are shown in Table 1.Myeloblast expression of ASPH was found in 38% of samples (n=16; 14 BM, 2 PB) with a mean fluorescent intensity (MFI) of 10 as a cutoff for ASPH surface expression positivity. ASPH expression was not seen on other non-neoplastic cells including CD34+ hematopoietic stem cells, B- or T-lymphocytes, and monocytes. A blinded, independent review of the data revealed a Cohen's kappa (κ) of 0.74 with standard error of the estimate of ± 0.11. Patients with AML with ASPH expression were clinically heterogeneous, with no correlation between ASPH expression and AML subtype, karyotype or mutation status (Table 1).ConclusionASPH is overexpressed in approximately 40% of patients with AML and serves as a promising therapeutic target. An ASPH nanoparticle vaccine is currently under clinical investigation and has shown promising results in solid tumors. We plan to expand clinical testing of targeting ASPH to AML. The ASPH positivity cutoff established via this work will serve as the eligibility criterion for the planned phase Ib/IIa of anti-ASPH vaccination in AML.. DisclosuresLebowitz:Sensei Biotherapeutics: Employment. Malhotra:Sensei Biotherapeutics: Employment. Fuller:Sensei Biotherapeutics: Employment. Shahlaee:Sensei Biotherapeutics: Equity Ownership; Sensei Biotherapeutics: Consultancy. Ghanbari:Sensei Biotherapeutics: Employment; Sensei Biotherapeutics: Equity Ownership. Emadi:NewLink Genetics: Research Funding.

A black phosphorus/manganese dioxide nanoplatform: Oxygen self-supply monitoring, photodynamic therapy enhancement and feedback

Selecting the timing of laser treatment is an important task for improving O2-dependent photodynamic therapy (PDT) efficiency. Here, a black phosphorus-based strategy was developed for dual-mode monitoring oxygen self-supply, enhancing photodynamic therapy, and feeding back therapeutic effect. The hybridized nanoplatform (R-MnO2-FBP) was prepared by assembly of Rhodamine B (RhB)-encapsulated manganese dioxide (R-MnO2) as O2 supplier and indicator, and fluorescein isothiocyanate (FITC)-labelled peptide-functionalized black phosphorus as the theranostic agent. The time-dependent assays suggested that the O2 release was proportional to the liberation of Mn2+ and RhB in the R-MnO2-FBP system. After specific delivery into cancer cells, R-MnO2-FBP was dissociated in the acidic and H2O2-rich environment and generated oxygen to overcome hypoxia-associated PDT resistance. In the meantime, it released both Mn2+and RhB dye, leading to dual-mode (magnetic resonance imaging/fluorescence imaging) monitoring of the oxygen self-supply process. More significantly, the imaging-guided PDT in hypoxic cells displayed 51.6% of cell apoptosis at optimizing timing of laser application, which could also be confirmed by the FITC fluorescence recovery induced by the activated caspase-3 in apoptotic cells. In vivo photonic therapy by R-MnO2-FBP further demonstrated the ability of R-MnO2-FBP to choose the timing of laser application, providing an efficient approach for the enhancement of PDT process.