Construction of a New Probe Based on Copper Chaperone Protein for Detecting Cu2+ in Cells.

Biomacromolecular probes have been extensively employed in the detection of metal ions for their prominent biocompatibility, water solubility, high selectivity, and easy modification of fluorescent groups. In this study, a fluorescent probe FP was constructed. The probe FP exhibited high specificity recognition for Cu2+. With the combination of Cu2+, the probe was subjected to fluorescence quenching. The research suggested that the probe FP carried out the highly sensitive detection of Cu2+ with detection limits of 1.7 nM. The fluorescence quenching of fluorescamine was induced by Cu2+ perhaps due to the PET (photoinduced electron transfer) mechanism. The FP-Cu2+ complex shows weak fluorescence, which is likely due to the PET quenching effect from Cu2+ to fluorescamine fluorophore. Moreover, the probe FP can be employed for imaging Cu2+ in living cells. The new fluorescent probe developed in this study shows the advantages of good biocompatibility and low cytotoxicity. It can be adopted for the targeted detection of Cu2+ in cells, and it has promising applications in the mechanism research and diagnosis of Cu2+-associated diseases.

Applicability of fluorescamine as a fluorogenic reagent for highly sensitive fluorimetric analysis of the tyrosine kinase inhibitor (avapritinib) in pharmaceuticals and biological samples.

Avapritinib (AVP) was the first precision drug to be approved by the US Food and Drug Administration (FDA) in 2020 for patients suffering from metastatic gastrointestinal stromal tumors (GISTs) and progressive systemic mastocytosis. The analysis of AVP in pharmaceutical tablets and human plasma was then carried out using a fast, efficient, sensitive, and simple fluorimetric method using a fluorescamine reagent. The procedure is based on the interaction between fluorescamine as a fluorogenic reagent and the primary aliphatic amine moiety in AVP using borate buffer solution at pH 8.8. The produced fluorescence was measured at 465 nm (Excitation at 395 nm). The calibration graph's linearity range was discovered to be 45.00-500.0 ng mL-1 . Utilizing the International Council for Harmonization (ICH) and US-FDA recommendations, the research technique was validated and bioanalytically validated. The proposed approach was effectively employed for determining the stated pharmaceuticals in plasma with a high percentage of recovery ranging from 96.87 to 98.09 and pharmaceutical formulations with a percentage of recovery equal to 102.11% ± 1.05%. In addition, the study was extended to a pharmacokinetic study of AVP with 20 human volunteers as a step for AVP management in therapeutic cancer centers.

Salvage parenteral antibiotics for multidrug-resistant (MDR) Gram-negative bacteria; a fluorescamine-based technique for ultrasensitive spectrofluorimetric measurement of Polymyxins; human plasma application.

Polymyxins (PMS), namely Colistin (CS) and polymyxin B (poly B), are antimicrobial drugs that have been recently used to treat multiresistant Gram-negative bacteria infections and their resurgence, owing to a lack of new antibiotics. A speedy, simple, and ultrasensitive spectrofluorimetric screening of PMS in pharmaceutical formulations and biological fluids was urgently required from this point forwards. A reaction between fluorescamine and the aliphatic amino moiety found in both drugs was performed in a slightly alkaline borate buffer (pH 8.5) resulted in highly fluorescent products measured at λem 460 (after λex 390.5 nm). Linear calibration curves were constructed over the concentration range 70-1800 ng ml-1 and 100 to 1400 ng ml-1 , with slope values of 0.273 and 0.286, correlation coefficients of 0.9998 and 0.9997, and determination coefficient of 0.9997 and 0.9994 for poly B and CS, respectively. The ultrasensitivity of the proposed method was demonstrated by the very low limit of quantification values of 67.56 ng ml-1 and 94.89 ng ml-1 for poly B and CS, respectively. The cited drugs were successfully determined in their intravenous market preparations by the prescribed method. Moreover, due to the high sensitivity, the suggested method was used to assay the investigated drugs in biological fluids.

In Vivo Femtosecond Laser Subsurface Cortical Microtransections Attenuate Acute Rat Focal Seizures.

Recent evidence shows that seizures propagate primarily through supragranular cortical layers. To selectively modify these circuits, we developed a new technique using tightly focused, femtosecond infrared laser pulses to make as small as ~100 µm-wide subsurface cortical incisions surrounding an epileptic focus. We use this "laser scalpel" to produce subsurface cortical incisions selectively to supragranular layers surrounding an epileptic focus in an acute rodent seizure model. Compared with sham animals, these microtransections completely blocked seizure initiation and propagation in 1/3 of all animals. In the remaining animals, seizure frequency was reduced by 2/3 and seizure propagation reduced by 1/3. In those seizures that still propagated, it was delayed and reduced in amplitude. When the recording electrode was inside the partially isolated cube and the seizure focus was on the outside, the results were even more striking. In spite of these microtransections, somatosensory responses to tail stimulation were maintained but with reduced amplitude. Our data show that just a single enclosing wall of laser cuts limited to supragranular layers led to a significant reduction in seizure initiation and propagation with preserved cortical function. Modification of this concept may be a useful treatment for human epilepsy.

Spectrofluorometric determination of alogliptin an antidiabetic drug in pure and tablet form using fluorescamine, a fluorogenic agent: application to content uniformity test.

Alogliptin is an antidiabetic drug that belongs to a group called dipeptidyl peptidase-4 enzyme inhibitors. As the drug contains a primary amino group in its structure, it readily reacts with fluorescamine in slightly alkaline medium (borate buffer, pH 8.8) to form a highly fluorescent product. Emission of this product was measured at 477 nm (λex = 387 nm). The linear range between the fluorescence intensity and the drug concentration was 0.1-0.5 µg ml-1 with a good correlation coefficient (0.9986). Limits of detection and quantitation were 22 and 72 ng ml-1 , respectively. Guidelines of the International Conference for Harmonisation were followed to validate the developed method with acceptable results. Alogliptin content was determined successfully in its commercial dosage form using the fluorescamine method with good recovery (98.60-101.26%). The method has excellent levels of accuracy and precision compared with the reported method as assessed using Student's t-test and Fisher's exact test. The method was applied successfully for the content uniformity test with high recovery and low relative standard deviation.

Optimisation of a high-throughput fluorescamine assay for detection of N-acyl-l-homoserine lactone acylase activity.

N-acyl-l-homoserine lactone (AHL) acylases are a well-known group of enzymes that disrupt quorum sensing in Gram-negative bacteria by degrading AHL signalling molecules. This degradation of signalling molecules (termed 'quorum quenching') has potential uses in the prevention or reduction of biofilm formation and/or bacterial infections. Therefore, there is a great deal of interest in the identification and characterisation of quorum quenching enzymes. Here, we present an optimised fluorescamine-based assay for the detection of AHL acylase activity and demonstrate it can be used in a high-throughput screening format.

The convenient use of fluorescamine for spectrofluorimetric determination of midodrine hydrochloride in pure form and its tablets formulation: Application to content uniformity testing.

A novel sensitive and simple spectrofluorimetric method was developed then validated for determination of midodrine in both its authentic pure form and its tablets. This method is based on the reaction between midodrine's aliphatic primary amine moiety with fluorescamine reagent, using borate buffer at pH 7.8 and yielding a highly fluorescent product whose fluorescence intensity was measured at 462 nm after excitation at 388 nm. This method represents the first attempt for determination of midodrine spectrofluorimetrically. A calibration curve was constructed showing that the linear range was 0.2-3.0 µg/ml. The limit of detection and limit of quantitation values were 0.06 and 0.19 µg/ml respectively. The correlation coefficient (r) and the determination coefficient (r2 ) values were 0.9992 and 0.9984 respectively. The proposed method was validated according to ICH guidelines and successfully applied for determination of midodrine in its tablets with an overall % recovery of 99.56 ± 0.95. Finally, the presented method was adapted to study the content uniformity test according to United States Pharmacopeia guidelines.

Determination of Sulfonamides in Feeds by High-Performance Liquid Chromatography after Fluorescamine Precolumn Derivatization.

A new multi-residue method for the analysis of sulfonamides (sulfadiazine, sulfamerazine, sulfamethazine, sulfaguanidine and sulfamethoxazole) in non-target feeds using high-performance liquid chromatography-fluorescence detection (HPLC-FLD) and precolumnderivatization was developed and validated. Sulfonamides (SAs) were extracted from feed with an ethyl acetate/methanol/acetonitrile mixture. Clean-up was performed on a Strata-SCX cartridge. The HPLC separation was performed on a Zorbax Eclipse XDB C18 column with a gradient mobile phase system of acetic acid, methanol, and acetonitrile. The method was validated according to EU requirements (Commission Decision 2002/657/EC). Linearity, decision limit, detection capability, detection and quantification limits, recovery, precision, and selectivity were determined, and adequate results were obtained. Using the HPLC-FLD method, recoveries were satisfactory (79.3⁻114.0%), with repeatability and reproducibility in the range of 2.7⁻9.1% to 5.9⁻14.9%, respectively. Decision limit (CCα) and detection capability (CCß) were 197.7⁻274.6 and 263.2⁻337.9 µg/kg, respectively, and limit of detection (LOD) and limit of quantification (LOQ) were 34.5⁻79.5 and 41.3⁻89.9 µg/kg, respectively, depending on the analyte. Results showed that this analytical procedure is simple, rapid, sensitive, and suitable for the routine control of feeds.

[Determination of Nonvolatile Amines in Foods by HPLC Following Fluorescamine Derivatization].

A method was developed for the determination of nonvolatile amines, such as histamine, tyramine, putrescine, and cadaverine, in foods. These nonvolatile amines were extracted from a sample with 5% trichloroacetic acid, and the extract was purified using an InertSep MC-1 cartridge column. The four amines were derivatized with fluorescamine, determined by HPLC with a fluorescence detector, and confirmed by LC-MS/MS. The average recoveries (n=5) and the relative standard deviations from 11 foods (pacific saury, dried mackerel, canned mackerel in brine, canned tuna in oil, fish sauce, surimi, rice-koji miso, soy sauce, gouda cheese, red wine, and beer) spiked at 100 mg/kg were 81-100% and 0.4-3.1%, respectively.

In-capillary derivatization with fluorescamine for the rapid determination of adamantane drugs by capillary electrophoresis with UV detection.

In-capillary derivatization using fluorescamine as the labeling reagent was proposed to enhance the detectability of adamantine drugs (memantine, amantadine and rimantadine) by spectrophotometric detection. Fluorescamine and the drugs were delivered to the capillary electrophoresis instrument at a ratio of 10:1 by zone injection. The derivatization reaction occurred following the application of voltage (20 kV). The derivatized products, hydrolyzed- fluorescamine and excess fluorescamine were separated in 7 min using 100 mM borate buffer (pH 10.0) containing 0.1% w/v of Brij®-35 and 20% v/v of acetonitrile. Validation data showed good linearity (r2  > 0.98), precision (%RSDs < 3.4), and accuracy (recoveries ranging from 98.0 to 102.0%). The detection and quantitation limits are in the range of 6.0-8.5 and 18-25 µM, respectively. The validation data is comparable to reported methods, however, the current method offers better precision with enhanced sensitivity (up to six times). Applications of the method show percent labeled amounts found in the studied samples within 100.6-109.3%, which complied with the United States Pharmacopeia limit (90.0-110.0%). The method was simple, rapid and, automated, which required no extra instrumentation or skillful operators.

[Determination of Histamine by a Rapid Fluorescamine-UPLC Method].

We have developed a rapid analysis method for determination of histamine using ultra-performance liquid chromatography and fluorescamine derivatization. Histamine was extracted from foods with trichloroacetic acid after homogenization. The supernatant obtained by centrifugation was filtered and derivatized without solid-phase extraction. The mobile phase was 10 mM phosphate-acetonitrile (75 : 25) without ion-pairing. Recovery tests of histamine from fishes, seasoning and fish products gave acceptable recovery (95.8-117.7%). This method should be useful for rapid evaluation of food poisoning outbreaks.

Fluorescamine Labeling for Assessment of Protein Conformational Change and Binding Affinity in Protein-Nanoparticle Interaction.

Protein adsorption alters the "biological identity" of nanoparticles (NPs) and could affect how biosystems respond to invading NPs. Study of protein-NP interaction can help understand how the physicochemical properties of NPs impact the interaction and thus potentially guide the design of safer and more effective NPs for biomedical or other applications. Binding affinity between proteins and NPs and the occurrence of protein conformational change upon binding to NPs are two important aspects to be learned, but few methods are currently available to assess both simultaneously in a simple way. Herein, we demonstrated that the fluorescamine labeling method developed by our group not only could reveal protein conformational change upon adsorption to NPs, owing to its capability to label the primary amines exposed on protein surface, but also could be applied to measure the binding affinity. By screening the interaction between a large number of proteins and four types of NPs, the present study also revealed that protein adsorption onto NPs could be strongly affected by structure flexibility. The proteins with high structure flexibility experienced high degrees of conformation change when binding to the polystyrene NPs, which could potentially influence protein function. Overall, we demonstrate that our assay is a quick, simple, and high-throughput tool to reveal potential impacts on protein activity and evaluate the strength of protein-NP binding.

Phase Transfer and Surface Functionalization of Hydrophobic Nanoparticle using Amphiphilic Poly(amino acid).

Functionalization of nanoparticles with chemical and biochemical is essential for their biomedical and other application. However, most of the high quality nanoparticles are hydrophobic in nature due to surfactant capping and their conversion into water-soluble functional nanoparticle via appropriate coating and conjugation chemistry is extremely critical issue. Here we report amphiphilic poly(amino acid)-based one-pot coating and conjugation approach that can transform hydrophobic nanoparticle into water-soluble nanoparticle functionalized with primary amine, thiol, and biomolecule. We have designed amphiphilic polyaspartimide that can anchor hydrophobic nanoparticle through octadecyl groups, leaving the polar polyethylene glycol and aspartimide groups exposed outwards. The aspartimide group is then reacted with primary amine containing chemical/biomolecule with the formation of water-soluble functional nanoparticle. This approach has been extended to different hydrophobic nanoparticles and biomolecules. The present approach has advantages over existing approaches as coating and functionalization can be performed in one pot and functional nanoparticles have <12 nm hydrodynamic size, high colloidal stability, and biocompartibility. This developed approach can be used to derive biocompatible nanobioconjugates for various biomedical applications.

High-throughput profiling of nanoparticle-protein interactions by fluorescamine labeling.

A rapid, high throughput fluorescence assay was designed to screen interactions between proteins and nanoparticles. The assay employs fluorescamine, a primary-amine specific fluorogenic dye, to label proteins. Because fluorescamine could specifically target the surface amines on proteins, a conformational change of the protein upon interaction with nanoparticles will result in a change in fluorescence. In the present study, the assay was applied to test the interactions between a selection of proteins and nanoparticles made of polystyrene, silica, or iron oxide. The particles were also different in their hydrodynamic diameter, synthesis procedure, or surface modification. Significant labeling differences were detected when the same protein incubated with different particles. Principal component analysis (PCA) on the collected fluorescence profiles revealed clear grouping effects of the particles based on their properties. The results prove that fluorescamine labeling is capable of detecting protein-nanoparticle interactions, and the resulting fluorescence profile is sensitive to differences in nanoparticle's physical properties. The assay can be carried out in a high-throughput manner, and is rapid with low operation cost. Thus, it is well suited for evaluating interactions between a larger number of proteins and nanoparticles. Such assessment can help to improve our understanding on the molecular basis that governs the biological behaviors of nanomaterials. It will also be useful for initial examination of the bioactivity and reproducibility of nanomaterials employed in biomedical fields.

Chitosan coated polylactic acid nanoparticle-mediated combinatorial delivery of cisplatin and siRNA/Plasmid DNA chemosensitizes cisplatin-resistant human ovarian cancer cells.

Development of resistance toward anticancer drugs results in ineffective therapy leading to increased mortality. Therefore, overriding resistance and restoring sensitivity to anticancer drugs will improve treatment efficacy and reduce mortality. While numerous mechanisms for drug resistance in cancer have previously been demonstrated, recent studies implicate a role for proteasome and the autophagy regulatory protein P62/SQSTM1 (P62) in contributing to drug resistance. Specifically, reduction in the expression of the ß5 subunit of the proteasome and/or enhanced P62 protein expression is known to contribute to cancer drug resistance such as cisplatin (CDDP) in ovarian cancer cells. Therefore, we hypothesized that restoration of ß5 expression and/or suppression of P62 protein expression in CDDP-resistant ovarian cancer cells will lead to restoration of sensitivity to CDDP and enhanced cell killing. To test our hypothesis we developed a biodegradable multifunctional nanoparticle (MNP) system that codelivered P62siRNA, ß5 plasmid DNA, and CDDP and tested its efficacy in CDDP resistant 2008/C13 ovarian cancer cells. MNP consisted of CDDP loaded polylactic acid nanoparticle as inner core and cationic chitosan (CS) consisting of ionically linked P62siRNA (siP62) and/or ß5 expressing plasmid DNA (pß5) as the outer layer. The MNPs were spherical in shape with a hydrodynamic diameter in the range of 280-350 nm, and demonstrated encapsulation efficiencies of 82% and 78.5% for CDDP and siRNA respectively. MNPs efficiently protected the siRNA and showed superior serum stability compared to naked siRNA as measured by gel retardation and spectrophotometry assays. The MNPs successfully delivered siP62 and pß5 to cause P62 knockdown and restoration of ß5 expression in 2008/C13 cells. Combined delivery of siP62, pß5, and CDDP using the MNPs resulted in a marked reduction in the IC50 value of CDDP in 2008/C13 cells from 125 ± 1.3 µM to 98 ± 0.6 µM (P < 0.05; 21.6% reduction) when compared to the reduction in the IC50 of CDDP observed in cells that had only siP62 delivered (IC50 = 106 ± 1.1 µM; P < 0.05; 15.2% reduction) or pß5 delivered (IC50 = 115 ± 2.8 µM; 8% reduction) via MNPs. Finally, our studies showed that the CDDP resistance index in 2008/C13 cells was reduced from 4.62 for free CDDP to 3.62 for MNP treatment. In conclusion our study results demonstrated the efficacy of our MNP in overcoming CDDP resistance in ovarian cancer cells.

Titration of lysine residues on adenine nucleotide translocase by fluorescamine induces permeability transition.

Chemical modification of primary amino groups of mitochondrial membrane proteins by the fluorescent probe fluorescamine induces non-specific membrane permeabilisation. Titration of the lysine ϵ-amino group promoted efflux of accumulated Ca(2+), collapse of transmembrane potential and mitochondrial swelling. Ca(2+) release was inhibited by cyclosporin A. Considering the latter, we assumed that fluorescamine induces permeability transition. Carboxyatractyloside also inhibited the reaction. Using a polyclonal antibody for adenine nucleotide translocase, Western blot analysis showed that the carrier appeared labelled with the fluorescent probe. The results point out the importance of the ϵ-amino group of lysine residues, located in the adenine nucleotide carrier, on the modulation of membrane permeability, since its blockage suffices to promote opening of the non-specific nanopore.

Spectrofluorimetric determination of tobramycin in human serum and pharmaceutical preparations by derivatization with fluorescamine.

A simple, sensitive and selective spectrofluorimetric method has been developed for the determination of tobramycin (TOB) in human serum and pharmaceutical preparations. The method is based on the reaction between the primary amino group of TOB and fluorescamine in borate buffer, pH 8.5, to give a highly fluorescent derivative which is measured at 469 nm after excitation at 388 nm. The fluorescence intensity was directly proportional to the concentration over the range 300-1500 ng/mL, with a limit of detection of 65 ng/mL and limit of quantitation of 215 ng/mL. All variables were investigated to optimize the reaction conditions. The method was validated according to International Conference on Harmonization guidelines in terms of specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. Good recoveries were obtained ranging from 97.4 to 100.64%, indicating that no interference was observed from concomitants usually present in pharmaceutical dosage forms. The method was successfully, applied for the analysis of the drug substance in its pharmaceutical preparations and spiked serum samples.

A lab-on-a-chip device for analysis of amlodipine in biological fluids using peroxyoxalate chemiluminescence system.

A highly sensitive, rapid and economical method for the determination of amlodipine (AM) in biological fluids was developed using a peroxyoxalate chemiluminescence (CL) system in a lab-on-a-chip device. Peroxyoxalate-CL is an indirect type of CL that allows the detection of native fluorophores or compounds derivatized with fluorescent labels. Here, fluorescamine was reacted with AM, and the derivatization product was used in a bis-(2,4,6-trichlorophenyl)oxalate-CL system. Fluorescamine reacts selectively with aliphatic primary amine at neutral or basic pH. As most of the calcium channel blocker and many cardiovascular drugs do not contain primary amine, the developed method is highly selective. The parameters that influenced the CL signal intensity were studied carefully. These included the chip geometry, pH, concentration of reagents used and flow rates. Moreover, we confirmed our previous observation about the effects of imidazole, which is commonly used in the bis-(2,4,6-trichlorophenyl)oxalate-CL system as a catalyst, and found that the signal was significantly improved when imidazole was absent. Under optimized conditions, a calibration curve was obtained with a linear range (10-100 µg/L). The limit of detection was 3 µg/L, while the limit of quantification was 10 µg/L. Finally the method was applied for the determination of AM in biological fluids successfully.

A novel spectrofluorimetric method for the determination of calcitonin in ampules through derivatization with fluorescamine.

In this study, a simple, sensitive and selective spectroflourimetric method has been developed for the determination of salmon calcitonin (sCT) in ampules. The method is based on the reaction between sCT and fluorescamine at pH 8.5 in borate buffer, resulting in a highly fluorescent derivative. Fluorescence of derivatized sCT solutions was measured by setting the excitation and emission monochromators and slit widths to 390, 484 and 10 nm, respectively. Sevaral derivatization parameters were optimized. A calibration graph was constructed using standard solutions of the derivatized calcitonin in the range 0.5-6.0 µg/mL. Limit of detection and limit of quantification values were determined to be 0.124 and 0.372 µg/mL, respectively. The proposed method was successfully applied for the determination of sCT in commercially available ampules. High recovery values (101.0-102.0 %), and a low relative standard deviation (RSD %) value (5.3-5.4) proved the accuracy and precision of the proposed method. An isocratic reversed-phase high-performance liquid chromatographic (HPLC) method, as a reference, was also developed for the determination of sCT. A reversed-phase Nucleosil® C18 column (250 mm × 4.6 mm i.d., 10 µm particle size, 120 Å pore size) was used and the detector was set at 210 nm. Statistical comparison of the results of the two methods showed clearly that there was no significant difference between them.

A simple high-throughput method for determination of antiepileptic analogues of γ-aminobutyric acid in pharmaceutical dosage forms using microplate fluorescence reader.

Pregabalin (PGB), gabapentin (GBP), and vigabatrin (VGB) are structural analogues of γ-aminobutyric acid used for the treatment of different forms of epilepsy. Their analytical determination is challenging since these molecules have no significant UV or visible absorption. Several derivatization methods have been developed and used for their determination in bulk or pharmaceutical dosage forms. We aimed to develop a high- throughput method using a microplate reader with fluorescence detection and simple derivatization with fluorescamine. Obtained method involves derivatization step of only 5 min at room temperature and simultaneous measurements of 96 samples (λex 395, λem 476 nm) thus rendering excellent high-throughput analysis. The method was found to be linear with r²>0.998 across investigated analytical ranges of 0.75 to 30.0 µg/mL for PGB, 2.00 to 80.0 µg/mL for GBP, and 1.50 to 60.0 µg/mL for VGB. Intraday and interday precision values did not exceed 4.93%. The accuracy was ranging between 96.6 to 103.5%. The method was also found to be specific since used excipients did not interfere with the method. The robustness study showed that derivatization procedure is more robust than spectrofluorimetric conditions. The developed high-throughput method was successfully applied for determination of drug content and dissolution profiles in pharmaceutical dosage forms of studied antiepileptic drugs.