RESUMO
PURPOSE: In principle, non-invasive mapping of the intracellular pH (pHi ) in vivo is possible using endogenous chemical exchange saturation transfer (CEST)-MRI of the amide and guanidyl signals. However, the application for cancer imaging is still impeded, as current state-of-the-art approaches do not allow for simultaneous compensation of concomitant effects that vary within tumors. In this study, we present a novel method for absolute pHi mapping using endogenous CEST-MRI, which simultaneously compensates for concentration changes, superimposing CEST signals, magnetization transfer contrast, and spillover dilution. THEORY AND METHODS: Compensation of the concomitant effects was achieved by a ratiometric approach (i.e. the ratio of one CEST signal at different B1 ) in combination with the relaxation-compensated inverse magnetization transfer ratio MTRRex and a separate first-order polynomial-Lorentzian fit of the amide and guanidyl signals at 9.4 T. Calibration of pH values was accomplished using in vivo-like model suspensions from porcine brain lysates. Applicability of the presented method in vivo was demonstrated in n = 19 tumor-bearing mice. RESULTS: In porcine brain lysates, measurement of pH was feasible over a broad range of physiologically relevant pH values of 6.2 to 8.0, while being independent of changes in concentration. A median pHi of approximately 7.2 was found in the lesions of 19 tumor-bearing mice. CONCLUSION: The presented method enables non-invasive mapping of absolute pHi values in tumors using CEST-MRI, which was so far prevented by concomitant effects. Consequently, pre-clinical studies on pHi changes in tumors are possible allowing the assessment of pHi in vivo as a biomarker for cancer diagnosis or treatment monitoring.
Assuntos
Amidas , Glioblastoma , Animais , Encéfalo , Concentração de Íons de Hidrogênio , Imageamento por Ressonância Magnética/métodos , Camundongos , SuínosRESUMO
BACKGROUND: Gene therapy has become a potential strategy for cancer treatment. However, the development of efficient gene vectors restricts the application for cancer gene treatment. Functionalization of polymers with functional groups can significantly improve their transfection efficacy. METHODS: Guanidyl can form bidentate hydrogen with the phosphate groups and phosphate groups are present in DNA and cell membranes, thus increasing DNA condensation and cellular uptake. Imidazolyl has high buffering capacity in endosomal/lysosomal acidic environment, facilitating endosome/lysosome escape. We designed a structure-integrated group of guanidyl and imidazolyl, 2-aminoimidazole (AM), which was conjugated to PAMAM generation 2 (G2) for gene therapy of gastric adenocarcinoma. RESULTS: Molecular docking results illustrated that G2-AM bound with DNA molecule effectively via multiple interactions. A quantitative luciferase assay showed that the transfection efficacy of G2-AM/pGL3 was approximately 100-fold greater than that of G2/pGL3, 90-fold greater than that of imidazolyl-modified G2 (G2-M) /pGL3 and 100-fold greater than that of G5/pGL3 without additional cytotoxicity. After introducing the pTRAIL gene into gastric adenocarcinoma cells, the apoptosis ratio of gastric adenocarcinoma cells treated with G2-AM/pTRAIL was 36.95%, which is much larger than the corresponding ratio of G2/pTRAIL (7.45%), G2-M/pTRAIL (11.33%) and G5/pTRAIL (23.2%). In a gastric adenocarcinoma xenograft model, the in vivo transfection efficacy of G2-AM/pRFP was much greater than that of G2/pRFP and G2-M/pRFP. CONCLUSIONS: These results demonstrate that AM could be modified with cationic polymers for potential application in gene delivery and gastric adenocarcinoma gene therapy.
Assuntos
Adenocarcinoma/terapia , Dendrímeros/farmacologia , Terapia Genética , Neoplasias Gástricas/terapia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Apoptose/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Vetores Genéticos/farmacologia , Células HEK293 , Células HeLa , Humanos , Camundongos , Simulação de Acoplamento Molecular , Poliaminas/farmacologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Amide-proton-transfer weighted (APTw) MRI has emerged as a non-invasive pH-weighted imaging technique for studies of several diseases such as ischemic stroke. However, its pH-sensitivity is relatively low, limiting its capability to detect small pH changes. In this work, computer simulations, protamine phantom experiments, and in vivo gas challenge and experimental stroke in rats showed that, with judicious selection of the saturation pulse power, the amide-CEST at 3.6ppm and guanidyl-CEST signals at 2.0ppm changed in opposite directions with decreased pH. Thus, the difference between amide-CEST and guanidyl-CEST can enhance the pH measurement sensitivity, and is dubbed as pHenh. Acidification induced a negative contrast in APTw, but a positive contrast in pHenh. In vivo experiments showed that pHenh can detect hypercapnia-induced acidosis with about 3-times higher sensitivity than APTw. Also, pHenh slightly reduced gray and white matter contrast compared to APTw. In stroke animals, the CEST contrast between the ipsilateral ischemic core and contralateral normal tissue was -1.85 ± 0.42% for APTw and 3.04 ± 0.61% (n = 5) for pHenh, and the contrast to noise was 2.9 times higher for pHenh than APTw. Our results suggest that pHenh can be a useful tool for non-invasive pH-weighted imaging.
Assuntos
Isquemia Encefálica/diagnóstico por imagem , Concentração de Íons de Hidrogênio , Aumento da Imagem/métodos , Imageamento por Ressonância Magnética/métodos , Acidente Vascular Cerebral/diagnóstico por imagem , Amidas , Animais , Simulação por Computador , Aumento da Imagem/normas , Imageamento por Ressonância Magnética/normas , Masculino , Imagens de Fantasmas , Ratos , Ratos Sprague-Dawley , Sensibilidade e EspecificidadeRESUMO
Microbial culture extracts are used for natural product screening to find antifungal lead compounds. A microbial culture extract library was constructed using 343 actinomycete isolates to examine the value of the adenylate kinase (AK) assay for screening to identify antifungal metabolites that disrupt cell integrity in plant pathogenic fungi. A culture extract of Streptomyces sp. strain KP6107 lysed cells of Fusarium oxysporum f.sp. lycopersici which resulted in high AK activity. The active ingredient N-1 was purified from the culture extract using various chromatographic procedures and identified to be the guanidyl-polyol macrolide antibiotic, niphimycin, which is a potent fungal cell membrane disruptor. Niphimycin showed broad-spectrum antifungal activity against Alternaria mali, Aspergillus oryzae, Colletotrichum coccodes, Colletotrichum gloeosporioides, Cercospora canescens, Cylindrocarpon destructans, F. oxysporum f.sp. cucumerinum, F. oxysporum f.sp. lycopersici, and Rhizoctonia solani at concentrations of 8-64 µg ml(-1). Anthracnose development in pepper plants was completely inhibited by treatment with 50 µg ml(-1) niphimycin, which was as effective as chlorothalonil. These results show that the AK assay is an efficient and selective tool in screening for cell membrane/wall disruptors of plant pathogenic fungi.
Assuntos
Adenilato Quinase/química , Fungicidas Industriais/química , Ascomicetos/crescimento & desenvolvimento , Bioensaio , Colletotrichum/crescimento & desenvolvimento , Fungicidas Industriais/isolamento & purificação , Fusarium/crescimento & desenvolvimento , Guanidinas/química , Guanidinas/isolamento & purificação , Testes de Sensibilidade Microbiana , Plantas/microbiologia , Streptomyces/crescimento & desenvolvimentoRESUMO
The present work aims to prepare efficient wound dressing with noncytotoxicity, proper mechanical strength, and the ability to preserve a hygienic environment over wounded skin tissue. To fulfill this goal, the synthesis of a novel silane crosslinking agent with antibacterial guanidinium chloride functional group is considered. The resulting reagent was applied to make a series of film-type stable crosslinked networks composed of poly(vinyl alcohol) and gelatin. The potential protection of wounds from external forces was confirmed, as these films had a very good tensile strength (16-31 MPa) and good elongation (54%-101%) under dry conditions. The good dimensional strength of dressings was preserved after hydration with simulated wound exudates. Based on the calculated fluid handling capacity of the prepared dressings (2.43-3.54 g 10-1cm-2d-1), they were suitable for treating wounds with 'light' to 'moderate' exudate volume. All the prepared dressings showed very good biocompatibility, as determined by the high viability of fibroblast cells directly contacted with dressing (over 80%) or leachates extracted from them (over 90%). In addition, dressings functionalized with guanidinium groups could effectively kill representative gram-positive and gram-negative bacterial strains.
Assuntos
Gelatina , Álcool de Polivinil , Silanos , Cicatrização , Antibacterianos , BandagensRESUMO
Protein therapeutics are highly promising for complex disease treatment. However, the lack of ideal delivery vectors impedes their clinical use, especially the carriers for in vivo delivery of functional cytosolic protein. In this study, we modified poly(ß amino ester)s (PAEs) with a phenyl guanidine (PG) group to enhance their suitability for cytosolic protein delivery. The effects of the PG group on protein binding, cell internalization, protein function protection, and endo/lysosomal escape were systematically evaluated. Compared to the unmodified PAEs (L3), guanidyl rich PAEs (L3PG) presented superior efficiency of protein binding and protein internalization, mainly via clathrin-mediated endocytosis. In addition, both PAEs showed robust capabilities to deliver cytosolic proteins with different molecular weight (ranging from 30 to 464 kDa) and isoelectric points (ranging from 4.3 to 9), which were significantly improved in comparison with the commercial reagents of PULsin and Pierce Protein Transection Reagent. Moreover, L3PG successfully delivered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas9 ribonucleoprotein (RNP) into HeLa cells expressing green fluorescent protein (GFP) and achieved more than 80% GFP expression knockout. These results demonstrated that guanidyl modification on PAEs can enhance its capabilities for intracellular delivery of cytosolic functional proteins and CRISPR/Cas9 ribonucleoprotein. The guanidyl-rich PAEs are promising nonviral vectors for functional protein delivery and potential use in protein and nuclease-based gene editing therapies.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Humanos , Sistemas CRISPR-Cas/genética , Células HeLa , Proteínas de Fluorescência Verde , Ésteres , Guanidina , GuanidinasRESUMO
The flexible and controlled synthesis of metal-organic framework (MOF)-derived hybrid nanostructures is of great significance in fine tuning of their enrichment performance in large-scale and in-depth phosphoproteome analysis. Herein, a magnetic guanidyl-functionalized MOF hybrid coating with multiaffinity sites, denoted as Fe3O4@G-ZIF-8, was fast fabricated via a one-pot epitaxial growth strategy for the first time and applied for selective and highly efficient enrichment of global phosphopeptides. The intrinsic unsaturated metal sites of ZIF-8 endow the surface-mounted MOF coatings with immobilized metal ion affinity chromatography interaction with multiphosphorylated peptides. The oriented anchoring of bifunctional guanidineacetic acid on the magnetic MOF nanospheres provides additional affinity sites (guanidyl groups) for specific recognition of phosphopeptides by "salt bridge" interaction, as well as active site carboxyl groups for the coordination with the metal ions. The as-prepared Fe3O4@G-ZIF-8 exhibits large surface area (382.5 m2 g-1), good superparamagnetic property (41.6 emu g-1) and stability, and size-exclusion effect (1.73 nm), which can serve as a specific adsorbent for global phosphopeptide analysis with satisfactory selectivity, great detection sensitivity (1 fmol), and rapid magnetic separation. Moreover, the successful application of Fe3O4@G-ZIF-8 for selective capture of both multi- and mono-phosphopeptides from human saliva and serum demonstrated the great potential of magnetic surface-mounted MOF coatings in effective identification of low-abundance phosphopeptides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry from complicated biological matrices.
Assuntos
Estruturas Metalorgânicas , Fosfopeptídeos , Cromatografia de Afinidade/métodos , Humanos , Íons/química , Fenômenos Magnéticos , Magnetismo , Estruturas Metalorgânicas/química , Fosfopeptídeos/análiseRESUMO
In this study, 6-deoxy-6-arginine modified chitosan (DAC), was synthesized and characterized by Fourier Transform Infrared Spectroscopy (FTIR), 1H and 13C nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC) and elemental analysis. The arginine was grafted onto C6 groups of chitosan. Antibacterial activity of DAC against gram-negative bacteria Escherichia coli (E. coli) and gram-positive bacteria Staphylococcus aureus (S. aureus) were investigated at concentration between 0.02 mg/mL and 10 mg/mL. Cell viability assessment was estimated in vitro with Caco-2 and L929 cells. Water solubility of DAC at different pH was also evaluated. The results showed that the minimum inhibitory concentration (MICs) of DAC against S. aureus and E. coli were 0.078 mg/mL and 0.312 mg/mL, respectively. The minimum bactericidal concentration (MBC) against S. aureus and E. coli was 0.625 mg/mL. The cytotoxicity of chitosan and DAC was not significantly different. It demonstrated that DAC might be a potential safe antibacterial agent.
Assuntos
Antibacterianos , Quitosana , Antibacterianos/química , Antibacterianos/farmacologia , Arginina/química , Células CACO-2 , Quitosana/análogos & derivados , Quitosana/química , Quitosana/farmacologia , Escherichia coli/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana/métodos , Solubilidade , Staphylococcus aureus/efeitos dos fármacosRESUMO
Protein phosphorylation plays vital roles in the regulation of various biological processes involving in protein folding, molecular recognition, cell growth, and metabolism. It is prerequisite to develop effective enrichment methods of trace phosphopeptides before mass spectrometry (MS) analysis. In this study, we proposed a facile strategy to synthesize magnetic ionic covalent organic frameworks (Fe3O4@iCOFs) for the capture of phosphopeptides with guanidyl as the ionic ligand instead of the post-synthetic functionalization strategy. The developed Fe3O4@iCOFs contain a large amount of amino groups, positive charge, as well as owned superparamagnetism. The enrichment of phosphopeptides is based on the electrostatic interaction and hydrogen bonds formed between phosphate groups and guanidyl groups. By combing with MS determinations, high sensitivity of phosphopeptides (the lowest detection amount being 0.4 fmol) was achieved. The obtained material provided selective enrichment capacity of phosphopeptides from non-fat milk digest and HeLa cells, showing great potential in the detection of low-abundance phosphopeptides in complex real samples.
Assuntos
Guanina/química , Nanopartículas de Magnetita/química , Estruturas Metalorgânicas/química , Fosfopeptídeos/análise , Células HeLa , Humanos , Íons/síntese química , Íons/química , Estruturas Metalorgânicas/síntese química , Estrutura Molecular , Tamanho da Partícula , Propriedades de Superfície , Células Tumorais CultivadasRESUMO
Cytosolic protein delivery is important for the development of protein therapeutics towards intracellular targets. Guanidyl polymers exhibit high binding affinity with cargo proteins, and thus were designed as carriers for intracellular protein delivery. However, the structure-activity relationship and mechanism of these polymers in cytosolic protein delivery remained to be investigated. In this study, we synthesized a total number of eighteen guanidyl-rich polymers by grafting various guanidyl containing compounds onto a polyethylenimine scaffold. The investigated guanidyl analogues were consisted of a guanidyl group and a hydrophobic component including cyclohexane, benzene, and alkanes with various chain lengths. It is surprising that only the polymers with both benzene and guanidyl possessed high efficiency in cytosolic protein delivery. Further results showed that all the synthesized polymers have efficient protein binding in water and high cellular uptake, but these polymers except the benzene-guanidyl based one enter the cytosol of cells without carrying their cargo proteins, suggesting poor stability of the polymer/protein complexes in culture medium. Paired guanidinium-π interactions between the ligands on benzene-guanidyl polymers are critical to the stabilization of polymer/protein complexes. In addition, a lead polymer in the library exhibited robust delivery efficacy to various cargo proteins, while maintaining their bioactivity after cell internalization. The results suggest that complex stability is a critical factor in polymer-mediated intracellular protein delivery systems, and provide new insights to guide design of polymeric protein vehicles.
Assuntos
Polímeros , Proteínas , Citosol , Interações Hidrofóbicas e Hidrofílicas , PolietilenoiminaRESUMO
Comprehensive mass spectrometry-based proteomics analysis is currently available but remains challenging, especially for post-translational modifications of phosphorylated proteins. Herein, multifunctional magnetic pillar[5]arene supramolecular organic frameworks were fabricated and immobilized with arginine (mP5SOF-Arg) for highly effective enrichment of global phosphopeptides. The specific phosphate-P5/phosphate-guanidine affinities and large surface area with regular porosity contribute to the high enrichment capacity. By coupling with mass spectrometry, high detection sensitivity (0.1 fmol), excellent selectivity (1:5000 molar ratios of ß-casein/cytochrome c), and high recyclability (seven cycles) were achieved for phosphopeptide analysis. mP5SOF-Arg can efficiently enrich phosphopeptides from practical samples, including defatted milk, egg yolk, and human saliva. Notably, a total of 450 phosphopeptides were explored for highly selective identification from A594 cells and 1445 phosphopeptides were identified from mouse liver tissue samples. mP5SOF-Arg exhibited great potential to serve as the basis for peptidomic research to identify phosphopeptides and provided insight for biomarker discovery.
Assuntos
Arginina/análogos & derivados , Calixarenos/química , Guanidinas/química , Fosfopeptídeos/isolamento & purificação , Células A549 , Adsorção , Animais , Caseínas , Citocromos c , Gema de Ovo/química , Humanos , Fenômenos Magnéticos , Leite/química , Fosfopeptídeos/química , Proteômica/métodos , Saliva/química , Extração em Fase Sólida/métodosRESUMO
PURPOSE: To investigate whether fat-corrected and relaxation-compensated amide proton transfer (APT) and guanidyl CEST-MRI enables the detection of signal intensity differences between breast tumors and normal-appearing fibroglandular tissue in patients with newly-diagnosed breast cancer. METHOD: Ten patients with newly-diagnosed breast cancer and seven healthy volunteers were included in this prospective IRB-approved study. CEST-MRI was performed on a 7â¯T-whole-body scanner followed by a multi-Lorentzian fit analysis. APT and guanidyl CEST signal intensities were quantified in the tumor and in healthy fibroglandular tissue after correction of B0/B1-field inhomogeneities, fat signal contribution, T1- and T2-relaxation; signal intensity differences of APT and guanidyl resonances were compared using Mann-Whitney-U-tests. Pearson correlations between tumor CEST signal intensities and the proliferation index Ki-67 were performed. RESULTS: APT CEST signal in tumor tissue (6.70⯱â¯1.38%Hz) was increased compared to normal-appearing fibroglandular tissue of patients (3.56⯱â¯0.54%Hz, pâ¯=â¯0.001) and healthy volunteers (3.70⯱â¯0.68%Hz, pâ¯=â¯0.001). Further, a moderate positive correlation was found between the APT signal and the proliferation index Ki-67 (R2â¯=â¯0.367, râ¯=â¯0.606, pâ¯=â¯0.11). Guanidyl CEST signal was also increased in tumor tissue (5.24⯱â¯1.85%Hz) compared to patients' (2.42⯱â¯0.45%Hz, pâ¯=â¯0.006) and volunteers' (2.36⯱â¯0.54%Hz, pâ¯<â¯0.001) normal-appearing fibroglandular tissue and a positive correlation with the Ki-67 level was observed (R2â¯=â¯0.365, râ¯=â¯0.604, pâ¯=â¯0.11). APT and guanidyl CEST signal in normal-appearing fibroglandular tissue was not different between patients and healthy volunteers (pâ¯=â¯0.88; pâ¯=â¯0.93). CONCLUSION: Relaxation-compensated and fat-corrected CEST-MRI allowed a non-invasive differentiation of breast cancer and normal-appearing breast tissue. Thus, this approach represents a contrast agent-free method that may help to increase diagnostic accuracy in MR-mammography.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Meios de Contraste , Aumento da Imagem/métodos , Imageamento por Ressonância Magnética/métodos , Adulto , Idoso , Amidas , Mama/diagnóstico por imagem , Diagnóstico Diferencial , Feminino , Humanos , Pessoa de Meia-Idade , Compostos Organometálicos , Estudos Prospectivos , Prótons , Sensibilidade e EspecificidadeRESUMO
A highly selective and efficient extraction material was synthesized through the functionalization of guanidyl onto the graphene oxide-grafted silica via a simple chemical modification, which was designed and proposed to improve the enrichment capacity for acidic herbicides. The extraction material was confirmed by scanning electron microscopy, Fourier transform infrared spectrometry, X-ray photoelectron spectrometry, thermal gravimetric analyzer and zeta potential analysis. Theoretical adsorption energies, static- and dynamic-state binding experiments, and comparative experiments with various adsorbents were investigated to elucidate the adsorption mechanism. The introduction of guanidyl endowed the sorbent with stronger Lewis base property and electron-donating ability, hence, excellent extraction recoveries for acidic herbicides could be obtained. Besides, electrostatic and π-π interactions were considered as two major driving impetuses in the adsorption process. Single-factor experiment and response surface methodology were utilized for the optimization of extraction and desorption conditions. Under the optimized conditions, the wide linearities were obtained with correlation coefficients ranging from 0.9904 to 0.9980, and the method detection limits were in the range of 0.5-2 µg L-1. The relative standard deviation values of the recoveries of five different extractions were 3.0-7.1%. Coupled with high performance liquid chromatography, the as-proposed method was successfully applied to detect five acidic herbicides in Lycium barbarum (Goji). It turned out that the proposed method provided a promising perspective for the selective extraction and determination of polar acidic compounds in complex samples.
Assuntos
Grafite/química , Herbicidas/isolamento & purificação , Lycium/química , Dióxido de Silício/química , Extração em Fase Sólida/métodos , Adsorção , Cromatografia Líquida de Alta Pressão , Limite de Detecção , Óxidos/química , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/isolamento & purificaçãoRESUMO
Antigen uptake by dendritic cells (DCs) is a key step for initiating antigen-specific T cell immunity. In the present study, novel synthetic polymeric nanoparticles were prepared as antigen delivery vehicles to improve the antigen uptake by DCs. Well-defined cationic and acid-responsive copolymers, monomethoxy poly(ethylene glycol)-block-poly(2-(diisopropyl amino) ethyl methacrylate)-block-poly(2-(guanidyl) ethyl methacrylate) (mPEG-b-PDPA-b-PGEM, PEDG) were synthesized by reversible addition-fragmentation chain transfer polymerization of 2-(diisopropylamino)ethyl methacrylate) and N-(tert-butoxycarbonyl) amino ethyl methacrylate monomers, followed by deprotection of tert-butyl protective groups and guanidinylation of obtained primary amines. 1H NMR, 13C NMR and GPC results indicated the successful synthesis of well-defined PEDG copolymers. PEDG copolymers could self-assemble into nanoparticles in aqueous solution, which were of cationic surface charges and showed acid-triggered disassembly contributed by PGEM and PDPA moieties, respectively. Significantly, PEDG nanoparticles could effectively condense with negatively charged model antigen ovalbumin (OVA) to form OVA/PEDG nanoparticle formulations with no influence on its secondary and tertiary structures demonstrating by far-UV circular dichroism and UV-vis spectra. In vitro antigen cellular uptake by bone marrow DCs (BMDCs) indicated using PEDG nanoparticles as antigen delivery vehicles could significantly improve the antigen uptake efficiency of OVA compared with free OVA or the commercialized Alum adjuvant. Moreover, as the surface cationic charges of OVA/PEDG nanoparticle formulations reduced, the uptake efficiency decreased correspondingly. Collectively, our work suggests that guanidinylated, cationic and acid-responsive PEDG nanoparticles represent a new kind of promising antigen delivery vehicle to DCs and hold great potential to serve as immunoadjuvants in the development of vaccines.
RESUMO
A label-free fluorescence assay for rapid and sensitive detection of heparin (Hep) or chondroitin sulfate (CS) was developed by guanidine-terminated poly (amidoanime) (PAMAM-Gu(+)) dendrimers induced aggregation of graphene quantum dots (GQDs). The fluorescence of GQDs was obviously quenched after mixing with PAMAM-Gu(+). However, the addition of highly negatively charged Hep or CS into the fluorescence sensing system resulted in the fluorescence recovery. Because the multi-positively charged PAMAM-Gu(+) would prefer to bind with highly negatively charged Hep or CS, resulting in the deaggregation of GQDs. Under the optimized experimental conditions, the recovery of fluorescence intensity ratio I/I0 (I0 and I were the fluorescence intensity of the sensing system in the absence or presence of target analytes, respectively) was proportional to the concentration of target analytes in the range of 0.04-1.6 µg mL(-1) for Hep and 0.1-2.5 µg mL(-1) for CS. In addition, this method afforded high sensitivity with the detection limit as low as 0.02 µg mL(-1) and 0.05 µg mL(-1) for Hep and CS, respectively. All results suggested that the fluorescence turn-on method could be successfully employed for sensitive and selective detection of heparin analogs.
Assuntos
Anticoagulantes/análise , Sulfatos de Condroitina/análise , Dendrímeros/química , Grafite/química , Heparina/análise , Nanopartículas/química , Pontos Quânticos/química , Corantes Fluorescentes/química , Guanidina/análogos & derivados , Espectrometria de Fluorescência/métodosRESUMO
The highly selective and efficient capture of heterogeneous types of phosphopeptides is critical for comprehensive and in-depth phosphoproteome analysis, but it still remains a challenge since the lack of affinity material with large binding capacity and controllable specificity. Here, a new affinity material was prepared to improve the enrichment capacity and endue the tunable specificity by introducing guanidyl onto poly(glycidyl methacrylate) (PGMA) modified Fe3O4 microsphere (denoted as Fe3O4@PGMA-Guanidyl). The thick polymer shell endows the composite microsphere with large amount of guanidyl and is beneficial to enhancing the affinity interaction between phosphopeptides and the material. Interestingly, the Fe3O4@PGMA-Guanidyl possesses tunable enriching ability for global phosphopeptides or only multiphosphopeptides through simple regulation of buffer composition. The composite has large enrichment capacity (200 mg g(-1)), extremely high detection sensitivity (0.5 fmol), high enrichment recovery (91.30%), great specificity, and rapid magnetic separation. Moreover, the result of the application to capture of phosphopeptides from tryptic digest of nonfat milk has demonstrated the great potential of Fe3O4@PGMA-Guanidyl in detection and identification of low-abundance phosphopeptides of interest in biological sample.