ABSTRACT
Pesticide contamination is a global concern, threatening human health and food safety. Herein, we developed heparin (HEP) functionalized upconversion nanoparticles (UCNPs)-based ratiometric nanosensor for the sensitive detection of 2,6-dichloro-4-nitroaniline (DCN) pesticide via inner filter effect. The strategy for HEP functionalization of UCNPs is based on adjusting the surface potentials of UCNPs with polyanionic HEP through the electrostatic interaction. UCNPs (NaYbF4:Gd/Y/Tm@NaYbF4@NaYF4) was designed with core-shell-shell structure and extra sensitizer layer for efficient and strong upconversion luminescence (UCL) in the range of UV to NIR. After incorporation of DCN, the upconverted UV emission of UCNPs-HEP ratiometric nanosensor was considerably quenched with the NIR UCL at 800 nm remaining unchanged as internal standard. The UCNPs-HEP ratiometric nanosensor can achieve outstandingly selective and sensitive detection of DCN at the wide linear range of 5-300 µM with a detection limit of 0.41 µM. The remarkable applicability of the UCNPs-HEP ratiometric nanosensor was verified in apple, cucumber and grapes samples. The developed UCNPs-HEP ratiometric nanosensor with excellent biocompatibility and water dispersion capability, is promising for convenient, selective and sensitive sensing of DCN towards food and aqueous samples.
Subject(s)
Heparin , Nanoparticles , Pesticides , Polyelectrolytes , Nanoparticles/chemistry , Pesticides/analysis , Heparin/analysis , Heparin/chemistry , Polyelectrolytes/chemistry , Polymers/chemistry , Limit of Detection , Polysaccharides/chemistry , Polysaccharides/analysisABSTRACT
Heparin is a highly negatively charged sulfated linear polymer glycosaminoglycan that has been widely used as an anticoagulant in medicine. Protamine is a cationic protein rich in arginine that is used to treat the blood-brain barrier during excess heparin surgery. Trypsin is the most important digestive enzyme-encoding generated by the pancreas and can specifically cleave the carboxyl ends of arginine and lysine residues. Heparin, protamine, and trypsin interact and constrain each other, and their fluctuations reflect the body's dysfunction. Therefore, it is necessary to develop a fast, sensitive, and highly selective assay for regularly monitoring the levels of heparin, protamine, and trypsin in serum. Herein, a fluorescent and colorimetric dual-mode upconversion nanoparticle (UCNP) biosensor was used for the determination of heparin, protamine, and trypsin based on the oxidase-mimicking activity of Ce4+ and electrostatic control. The biosensor exhibited sensitive detection of heparin, protamine, and trypsin with low limits of detection (LODs) of 16 ng/mL, 87 ng/mL and 31 ng/mL, respectively. Furthermore, the designed biosensor could eliminate autofluorescence, which not only effectively increased the accuracy of the sensor but also provided a new sensing pathway for the detection of differently charged biotargets.
Subject(s)
Biosensing Techniques , Heparin , Protamines , Static Electricity , Trypsin , Protamines/chemistry , Protamines/metabolism , Biosensing Techniques/methods , Heparin/chemistry , Heparin/metabolism , Heparin/analysis , Trypsin/metabolism , Trypsin/chemistry , Nanoparticles/chemistry , Humans , Limit of Detection , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Colorimetry/methods , Spectrometry, Fluorescence/methodsABSTRACT
In this strategy, the fluorescence sensor Nap-Co-T1 employing the fluorescence resonance energy transfer (FRET) mechanism was designed and synthesized to have an efficient response to Heparin, and the FRET mechanism was explored for different excitation-emission wavelengths with different distances between the energy acceptor and the energy donor (comparing with fluorescence sensor Nap-TPA-T2). Upon the addition of Heparin, the fluorescence emission of Nap-Co-T1 was turned on at 565 nm, and the fluorescence color changed of the solution from colorless to bright yellow. The limit of detection (LOD) was as low as 0.04 µg/mL. With the addition of antagonistic protamine (PRTM) to the sensor complex with Heparin, the fluorescence emission was turned off to a certain extent, and the reversibility of the "off-on-off" system was maintained for five cycles or more. In addition, Nap-Co-T1 provides rapid and sensitive detection of Heparin in human serum albumin solution and artificial urine and is highly sensitive to environmental viscosity.
Subject(s)
Fluorescence Resonance Energy Transfer , Heparin , Limit of Detection , Heparin/analysis , Heparin/chemistry , Fluorescence Resonance Energy Transfer/methods , Humans , Fluorescent Dyes/chemistry , Protamines/analysis , Protamines/chemistry , Spectrometry, Fluorescence/methodsABSTRACT
BACKGROUND: Infant formulas, and pediatric and adult nutritional products, are being fortified with bovine lactoferrin (bLF) due to its beneficial impacts on immune development and gut health. Lactoferrin supplementation into these products requires an analytical method to accurately quantify the concentrations of bLF to meet global regulatory and quality standards. OBJECTIVE: To develop and validate a lactoferrin method capable of meeting the AOAC INTERNATIONAL Standard Method Performance Requirements (SMPR®) 2020.005. METHODS: Powder formula samples are extracted using warm dibasic phosphate buffer, pH 8, then centrifuged at 4°C to remove insoluble proteins, fat, and other solids. The soluble fraction is further purified on a HiTrap heparin solid-phase extraction (SPE) column to isolate bLF from interferences. Samples are filtered, then analyzed by LC-UV using a protein BEH C4 analytical column and quantitated using an external calibrant. RESULTS: The LOQ (2 mg/100 g), repeatability (RSD: 2.0-4.8%), recovery (92.1-97.7%), and analytical range (4-193 mg/100 g) all meet the method requirements as stated in SMPR 2020.005 for lactoferrin. CONCLUSION: The reported single-laboratory validation (SLV) results demonstrate the ability of this lactoferrin method to meet or exceed the method performance requirements to measure soluble, intact, non-denatured bLF in infant and adult nutritional powder formulas. HIGHLIGHTS: The use of a heparin affinity column to isolate lactoferrin from bovine milk products combined with a selective analytical chromatographic column provides suitable analyte specificity without requiring proprietary equipment or reagents.
Subject(s)
Infant Formula , Lactoferrin , Lactoferrin/analysis , Cattle , Infant Formula/chemistry , Animals , Chromatography, High Pressure Liquid/methods , Heparin/analysis , Heparin/chemistry , Adult , Infant , Humans , Powders/chemistry , Solid Phase Extraction/methods , Chromatography, Reverse-Phase/methods , Spectrophotometry, Ultraviolet/methods , Food, Formulated/analysis , Reproducibility of Results , Chromatography, Affinity/methodsABSTRACT
Developing "turn on" fluorescent probes was desirable for the detection of the effective anticoagulant agent heparin in clinical applications. Through combining the aggregation induced emission (AIE) fluorogen tetraphenylethene (TPE) and heparin specific binding peptide AG73, the promising "turn on" fluorescent probe TPE-1 has been developed. Nevertheless, although TPE-1 could achieve the sensitive and selective detection of heparin, the low proteolytic stability and undesirable poor solubility may limit its widespread applications. In this study, seven TPE-1 derived fluorescent probes were rationally designed, efficiently synthesized and evaluated. The stability and water solubility were systematically estimated. Especially, to achieve real-time monitoring of proteolytic stability, the novel Abz/Dnp-based "turn on" probes that employ the internally quenched fluorescent (IQF) mechanism were designed and synthesized. Moreover, the detection ability of synthetic fluorescent probes for heparin were systematically evaluated. Importantly, the performance of d-type peptide fluorescent probe XH-6 indicated that d-type amino acid substitutions could significantly improve the proteolytic stability without compromising its ability of heparin sensing, and attaching solubilizing tag 2-(2-aminoethoxy) ethoxy) acid (AEEA) could greatly enhance the solubility. Collectively, this study not only established practical strategies to improve both the water solubility and proteolytic stability of "turn on" fluorescent probes for heparin sensing, but also provided valuable references for the subsequent development of enzymatic hydrolysis-resistant d-type peptides based fluorescent probes.
Subject(s)
Fluorescent Dyes , Heparin , Peptides , Fluorescent Dyes/chemistry , Fluorescent Dyes/chemical synthesis , Heparin/analysis , Heparin/chemistry , Peptides/chemistry , Peptides/chemical synthesis , Molecular Structure , Humans , Spectrometry, FluorescenceABSTRACT
A novel heparinase III from Pedobacter schmidteae (PsHep-III) with high activity and good stability was successfully cloned, expressed, and characterized. PsHep-III displayed the highest specific activity ever reported of 192.8 U mg-1 using heparin as the substrate. It was stable at 25 °C with a half-life of 323 h in an aqueous solution. PsHep-III was employed for the depolymerization of heparin, and the enzymatic hydrolyzed products were analyzed with gel permeation chromatography and high-performance liquid chromatography. PsHep-III can break glycosidic bonds in heparin like â4]GlcNAc/GlcNAc6S/GlcNS/GlcNS6S/GlcN/GlcN6S(1 â 4)ΔUA/ΔUA2S[1 â and efficiently digest heparin into seven disaccharides including N-acetylated, N-sulfated, and N-unsubstituted modification, with molecular masses of 503, 605, 563, 563, 665, 360, and 563 Da, respectively. These results indicated that PsHep-III with broad substrate specificity could be combined with heparinase I to overcome the low selectivity at the N-acetylated modification binding sites of heparinase I. This work will contribute to the application of PsHep-III for characterizing heparin and producing low-molecular-weight heparin effectively.
Subject(s)
Heparin , Polysaccharide-Lyases , Heparin/analysis , Heparin/chemistry , Heparin/metabolism , Heparin Lyase/genetics , Heparin Lyase/chemistry , Heparin Lyase/metabolism , Polysaccharide-Lyases/genetics , Polysaccharide-Lyases/metabolism , Binding SitesABSTRACT
AIM: The study objective was to evaluate the performance of sthemO 301 system and to compare it with the analyzer used in our university hospital laboratory (STA R Max® 2), for a selection of hemostasis parameters. METHODS: Method comparison (according to CLSI EP09-A3), carryover (according to CLSI H57-A), APTT sensitivity to heparin (according to CLSI H47-A2), HIL level assessment, and productivity were performed using leftover samples from our laboratory (n > 1000). Commercial quality control materials were used to evaluate precision (according to CLSI EP15-A3) and accuracy. The assays tested on sthemO 301 were: PT, APTT (silica and kaolin activators), fibrinogen (Fib), thrombin time (TT), chromogenic and clotting protein C (PC) activity, and von Willebrand factor antigen (VWF:Ag) levels. RESULTS: All intra-assay and inter-assay precision CVs were below the maximal precision limit proposed by the French Group for Hemostasis and Thrombosis (GFHT). Accuracy was verified with bias below GFHT criteria and most Z-scores were between -2 and +2. No clinically relevant carryover was detected. Silica APTT reagent sensitivity to unfractionated heparin was moderate, as expected. Productivity results were consistent over the 10 repeats performed. The overall agreement between the two systems was excellent for all assays, with Spearman rank correlation coefficient all above 0.9 and slopes of Passing-Bablok correlation near 1 and intercepts close to 0. CONCLUSION: For the methods tested, sthemO 301 system met all the criteria to implement a novel coagulation analyzer in the laboratory and result comparability with STA R Max® 2 was good.
Subject(s)
Blood Coagulation Tests , Laboratories, Clinical , Humans , Blood Coagulation Tests/instrumentation , Heparin/analysis , Quality Control , Reproducibility of Results , Sensitivity and Specificity , Laboratories, Clinical/standardsABSTRACT
Purifying extracellular vesicles (EVs) has been challenging because EVs are heterogeneous in cargo yet share similar sizes and densities. Most surface marker-based affinity separation methods are limited to research or diagnostic scales. We report that heparin chromatography can separate purified EVs into two distinct subpopulations as ascertained by MS/MS: a non-heparin-binding (NHB) fraction that contains classical EV markers such as tetraspanins and a heparin-binding (HB) fraction enriched in fibronectins and histones. Both fractions were similarly fusogenic but induced different transcriptional responses in endothelial cells. While EVs that were purified by conventional, non-affinity methods alone induced ERK1/2 phosphorylation and Ki67, the NHB fraction did not. This result suggests heparin chromatography as an additional novel fractionation step that is inherently scalable, does not lead to loss of material, and separates inflammatory and pyrogenic EVs from unreactive EVs, which will improve clinical applications.
Subject(s)
Extracellular Vesicles , Heparin , Heparin/pharmacology , Heparin/analysis , Heparin/chemistry , Tandem Mass Spectrometry , Endothelial Cells , Extracellular Vesicles/chemistry , Chromatography, Affinity/methodsABSTRACT
Heparin, an anionic biomacromolecule, is routinely used as an anticoagulant during medical surgery to prevent blood clot formation and in the treatment of several heart, lung, and circulatory disorders having a higher risk of blood clotting. We herein report supramolecular polymeric nanoassemblies of cationic pyrene-tagged bis-imidazolium amphiphiles for heparin detection with high sensitivity and selectivity in aqueous buffer, plasma, and serum media. The nano-assemblies exhibited cyan-green excimeric emission in aqueous media, and their multivalent array of positive surface charges allowed them to form co-assemblies with heparin, resulting in significantly enhanced emission. This provided a convenient method for heparin detection in buffer at nanomolar concentrations, and most notably, a ratiometric fluorescence response was obtained even in highly competitive 100% human serum and 100% human plasma in a clinically relevant concentration range. Moreover, using the heparin-based luminescent co-assemblies, protamine sulfate, a clinically administered antidote to heparin, was also detected in 100% human serum and 100% human plasma at sub-micromolar concentrations.
Subject(s)
Blood Chemical Analysis , Heparin , Protamines , Humans , Anticoagulants/pharmacology , Blood Coagulation , Heparin/analysis , Heparin/pharmacology , Luminescence , Polymers/pharmacology , Protamines/analysis , Protamines/pharmacology , Blood Chemical Analysis/methods , Serum/chemistry , Plasma/chemistry , BuffersABSTRACT
Mitochondrial content has been reported outside of cells either within extracellular vesicles (EVs) or as free mitochondria. Mitochondrial EVs can potentially play multiple physiological and pathophysiological roles. To understand their functions, isolation protocols to separate mitochondrial EVs from other mitochondrial content need to be established. In the present work, we use a multiple reaction monitoring assay with isotope labeled internal standards to quantify 11 mitochondrial, 6 plasma membrane-specific, 4 endosomal membrane-specific, and 2 soluble proteins to evaluate the efficiency of chromatographic isolation of mitochondrial EVs. The isolation protocol includes ultracentrifugation, size exclusion chromatography, and chromatography on immobilized heparin. All protein concentrations were normalized to the concentration of ATP synthase alpha subunit to generate a ratio that allows comparison of different samples obtained during the isolation. We have shown that initial samples after ultracentrifugation are contaminated with non-EV mitochondrial content that cannot be separated from EVs using size exclusion chromatography, but can be efficiently separated from EVs on the column with immobilized heparin.
Subject(s)
Extracellular Vesicles , Extracellular Vesicles/chemistry , Chromatography, Gel , Mitochondria , Heparin/analysis , UltracentrifugationABSTRACT
Naturally occurring adeno-associated virus (AAV) serotypes that bind to ligands such as AVB sepharose or heparin can be purified by affinity chromatography, which is a more efficient and scalable method than gradient ultracentrifugation. Wild-type AAV8 does not bind effectively to either of these molecules, which constitutes a barrier to using this vector when a high throughput design is required. Previously, AAV8 was engineered to contain a SPAKFA amino acid sequence to facilitate purification using AVB sepharose resin; however, in vivo studies were not conducted to examine whether these capsid mutations altered the transduction profile. To address this gap in knowledge, a mutant AAV8 capsid was engineered to bind to AVB sepharose and heparan sulfate (AAV8-AVB-HS), which efficiently bound to both affinity columns, resulting in elution yields of >80% of the total vector loaded compared to <5% for wild-type AAV8. However, in vivo comparison by intramuscular, intravenous, and intraperitoneal vector administration demonstrated a significant decrease in AAV8-AVB-HS transduction efficiency without alteration of the transduction profile. Therefore, although it is possible to engineer AAV capsids to bind various affinity ligands, the consequences associated with mutating surface exposed residues have the potential to negatively impact other vector characteristics including in vivo potency and production yield. This study demonstrates the importance of evaluating all aspects of vector performance when engineering AAV capsids.
Subject(s)
Capsid , Heparin , Capsid/metabolism , Sepharose/analysis , Sepharose/metabolism , Transduction, Genetic , Heparin/analysis , Heparin/metabolism , Genetic Vectors/genetics , Capsid Proteins/genetics , Capsid Proteins/metabolism , Dependovirus/geneticsABSTRACT
The development of cell-free regenerative biomaterials is among the current approaches of tissue engineering (TE). While materials targeting homogeneous continuous tissues can be produced more easily, significant difficulties are encountered in composite tissue interfaces such as the tendon-bone. The complex bioactive and chemical contents of the microenvironment in neighboring tissues make this situation even more difficult. While target tissue can be significantly mimicked with decellularized tissues, there is a need for incorporation of inorganic components into composite interfaces. The regenerative properties of biomaterials can be regulated by enriching them with growth factors, and the mechanical properties can be imparted by developing nanocomposites of right composition. In the first phase of the study, protocols were optimized to obtain bovine tendon-based scaffolds with high bioactive content from decellularized hydrogels. The results showed that DNA could be removed from tissues (<50 ng/mg ECM) using 0.1% SDS and 0.1% EDTA after freeze-thaw, and the content of sGAGs, an important component for tendon tissue repair, was preserved in the final product at >50%. In the second stage, the scaffolds were produced in composite form containing different amounts of nanohydroxyapatite (HAp). In the final stage, tendon-based nanocomposite scaffolds were activated with heparin to impart growth factor binding affinity. The physical, chemical, thermal, mechanical and in vitro biological properties of the scaffolds were studied in detail. The findings revealed that HAp increased the thermal stability and compressive strength; and heparin could be successfully integrated into scaffolds. Nanocomposite scaffolds were found to be highly hemocompatible.
Subject(s)
Nanocomposites , Tissue Scaffolds , Animals , Biocompatible Materials/chemistry , Cattle , Extracellular Matrix/chemistry , Heparin/analysis , Heparin/metabolism , Heparin/pharmacology , Prospective Studies , Tendons , Tissue Engineering/methods , Tissue Scaffolds/chemistryABSTRACT
Nuclear magnetic resonance (NMR) spectrometric methods for the quantitative analysis of pure heparin in crude heparin is proposed. For quantification, a two-step routine was developed using a USP heparin reference sample for calibration and benzoic acid as an internal standard. The method was successfully validated for its accuracy, reproducibility, and precision. The methodology was used to analyze 20 authentic porcine heparinoid samples having heparin content between 4.25 w/w % and 64.4 w/w %. The characterization of crude heparin products was further extended to a simultaneous analysis of these common ions: sodium, calcium, acetate and chloride. A significant, linear dependence was found between anticoagulant activity and assayed heparin content for thirteen heparinoids samples, for which reference data were available. A Diffused-ordered NMR experiment (DOSY) can be used for qualitative analysis of specific glycosaminoglycans (GAGs) in heparinoid matrices and, potentially, for quantitative prediction of molecular weight of GAGs. NMR spectrometry therefore represents a unique analytical method suitable for the simultaneous quantitative control of organic and inorganic composition of crude heparin samples (especially heparin content) as well as an estimation of other physical and quality parameters (molecular weight, animal origin and activity).
Subject(s)
Heparin , Heparinoids , Animals , Glycosaminoglycans , Heparin/analysis , Ions , Magnetic Resonance Spectroscopy/methods , Quality Control , Reproducibility of Results , SwineABSTRACT
The purification of extracellular vesicles (EVs) remains a major hurdle in the progression of fundamental research and the commercial application of EV-based products. In this study, we evaluated the potential of heparin affinity chromatography (HAC) to purify neural stem cell-derived EVs as part of a multistep process. Bind-elute chromatography, such as HAC, is an attractive method of purification because it is highly scalable, robust and can be automated. Our findings support an interaction between EVs and heparin. The recovery of EVs using HAC based on particle counts was a minimum of 68.7%. We found HAC could remove on average 98.8% and 99.0% of residual protein and DNA respectively. In addition to EV purification, HAC was used to separate EVs into three populations based on their affinity to the heparin column. Within these populations, we detected differences in the expression of the exosome-associated protein TSG101 and the tetraspanin immunophenotype. However, the significance of these observations is not clear. Overall HAC shows promise as a potential purification method to capture EVs and this study proposes a novel application of HAC for EV fractionation. Moving forward, a better understanding of the heparin-EV interaction would be required before HAC can be more widely adopted for these applications.
Subject(s)
Exosomes , Extracellular Vesicles , Chemical Fractionation , Chromatography, Affinity/methods , Extracellular Vesicles/chemistry , Heparin/analysisABSTRACT
Heparin has been used successfully as a clinical antithrombotic for almost one century. Its isolation from animal sources (mostly porcine intestinal mucosa) involves multistep purification processes starting from the slaughterhouse (as mucosa) to the pharmaceutical plant (as the API). This complex supply chain increases the risk of contamination and adulteration, mainly with non-porcine ruminant material. The structural similarity of heparins from different origins, the natural variability of the heparin within samples from each source as well as the structural changes induced by manufacturing processes, require increasingly sophisticated methods capable of detecting low levels of contamination. The application of suitable multivariate classification approaches on API 1H NMRspectra serve as rapid and reliable tools for product authentication and the detection of contaminants. Soft Independent Modeling of Class Analogies (SIMCA), Discriminant Analysis (DA), Partial Least Square Discriminant Analysis (PLS-DA) and local classification methods (kNN, BNN and N3) were tested on about one hundred certified heparin samples produced by 14 different manufacturers revealing that Partial Least Squares Discriminant Analysis (PLS-DA) provided the best discrimination of contaminated batches, with a balanced accuracy of 97%.
Subject(s)
Heparin , Ruminants , Animals , Discriminant Analysis , Heparin/analysis , Least-Squares Analysis , Magnetic Resonance Spectroscopy/methods , Pharmaceutical Preparations , SwineABSTRACT
Low molecular weight heparins (LMWHs), depolymerized from unfractionated heparin (UFH), are widely used as anticoagulant drugs in clinic. The variable degradation methods result the different types of LMWHs, such as enoxaparin prepared by alkaline degradation following benzylation and nadroparin degraded by nitrous acid and subsequent reduction. They have different anticoagulant activities, molecular weight and special oligosaccharide sequences. Oligosaccharide analysis of the heparinase-catalyzed digestion products of heparin and LMWHs is an important way to explore the fine structural composition. In this work, a MHC-2D-LC-MS system using SAX followed by SEC and tandem to MS was applied to analyze the heparinase-products of LMWHs. 15 components of enoxaparin and 20 components of nadroparin were separated and unambiguously characterized with mass spectrum, including eight common disaccharides, and the special structural domains resistant to enzyme digestion which have the 3-O sulfated residue and/or characteristic terminal residues and the linkage region tetrasaccharides.
Subject(s)
Heparin, Low-Molecular-Weight , Heparin , Anticoagulants/chemistry , Enoxaparin/chemistry , Heparin/analysis , Heparin Lyase/metabolism , Heparin, Low-Molecular-Weight/analysisABSTRACT
Antiplatelet and anticoagulant drugs are classified antithrombotic agents with the purpose to reduce blood clot formation. For a successful treatment of many known complex cardiovascular diseases driven by platelet and/or coagulation activity, the need of more than one antithrombotic agent is inevitable. However, combining drugs with different mechanisms of action enhances risk of bleeding. Dual anticoagulant and antiplatelet (APAC), a novel semisynthetic antithrombotic molecule, provides both anticoagulant and antiplatelet properties in preclinical studies. APAC is entering clinical studies with this new exciting approach to manage cardiovascular diseases. For a better understanding of the biological function of APAC, comprehensive knowledge of its structure is essential. In this study, atomic force microscopy (AFM) was used to characterize APAC according to its structure and to investigate the molecular interaction of APAC with von Willebrand factor (VWF), since specific binding of APAC to VWF could reduce platelet accumulation at vascular injury sites. By the optimization of drop-casting experiments, we were able to determine the volume of an individual APAC molecule at around 600 nm3, and confirm that APAC forms multimers, especially dimers and trimers under the experimental conditions. By studying the drop-casting behavior of APAC and VWF individually, we depictured their interaction by using an indirect approach. Moreover, in vitro and in vivo conducted experiments in pigs supported the AFM results further. Finally, the successful adsorption of APAC to a flat gold surface was confirmed by using photothermal-induced resonance, whereby attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) served as a reference method.
Subject(s)
Anticoagulants/analysis , Heparin/analogs & derivatives , Microscopy, Atomic Force/methods , Platelet Aggregation Inhibitors/analysis , Proteoglycans/analysis , Heparin/analysis , Humans , Spectroscopy, Fourier Transform Infrared/methodsABSTRACT
For heparin sensing, Mudliar and Singh developed fluorescence and absorption spectroscopic approaches by utilizing emissive H-aggregates of thioflavin T (ThT) formed upon heparin binding. It has been proposed that the methods work not only in pure aqueous solution but also in complex biological media such as human serum. However, the optical features used to detect and quantify heparin are very sensitive to the ionic strength of the solution and completely vanish at 1.45 mM NaCl. Curiously, the authors were able to determine the heparin content of 1% serum samples containing the same level of electrolyte. In addition, the experimental conditions employed for heparin detection in serum samples were substantially modified, reducing the optical path length from 1 to 0.1 cm and increasing the dye concentration by an unknown measure. ThT shows a concentration-dependent tendency for aqueous aggregation, which markedly modifies its absorption and fluorescence properties. The authors have failed to verify that spectral characteristics of the ThT-heparin system observed in pure aqueous medium remain unchanged at higher dye concentrations and in the presence of serum components. Taking these issues into consideration, the heparin detection scheme offered for serum samples cannot be reproduced.
Subject(s)
Benzothiazoles/chemistry , Fluorescent Dyes/chemistry , Heparin/analysis , Humans , Molecular Structure , Spectrometry, FluorescenceABSTRACT
A new fluorene-pyridine oligoelectrolyte (OFP) is rationally proposed and readily synthesized via a simple one-pot Sonogashira approach. Hence, an unexpectedly small cationic oligomer nanosensor (i.e. OFPNPs, â¼ 1.2 nm in diameter) was conveniently fabricated owing to the enhanced flexibility endowed by the meta-substituted pyridyl unit. Inspiringly, this facile nanoplatform with low cytotoxicity favors the ultrasensitive fluorescence assay for heparin and protamine with a detection limit (LOD, S/N = 3) as low as 1.2 ng mL-1 and 0.5 ng mL-1, respectively, involving heparin-induced aggregation of OFPNPs through electrostatic interaction or competitive rebinding of protamine to heparin.
Subject(s)
Fluorenes/chemistry , Fluorescent Dyes/chemistry , Heparin/analysis , Nanoparticles/chemistry , Protamines/analysis , Pyridines/chemistry , Binding, Competitive , Electrolytes/chemistry , Humans , Limit of Detection , MCF-7 Cells , Optical Imaging , Spectrometry, Fluorescence , Static Electricity , Structure-Activity RelationshipABSTRACT
Heparin is a linear sulfated polysaccharide. It is composed of a repeating disaccharide unit with different sulfo patterns. The compositional analysis after heparin was decomposed to disaccharides and enzyme resistant domains is an important way to delve into its structure. Strong anion exchange (SAX) chromatography is commonly used for the compositional analysis due to its high resolution, stability and capability of quantitation. However, nonvolatile salt in mobile phase is not compatible with MS, then the structural domains cannot be identified without standards. Here, a new two-dimensional liquid chromatography system, multiple heart cut (MHC), was developed and linked to mass spectrometry (MS) directly to provide a comprehensive analysis of enzyme digested heparin. SAX was applied as the first dimensional chromatography, in which 17 peaks were observed and integrated in the digested heparin. Size-exclusion chromatography (SEC) was used as the second dimensional chromatography to desalt efficiently. Structural information of each component was then obtained with MS, including eight common disaccharides, eight enzyme resistant tetrasaccharides and a heparin-core protein linkage domain. The comparison of enzyme digested heparins obtained from different vendors using this system suggested their similar major structure and activity, but slightly different production processes.