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Electrochemically modulated liquid chromatography (EMLC) uses electrical potentials, applied to a conductive chromatographic stationary phase (e.g., porous graphitic carbon [PGC]), to manipulate analyte retention. This paper reports the design of a capillary EMLC column with a smaller internal diameter (ID; 250 µm) than that of the standard bore predecessor (3.3 mm ID). The new capillary EMLC columns are configured so that the PGC stationary phase serves as the working electrode in a two-electrode electrochemical cell and simplifies electrode placement by obviating the need for a counter electrode. This configuration also eliminates the internal Nafion sleeve that is critical to operation for the standard bore columns, thereby avoiding Nafion deformation as a source of chromatographic band broadening and rupturing as a mode of column failure. Indeed, values for chromatographic efficiency obtained on the capillary columns meet or exceed those measured for the standard columns (20â¯000-40â¯000 vs 14â¯000 plates/m, respectively) with near symmetric elution bands (asymmetry factors of 1.1-1.4 for well-packed capillaries) that surpass band symmetries observed in all prior studies. A test suite of aromatic sulfonates was used to characterize the chromatographic performance of the capillary EMLC columns. Separations of this test mixture showed that retention factors for individual analytes could be manipulated by as much as 21× by changing the applied potential at the PGC stationary phase. Changes in retention behavior at different potential ranges, hypothesized to result from differences in adsorption orientation, were also observed and are consistent with past work. Collectively, the retention behavior unique to EMLC is operative in this new capillary configuration and promises to open new avenues in tuning LC separations.
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This paper presents a method for immunometric biomarker quantitation that uses standard flow-through assay reagents and obviates the need for constructing a calibration curve. The approach relies on a nitrocellulose immunoassay substrate with multiple physical addresses for analyte capture, each modified with different amounts of an analyte-specific capture antibody. As such, each address generates a distinctly different readout signal that is proportional to the analyte concentration in the sample. To establish the feasibility of this concept, equations derived from antibody-antigen binding equilibrium were first applied in modeling experiments. Next, nitrocellulose membranes with multiple capture antibody addresses were fabricated for detection of a model analyte, human Immunoglobulin G (hIgG), by a heterogeneous sandwich immunoassay using antibody-modified gold nanoparticles (AuNPs) as the immunolabel. Counting the number of colored capture addresses visible to the unassisted eye enabled semiquantitative hIgG determination. We then demonstrated that, by leveraging the localized surface plasmon resonance of the AuNPs, surface-enhanced Raman spectroscopy (SERS) can be used for quantitative readout. By comparing the SERS signal intensities from each capture address with values predicted using immunoassay equilibrium theory, the concentration of hIgG can be determined (â¼30% average absolute deviation) without reference to a calibration curve. This work also demonstrates the ability to manipulate the dynamic range of the assay over â¼4 orders of magnitude (from 2 ng mL-1 to 10 µg mL-1). The potential prospects in applying this concept to point-of-need diagnostics are also discussed.
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Imunoensaio/métodos , Imunoglobulina G/análise , Biomarcadores/análise , Calibragem , Humanos , Análise Espectral Raman , Propriedades de SuperfícieRESUMO
The in vivo analytical performance of percutaneously implanted nitric oxide (NO)-releasing amperometric glucose biosensors was evaluated in swine for 10 d. Needle-type glucose biosensors were functionalized with NO-releasing polyurethane coatings designed to release similar total amounts of NO (3.1 µmol cm(-2)) for rapid (16.0 ± 4.4 h) or slower (>74.6 ± 16.6 h) durations and remain functional as outer glucose sensor membranes. Relative to controls, NO-releasing sensors were characterized with improved numerical accuracy on days 1 and 3. Furthermore, the clinical accuracy and sensitivity of rapid NO-releasing sensors were superior to control and slower NO-releasing sensors at both 1 and 3 d implantation. In contrast, the slower, extended, NO-releasing sensors were characterized by shorter sensor lag times (<4.2 min) in response to intravenous glucose tolerance tests versus burst NO-releasing and control sensors (>5.8 min) at 3, 7, and 10 d. Collectively, these results highlight the potential for NO release to enhance the analytical utility of in vivo glucose biosensors. Initial results also suggest that this analytical performance benefit is dependent on the NO-release duration.
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Técnicas Biossensoriais/métodos , Glucose/análise , Doadores de Óxido Nítrico/química , Animais , Técnicas Biossensoriais/instrumentação , Glicemia/análise , Nanopartículas/química , Óxido Nítrico , Poliuretanos/química , Próteses e Implantes , Sensibilidade e Especificidade , Sus scrofaRESUMO
For mammalian cell-derived recombinant biotherapeutics, controlling host cell DNA levels below a threshold is a regulatory requirement to ensure patient safety. DNA removal during drug substance manufacture is accomplished by a series of chromatography-based purification steps and a qPCR-based analytical method is most used to measure DNA content in the purified drug substance to enable material disposition. While the qPCR approach is mature and its application to DNA measurement is widespread in the industry, it is susceptible to trace levels of process-related contaminants that are carried forward. In this study, we observed failures in spike recovery studies that are an integral component of the qPCR-based DNA testing, suggesting the presence of an inhibitory compound in the sample matrix. We generated hypotheses around the origin of the inhibitory compound and generated multiple sample matrices and deployed a suite of analytical techniques including Raman and NMR spectroscopy to determine the origin and identity of the inhibitory compound. The caustic wash step and depth filter extractables were ruled out as root causes after extensive experimentation and DNA testing. Subsequently, 2-(N-morpholino)ethanesulfonic acid (MES), a buffer used in the chromatography unit operations, was identified as the source of the contaminant. A 500-fold concentration followed by Raman and NMR spectroscopy analysis revealed the identity of the inhibitory compound as polyvinyl sulfone (PVS), an impurity that originates in the MES manufacturing process. We have implemented PVS concentration controls for incoming MES raw material, and our work highlights the need for rigor in raw material qualification and control.
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Cromatografia , DNA , Animais , Humanos , Espectroscopia de Ressonância Magnética/métodos , DNA/genética , MamíferosRESUMO
The foreign body response (FBR) to nitric oxide (NO)-releasing subcutaneous implants was compared between healthy and streptozotocin-induced diabetic swine by evaluating inflammation, collagen capsule formation, and angiogenesis. Steel wire substrates were first modified with polyurethane membranes capable of diverse NO-release kinetics (NO fluxes and release durations of 0.8-630.0 pmol cm-2 s-1 and 2-13 d, respectively). The NO-releasing materials were implanted in the subcutis for 3, 10, or 25 d for histological and immunohistochemical evaluation of the FBR. A delayed, more severe inflammatory response to control (i.e., non-NO-releasing) implants was observed in diabetic pigs relative to healthy swine. Regardless of the animal disease state, each NO-releasing implant tested elicited reduced inflammation compared to controls at both 3 and 10 d. However, only the NO-release materials capable of releasing low NO fluxes (0.8-3.3 pmol cm-2 s-1) for 7-13 d durations mitigated the inflammatory response at 25 d. Using immunohistochemical staining for the endothelial cell surface marker CD-31, we also observed poor blood vessel development at non-NO-releasing implants in diabetic swine. Relative to controls, NO-releasing implants with the longest NO-release duration (13 d) increased blood vessel densities by 47.1 and 70.4% in the healthy and diabetic pigs, respectively. In the healthy model, tissues surrounding the long NO-release materials contained sparse amounts of collagen, whereas implants with shorter NO-release durations (2, 3, and 7 d) were characterized with a dense collagen encapsulation layer, similar to controls. Collagen deposition in diabetic swine was inhibited, and unaffected by NO. These results emphasize several key differences in the FBR in the setting of acute onset diabetes. The observation that NO release counteracts the more severe FBR in diabetic swine while simultaneously promoting tissue integration may help guide the design of medical implants (e.g., glucose sensors) with improved performance for diabetes management.
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Diabetes Mellitus Experimental/fisiopatologia , Reação a Corpo Estranho/patologia , Implantes Experimentais , Inflamação/patologia , Neovascularização Patológica/patologia , Óxido Nítrico/metabolismo , Poliuretanos/química , Animais , Colágeno/metabolismo , Feminino , Reação a Corpo Estranho/metabolismo , Masculino , Neovascularização Patológica/metabolismo , Tela Subcutânea/metabolismo , Tela Subcutânea/patologia , SuínosRESUMO
Chitosan was selectively monophosphorylated via reaction with phosphorus oxychloride (POCl3) to enhance water solubility while avoiding polyphosphate formation. The use of POCl3 resulted in negligible product degradation (i.e., breakdown of O-glycosidic bonds) even after a 3 d reaction period (<5% weight loss). X-ray photoelectron spectroscopy (XPS) characterization of the POCl3-phosphorylated chitosan (P-chitosan) revealed a phosphorus to nitrogen (P/N) atomic ratio of 0.30. Phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy verified the monophosphorylation of chitosan's primary and secondary alcohols, and primary amines. The calcium chelation efficiency for the phosphorylated product approached 0.05 mg Ca2+ per mg of P-chitosan as measured by inductively coupled plasma-optical emission spectrometry (ICP-OES), indicating improved chelation over native chitosan. This selective monophosphorylation approach proved useful for modifying other biopolymers, including cellulose and alginate.
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Nitric oxide (NO)-releasing polymers have proven useful for improving the biocompatibility of in vivo glucose biosensors. Unfortunately, leaching of the NO donor from the polymer matrix remains a critical design flaw of NO-releasing membranes. Herein, a toolbox of NO-releasing silica nanoparticles (SNPs) was utilized to systematically evaluate SNP leaching from a diverse selection of biomedical-grade polyurethane sensor membranes. Glucose sensor analytical performance and NO-release kinetics from the sensor membranes were also evaluated as a function of particle and polyurethane (PU) chemistries. Particles modified with N-diazeniumdiolate NO donors were prone to leaching from PU membranes due to the zwitterionic nature of the NO donor modification. Leaching was minimized (<5% of the entrapped silica over 1 month) in low water uptake PUs. However, SNP modification with neutral S-nitrosothiol (RSNO) NO donors lead to biphasic leaching behavior. Particles with low alkanethiol content (<3.0 wt % sulfur) leached excessively from a hydrogel PU formulation (HP-93A-100 PU), while particles with greater degrees of thiol modification did not leach from any of the PUs tested. A functional glucose sensor was developed using an optimized HP-93A-100 PU membrane doped with RSNO-modified SNPs as the outer, glucose diffusion-limiting layer. The realized sensor design responded linearly to physiological concentrations of glucose (minimum 1-21 mM) over 2 weeks incubation in PBS and released NO at >0.8 pmol cm-2 s-1 for up to 6 days with no detectable (<0.6%) particle leaching.
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Nitric oxide-releasing mesoporous silica nanoparticles (MSNs) were prepared using an aminosilane-template surfactant ion exchange reaction. Initially, bare silica particles were synthesized under basic conditions in the presence of cetyltrimethylammonium bromide (CTAB). These particles were functionalized with nitric oxide (NO) donor precursors (i.e., secondary amines) via the addition of aminosilane directly to the particle sol and a commensurate ion exchange reaction between the cationic aminosilanes and CTAB. N-Diazeniumdiolate NO donors were formed at the secondary amines to yield NO-releasing MSNs. Tuning of the ion exchange-based MSN modification approach allowed for the preparation of monodisperse particles ranging from 30 to 1100 nm. Regardless of size, the MSNs stored appreciable levels of NO (0.4-1.5 µmol mg(-1)) with tunable NO release durations (1-33 h) dependent on the aminosilane modification. Independent control of NO release properties and particle size was achieved, demonstrating the flexibility of this novel MSN synthesis over conventional co-condensation and surface grafting strategies.
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Aminas/química , Óxido Nítrico/química , Silanos/química , Dióxido de Silício/química , Tensoativos/química , Troca Iônica , Cinética , Medições Luminescentes , Espectroscopia de Ressonância Magnética , Nanopartículas/química , Nanopartículas/ultraestrutura , Nitrogênio/química , Tamanho da Partícula , PorosidadeRESUMO
The fabrication of electrospun composite polyurethane fibers capable of dual-action antibacterial dendrimer release is reported. Generation 4 (G4) poly(amidoamine) dendrimers were functionalized with octyl alkyl chain or quaternary ammonium (QA) moieties followed by modification of the resulting secondary amines with N-diazeniumdiolate nitric oxide (NO) donors to produce dual-action antibacterial dendrimers. Control and NO-releasing dendrimers were doped into polyurethane solutions prior to electrospinning of the polymer to yield well-defined dendrimer-doped composite polyurethane fibers. The fiber mats were semi-porous (≥30% porosity) and exhibited high water uptake (>100% relative to fiber mass). Dendrimer- and NO-release characteristics (rates and totals) were dependent on the dendrimer modification and polyurethane composition, with total dendrimer- and NO-release amounts ranging from 10 - 80 µg/mg and 0.027 - 0.072 µmol NO/mg, respectively. The antibacterial action of the fibers was evaluated against Gram-negative and Gram-positive bacterial strains. Nitric oxide-releasing fibers demonstrated broad-spectrum bactericidal action at short (2 h) and long (24 h) timescales.
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Hyperbranched polyesters with a range of exterior thiol modifications were synthesized through a Michael addition thiol-ene reaction. S-Nitrosothiol nitric oxide (NO) donors were subsequently introduced onto the scaffolds to yield NO-releasing polyesters with total NO storage of ~2.0 µmol mg-1. Multiple decomposition pathways (i.e., use of light, copper ions, and heat) triggered S-nitrosothiol NO donor breakdown and NO release under physiological conditions (37 °C, pH 7.4). The NO-releasing polyesters were characterized as a function of chemical modification and scaffold size or generation. The approaches described herein expand the scope of biodegradable NO-releasing materials with large NO payloads.
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The utility of continuous glucose monitoring devices remains limited by an obstinate foreign body response (FBR) that degrades the analytical performance of the in vivo sensor. A number of novel materials that resist or delay the FBR have been proposed as outer, tissue-contacting glucose sensor membranes as a strategy to improve sensor accuracy. Traditionally, researchers have examined the ability of a material to minimize the host response by assessing adsorbed cell morphology and tissue histology. However, these techniques do not adequately predict in vivo glucose sensor function, necessitating sensor performance evaluation in a relevant animal model prior to human testing. Herein, the effects of critical experimental parameters, including the animal model and data processing methods, on the reliability and usefulness of preclinical sensor performance data are considered.
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Materiais Biocompatíveis , Técnicas Biossensoriais/instrumentação , Glicemia/análise , Animais , Modelos Animais de Doenças , Teste de MateriaisRESUMO
S-Nitrosothiol-modified chitosan oligosaccharides were synthesized by reaction with 2-iminothiolane hydrochloride and 3-acetamido-4,4-dimethylthietan-2-one, followed by thiol nitrosation. The resulting nitric oxide (NO)-releasing chitosan oligosaccharides stored â¼0.3µmol NO mg(-1) chitosan. Both the chemical structure of the nitrosothiol (i.e. primary and tertiary) and the use of ascorbic acid as a trigger for NO donor decomposition were used to control the NO-release kinetics. With ascorbic acid, the S-nitrosothiol-modified chitosan oligosaccharides elicited a 4-log reduction in Pseudomonas aeruginosa viability. Confocal microscopy indicated that the primary S-nitrosothiol-modified chitosan oligosaccharides associated more with the bacteria relative to the tertiary S-nitrosothiol system. The primary S-nitrosothiol-modified chitosan oligosaccharides elicited minimal toxicity towards L929 mouse fibroblast cells at the concentration necessary for a 4-log reduction in bacterial viability, further demonstrating the potential of S-nitrosothiol-modified chitosan oligosaccharides as NO-release therapeutics.