RESUMEN
This work investigates the micellar titration of phenytoin (a weakly acidic drug) with cetyltrimethylammonium hydroxide (CTAH) to form a hydrophobic ion-pair to enhance oil solubility of phenytoin, followed by an effort to formulate nanoemulsion that could potentially prevent precipitation of phenytoin at physiological pH. The ion-pair formulated in nanoemulsion was evaluated for in vitro precipitation during serial dilution at physiological pH. The formation of ion-pair during titration was explained in context of pH-solubility data. The mathematical model successfully integrated ionization and micellization equilibria to reflect on dominant mechanisms for solubilization. The micellar phenomenon during titration was confirmed using Dynamic Light Scattering (DLS). The phase changes of the excess undissolved solids during titration were evident from X-Ray Powder Diffraction (XRPD) and Fourier Transform Infrared Spectroscopy (FTIR). This analysis confirmed the conversion of phenytoin into ionized state and its subsequent ionic interaction with CTAH forming hydrophobic ion-pair complex (HIP). The complete ion pair formation was evident at pHmax (8.8 to 9.2), and its 1:1 stoichiometry was confirmed using HPLC (Phenytoin and CTAH) and H1 NMR, hence could also be called as a lipophilic salt. The ion-pair (salt) was insoluble in water and showed remarkably high partition coefficient (log P) in octanol/water. As characterized by Hot Stage Microscopy (HSM), the melting point of the ion-pair complex was lowered to 150.8°C compared to the free acid (> 300οC), this was even further lowered to 81.1 °C when evaluated in castor oil. This led to approximately eight-fold higher solubility of hydrophobic ion pair (HIP) in castor oil compared to the free acid form. The high miscibility in castor oil was suitable to formulate a high drug load injectable dispersed system. This was successfully achieved with lecithin and polysorbate as emulsifiers without leaching drug into continuous phase at pH 7.4. This nanoemulsion (<300 nm, and > +30 mV zeta potential) remain stable when evaluated over a period of one month. A serial dilution study of the nanoemulsion was performed in PBS buffer, microscopic observations suggested no birefringence despite incubation at 25°C for several hours. This result indicated that Phenytoin remained strongly partitioned within dispersed oily phase with a higher drug loading when ion-paired phenytoin was used. The higher drug load could enable a small volume slow bolus injection to meet 50 mg/min or lower delivery rate criteria for Phenytoin in the clinical set up. This provided a pathway to further explore potential injectable nano-emulsion formulations that could alleviate typical phlebitis issue associated with the injectable phenytoin solution administration at physiological pH.
Asunto(s)
Aceite de Ricino , Fenitoína , Solubilidad , Emulsiones , Micelas , Concentración de Iones de Hidrógeno , Agua/químicaRESUMEN
A phase I trial (NCT03447314; 204686) evaluated the safety and efficacy of GSK1795091, a Toll-like receptor 4 (TLR4) agonist, in combination with immunotherapy (GSK3174998 [anti-OX40 monoclonal antibody], GSK3359609 [anti-ICOS monoclonal antibody], or pembrolizumab) in patients with solid tumors. The primary endpoint was safety; other endpoints included efficacy, pharmacokinetics, and pharmacodynamics (PD). Manufacturing of GSK1795091 formulation was modified during the trial to streamline production and administration, resulting in reduced PD (cytokine) activity. Fifty-four patients received GSK1795091 with a combination partner; 32 received only the modified GSK1795091 formulation, 15 received only the original formulation, and seven switched mid-study from the original to the modified formulation. Despite the modified formulation demonstrating higher systemic GSK1795091 exposure compared with the original formulation, the transient, dose-dependent elevations in cytokine and chemokine concentrations were no longer observed (e.g., IP-10, IL10, IL1-RA). Most patients (51/54; 94%) experienced ≥1 treatment-emergent adverse event (TEAE) during the study. Safety profiles were similar between formulations, but a higher incidence of TEAEs associated with immune responses (chills, fatigue, pyrexia, nausea, and vomiting) were observed with the original formulation. No conclusions can be made regarding GSK1795091 anti-tumor activity due to the limited data collected. Manufacturing changes were hypothesized to have caused the change in biological activity in this study. Structural characterization revealed GSK1795091 aggregate size in the modified formulation to be twice that in the original formulation, suggesting a negative correlation between GSK1795091 aggregate size and PD activity. This may have important clinical implications for future development of structurally similar compounds.
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Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias , Humanos , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Citocinas , Lípido A/uso terapéutico , Neoplasias/tratamiento farmacológico , Receptor Toll-Like 4/agonistas , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversosRESUMEN
The loss of active substance, both small and large molecules, from sterile liquid drug products after contact with an administration kit has been extensively reported in the literature. This loss has been reported to be caused by incompatibility of the active substances with the contact surfaces of the administration kit and adsorption or sticking of the active substance to the surfaces of the administration kit. This paper investigates the mechanism for loss of a highly potent active substance based on the type and design of the administration kit. Two administration kits (syringe/Insyte Catheter and syringe/Nexiva Catheter) of different designs were used to administer a solution formulation of an ultra-low dose (nanograms) of a model hydrophobic active substance Compound X. The Nexiva Catheter was longer with tubing and Y connectors while the Insyte Catheter was shorter with no split septum tubing. Dose recovery from both administration kits was determined using high pressure liquid chromatography. The results indicated that the full dose was recovered from the syringes and Insyte Catheter. However, there was a significant loss of active substance from the Nexiva Catheter configuration even after post administration flush, which was due to holdup volume of the formulation within dead spaces of the Nexiva Catheter. It was also demonstrated that the dose recovery from the Nexiva Catheter can be significantly increased with increase in the post administration flush volume, which further confirms that the observed loss of active substance was not due to incompatibility or surface adsorption. The significance of this work is to provide awareness to formulation scientists that closed system Catheter design with Y connectors can be the main contributor for the loss in active substance, especially at ultra-low doses, and therefore dose recovery experiments should be expanded to include proper flushing of the Y connectors to expel any holdup volume from the Catheter.
Asunto(s)
Jeringas , Administración Intravenosa , Infusiones IntravenosasRESUMEN
The aim of this work was to identify stable topical platform cream formulations (placebo creams without active drug substance) using the quality attributes of cream consistency, droplet size distribution (<1 µm), and separation or instability index of <0.1 to accelerate the development of topical cream drug product. The formulations were developed with six emulsifier systems that were screened in three different solvent systems across a range of emulsifier ratios. Each formulation was characterized by microscopy, separation index, and consistency. The results showed that there are three emulsifier combination (PEG 40 stearate:GMS, S21:S2, and PEG 40 stearate:Span 60) that works well with the solvent systems. Platform cream formulations F4, F15, F33, F40, F52, F69, F77, F87, and F106 were found to meet the three criteria for a long-term stable platform cream formulation. Formulation development for topical administered drug product can be very time consuming, expensive, and resourceful in identifying a chemically and physically stable product. In early development, where it can take 1-2 years to develop a first time in human (FTIH) formulation for a new chemical entity. The use of the platform base cream formulations will expedite the early development timeline for new chemical entity by 3-6 months.
Asunto(s)
Química Farmacéutica/métodos , Composición de Medicamentos/métodos , Desarrollo de Medicamentos/métodos , Crema para la Piel/síntesis química , Administración Tópica , Emulsionantes/administración & dosificación , Emulsionantes/síntesis química , Crema para la Piel/administración & dosificaciónRESUMEN
This study investigates the influence of surfactant sodium lauryl sulfate (SLS) on the solubility of poorly-water soluble drug substances, model Compound X and Compound Y, used in a fixed dose combination oral solid dosage form. To determine the impact of SLS concentration on the solubility of compounds X and Y, we experimentally determined the critical micelle concentration (CMC) of SLS in water, simulated gastric fluid (SGF), and fed state simulated intestinal fluid (FeSSIF) in the presence of Compound X and Compound Y using UV/Visible spectrophotometry at 25°C. The aggregation of SLS was characterized by calculating the standard Gibbs free energy of micellization in all the media investigated. To enhance the understanding of SLS aggregation, high throughput experiments and in-vivo mechanistic modelling were used to determine the effect of increasing levels of SLS on the solubility of compounds X and Y as both single agent and combination products to be formulated into a suitable oral solid dosage form. Micellar formation of SLS is a spontaneous process as shown by the negative values of the standard free energy of micellization. The CMC of SLS in the various media investigated in the presence of compounds X and Y decreases in the following order: water> FeSSIF> SGF. However, the aggregation of SLS in the various media is overall more spontaneous in the following order: SGF>FeSSIF>water. Using high throughput experimentation and in-vivo mechanistic modelling, it was determined that a combination oral solid product of compounds X and Y will have optimum solubility and in-vivo absorption if 2 mg of SLS was used in the oral solid dosage form. The results obtained from this study will help broaden the understanding of the micellization process involving SLS and poorly-water soluble drugs used in combination oral solid dosage forms.
Asunto(s)
Absorción Intestinal , Modelos Biológicos , Dodecil Sulfato de Sodio/química , Dodecil Sulfato de Sodio/farmacocinética , Tensoactivos/química , Tensoactivos/farmacocinética , Células CACO-2 , Humanos , Secreciones Intestinales/química , Micelas , Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/metabolismo , Solubilidad , Agua/químicaRESUMEN
The packaging presentation of oral liquid pediatric medicines is a critical step in maintaining chemical and physical stability, compliance, adherence, and proper handling by the target patient population, guardians, caregivers, and health-care professionals. The common packaging presentations for commercial oral liquid pediatric drug products are glass bottle, plastic bottle, sachet, and stick pack configurations. The type of pack presentation selected is driven by the quality target product profile (QTPP) that is designed around the physicochemical properties of the drug substance and the desired drug product suitability for the target population. The QTPP defines the intended use of the drug product, drug product quality criteria, dose strength, dosage form, container closure system, storage conditions, stability criteria, dosing device, shelf life, and attributes affecting the pharmacokinetic characteristics. Oral liquid pediatric formulations are typically prepared from a powder that is constituted at the time of use as a suspension or a solution for single or multiple use depending on the stability of the constituted formulation. Active ingredients with high aqueous solubility can be developed as a powder for oral solution and presented in a bottle for multiple use product and a stick pack, packet, or sachet for single-use product. Active ingredients with low aqueous solubility can be developed as a powder for oral suspension and presented in a bottle for multiple use product and a stick pack or sachet for single-use product. A secondary package may be used in cases where the primary pack failed to provide adequate protection against light degradation. This work will help formulation scientists select the most appropriate pack presentation in the early stages of pediatric clinical development.
Asunto(s)
Embalaje de Medicamentos , Drogas en Investigación/química , Pediatría/métodos , Farmacología Clínica/métodos , Administración Oral , Adolescente , Fenómenos Químicos , Química Farmacéutica , Niño , Preescolar , Composición de Medicamentos , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Drogas en Investigación/administración & dosificación , Humanos , Lactante , Soluciones Farmacéuticas , Polvos , Solubilidad , Suspensiones , Gestión de la Calidad TotalRESUMEN
Piezoelectric-excited millimeter-sized cantilever (PEMC) biosensors were fabricated and functionalized with immunoglobulin M (IgM) for the detection of Cryptosporidium parvum oocyst in a flow configuration at 1 mL/min. The detection of 100, 1000, and 10,000 oocysts/mL was achieved with a positive sensor response in less than 1 min. Bovine serum albumin (BSA) was used as a blocking agent in each experiment and was shown to eliminate non-specific binding. The sensor's resonance frequency response correlates with C. parvum oocyst concentration logarithmically. The oocyst attachment rate was found to increase by an order of magnitude in increasing concentration from 100 to 10,000 oocysts/mL. The significance of these results is that IgM-functionalized PEMC sensors are highly selective and sensitive to C. parvum oocyst and therefore, have the potential to accurately identify and quantify C. parvum oocyst in drinking water.
Asunto(s)
Técnicas Biosensibles/instrumentación , Cryptosporidium/aislamiento & purificación , Inmunoensayo/instrumentación , Inmunoglobulina M/inmunología , Oocistos/inmunología , Transductores , Contaminantes del Agua/análisis , Animales , Técnicas Biosensibles/métodos , Sistemas de Computación , Inmunoensayo/métodos , Inmunoglobulina M/química , MiniaturizaciónRESUMEN
Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors immobilized with antibody specific to Escherichia coli (EC) 0157:H7 is used to detect EC at 1 cell/mL in 1 mL and 1 L samples in a batch and flow mode, respectively. Two sensor designs were used. The first design (PEMC-a) has both the piezoelectric and non-piezoelectric layer anchored, while in the second design (PEMC-b) had only the piezoelectric layer anchored. PEMC-a, used in batch mode with 1 mL sample, showed limit of detection at 10 cells/ mL using the second bending mode at 85.5 kHz in air. PEMC-b exhibited resonant frequencies at 186.5, 883.5, and 1778.5 kHz in air and 162.5, 800.0, and 1725.5 kHz in sample flow conditions. A one-liter sample containing 1000 EC cells was introduced at 1.5, 2.5, 3, and 17 ml/min, and the change in resonant frequency was monitored. The total frequency change observed for the mode at 800 kHz and sample flow rates of 1.5, 2.5, 3, and 17 mL/min were 2230+/-11, 3069 +/-47, 4686+/-97, and 7188+/-52 Hz, respectively. Each detection experiment was confirmed by exposing the sensor to a low pH solution followed by a phosphate buffered saline (PBS) rinse, which caused the release of the attached EC. The final frequency change observed was nearly identical to the value prior to EC attachment. Kinetic analysis showed that the observed binding rate constant at 1.5, 2.5, 3 mL/min were 0.009, 0.015, and 0.021 min(-1), respectively. The significance of these results is that very low concentration of pathogens in large sample volumes can be measured in a short time period without the need for filtration or enrichment.
Asunto(s)
Escherichia coli O157/aislamiento & purificación , ElectricidadRESUMEN
A sensitive and reliable method for the detection of Bacillus anthracis (BA; Sterne strain 7702) spores in presence of large amounts of Bacillus thuringiensis (BT) and Bacillus cereus (BC) is presented based on a novel PZT-anchored piezoelectric excited millimeter-sized cantilever (PAPEMC) sensor with a sensing area of 1.5 mm2. Antibody (anti-BA) specific to BA spores was immobilized on the sensing area and exposed to various samples of BA, BT, and BC containing the same concentration of BA at 333 spores/mL, and the concentration of BT + BC was varied in concentration ratios of (BA:BT + BC) 0:1, 1:0, 1:1, 1:10, 1:100, and 1:1000. In each case, the sensor responded with an exponential decrease in resonant frequency and the steady-state frequency changes reached were 14 +/- 31 (n = 11), 2742 +/- 38 (n = 3), 3053 +/- 19 (n = 2), 2777 +/- 26 (n = 2), 2953 +/- 24 (n = 2), and 3105 +/- 27 (n = 2) Hz, respectively, in 0, 27, 45, 63, 154, and 219 min. The bound BA spores were released in each experiment, and the sensor response was nearly identical to the frequency change during attachment. These results suggest that the transport of BA spores to the antibody immobilized surface was hindered by the presence of other Bacillus species. The observed binding rate constant, based on the Langmuir kinetic model, was determined to be 0.15 min-1. A hindrance factor (alpha) is defined to describe the reduced attachment rate in the presence of BT + BC and found to increase exponentially with BT and BC concentration. The hindrance factor increased from 3.52 at 333 BT + BC spores/mL to 11.04 at 3.33 x 105 BT + BC spores/mL, suggesting that alpha is a strong function of BT and BC concentration. The significance of these results is that anti-BA functionalized PEMC sensors are highly selective to Bacillus anthracis spores and the presence of other Bacillus species, in large amounts, does not prevent binding but impedes BA transport to the sensor.
Asunto(s)
Bacillus/citología , Técnicas Biosensibles/métodos , Esporas Bacterianas , Anticuerpos Antibacterianos , Bacillus anthracis/citología , Bacillus cereus/citología , Bacillus thuringiensis/citología , Cinética , MétodosRESUMEN
Rapid (approximately 10 min) measurement of very low concentration of pathogens (approximately 10 cells/mL) and protein (approximately fg/mL) has widespread use in medical diagnostics, monitoring biothreat agents, and in a broader context as a research method. For low-level pathogen, we currently use culture enrichment methods and, thus, rapid analysis is not possible. For low protein concentration, no direct method is currently available. We report here a novel macrocantilever design whose high-order resonant mode near 1 MHz exhibits mass detection sensitivity of 10 cells/mL for cells and 100 fg/mL for protein. The sensor is 1x3 mm and uses a piezoelectric layer for both actuation and sensing resonance. Sample is flowed (approximately 1 mL/min) past the antibody-immobilized sensor, and as antigen binds to the sensor, resonance frequency decreases in proportion to antigen concentration. The sensor showed selectivity to the pathogen even though copious nonpathogenic variant was simultaneously present.
Asunto(s)
Escherichia coli/química , Proteínas del Tejido Nervioso/análisis , Albúmina Sérica Bovina/análisis , Animales , Anticuerpos/química , Anticuerpos/inmunología , Reacciones Antígeno-Anticuerpo , Bovinos , Electrodos , Escherichia coli/inmunología , Escherichia coli/patogenicidad , Proteínas del Tejido Nervioso/inmunología , Sensibilidad y Especificidad , Albúmina Sérica Bovina/inmunología , Factores de TiempoRESUMEN
Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors consisting of a piezoelectric and a borosilicate glass layer with a sensing area of 4 mm2 were fabricated. An antibody specific to Escherichia coli (anti-E. coli) O157:H7 was immobilized on PEMC sensors, and exposed to samples containing E. coli O157:H7 (EC) prepared in various matrices: (1) broth, broth plus raw ground beef, and broth plus sterile ground beef without inoculation of E. coli O157:H7 served as controls, (2) 100 mL of broth inoculated with 25 EC cells, (3) 100 mL of broth containing 25 g of raw ground beef and (4) 100 mL of broth with 25 g of sterile ground beef inoculated with 25 EC cells. The total resonant frequency change obtained for the broth plus EC samples were 16+/-2 Hz (n=2), 30 Hz (n=1), and 54+/-2 Hz (n=2) corresponding to 2, 4, and 6h growth at 37 degrees C, respectively. The response to the broth plus 25 g of sterile ground beef plus EC cells were 21+/-2 Hz (n=2), 37 Hz (n=1), and 70+/-2 Hz (n=2) corresponding to 2, 4, and 6 h, respectively. In all cases, the three different control samples yielded a frequency change of 0+/-2 Hz (n=6). The E. coli O157:H7 concentration in each broth and beef samples was determined by both plating and by pathogen modeling program. The results indicate that the PEMC sensor detects E. coli O157:H7 reliably at 50-100 cells/mL with a 3 mL sample.
Asunto(s)
Técnicas Biosensibles/instrumentación , Escherichia coli O157/aislamiento & purificación , Microbiología de Alimentos , Carne/microbiología , Animales , BovinosRESUMEN
In this paper, we describe a new modality of measuring human serum albumin (HSA) adsorption continuously on CH3-, COOH-, and OH-terminated self-assembled monolayers (SAMs) of C11-alkanethiols and the direct quantification of the adsorbed amount. A gold-coated piezoelectric-excited millimeter-sized cantilever (PEMC) sensor of 6-mm2 sensing area was fabricated, where resonant frequency decreases upon mass increase. The resonant frequency in air of the detection peak was 45.5 +/- 0.01 kHz. SAMs of C11-thiols (in absolute ethanol) with different end groups was prepared on the PEMC sensor and then exposed to buffer solution containing HSA at 10 microg/mL. The resonant frequency decreased exponentially and reached a steady-state value within 30 min. The decrease in resonant frequency indicates that the mass of the sensor increased due to HSA adsorption onto the SAM layer. The frequency change obtained for the HSA adsorption on CH3-, COOH-, and OH-terminated SAM were 520.8 +/- 8.6 (n = 3), 290.4 +/- 6.1 (n = 2), and 210.6 +/- 8.1 Hz (n = 3), respectively. These results confirm prior conclusions that albumin adsorption decreased in the order, CH(3) > COOH > OH. Observed binding rate constants were 0.163 +/- 0.003, 0.248 +/- 0.006, and 0.381 +/- 0.001 min(-1), for methyl, carboxylic, and hydroxyl end groups, respectively. The significance of the results reported here is that both the formation of self-assembled monolayers and adsorption of serum protein onto the formed layer can be measured continuously, and quantification of the adsorbed amount can be determined directly.
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Albúminas/análisis , Alcanos/química , Materiales Biocompatibles/química , Técnicas Biosensibles/métodos , Oro/química , Compuestos de Sulfhidrilo/química , Adsorción , Albúminas/metabolismo , Técnicas Biosensibles/instrumentación , Electroquímica , Diseño de Equipo , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Propiedades de Superficie , Factores de TiempoRESUMEN
Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors of 4mm(2) sensing area were immobilized with antibody specific to Bacillus anthracis (anti-BA) spores or bovine serum albumin (anti-BSA). Detection of pathogen (Bacillus anthracis (BA) at 300 spores/mL) and BSA (1 mg/mL) were investigated under both stagnant and flow conditions. Two flow cell designs were evaluated by characterizing flow-induced resonant frequency shifts. One of the flow cells labeled SFC-2 (hold-up volume of 0.3 mL), showed small fluctuations (+/-20 Hz) around a common resonant frequency response of 217 Hz in the flow rate range of 1-17 mL/min. The total resonant frequency change obtained for the binding of 300 spores/mL in 1h was 90+/-5 Hz (n=2), and 162+/-10 Hz (n=2) under stagnant and flow conditions, respectively. Binding of antibodies, anti-BA and anti-BSA, were more rapid under flow than under stagnant conditions. The sensor was repeatedly exposed to BSA with an intermediate release step. The first and second responses to BSA were nearly identical. The total resonant frequency response to BSA was 388+/-10 (n=2) Hz under flow conditions. Kinetic analysis is carried out to quantify the effect of flow rate on antibody immobilization and the two types of detection experiments.
Asunto(s)
Bacillus anthracis/aislamiento & purificación , Técnicas Biosensibles , Albúmina Sérica Bovina/análisis , Animales , Bacillus anthracis/inmunología , Técnicas Biosensibles/instrumentación , Bovinos , Esporas Bacterianas/inmunología , Esporas Bacterianas/aislamiento & purificaciónRESUMEN
To enhance the mass change sensitivity of the resonating piezoelectric-excited millimeter-sized cantilever (PEMC) sensors, we reduced its length and eliminated one layer of its composite structure. As a result the mass sensitivity of the second flexural mode increased by two orders of magnitude (from 10(-9) to 10(-11)g/Hz) and the resonant frequency increased by more than 5 kHz. We demonstrate the effects of modification by detecting a model pathogen Group A Streptococcus (GAS) at 700 cells/mL. The resonant frequency change of the second mode at concentrations of 700, 7 x 10(3), 7 x 10(5), 7 x 10(6), 7 x 10(7), and 7 x 10(9)cells/mL resulted in, respectively, 3.1+/-0.5, 11.6+/-1, 15.7+/-1, 25.7+/-0.15, 28.5+/-2, and 40.5+/-3 ng (n=3 for all) of pathogen attachment. A kinetic model for the binding is proposed and verified. The observed binding rate constant was found to be in the range of 0.051-0.166 min(-1). The significance of the results we report is that the modified PEMC sensors have high mass sensitivity that pathogens can be detected at very low concentration under liquid immersion conditions.
Asunto(s)
Técnicas Biosensibles/instrumentación , Streptococcus pyogenes/química , Aire , Plomo , Titanio , CirconioRESUMEN
Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors consisting of a piezoelectric and a borosilicate glass layer with a sensing area of 2.48 mm2 were fabricated. Antibody specific to Bacillus anthracis (BA, Sterne strain 7702) spores was immobilized on PEMC sensors, and exposed to spores (300 to 3x10(6) spores/mL). The resonant frequency decreased at a rate proportional to the spore concentration and reached a steady state frequency change of 5+/-5 Hz (n=3), 92+/-7 Hz (n=3), 500+/-10 Hz (n=3), 1030+/-10 Hz (n=2), and 2696+/-6 Hz (n=2) corresponding to 0, 3x10(2), 3x10(3), 3x10(4), and 3x10(6) spores/mL, respectively. The reduction in resonant frequency is proportional to the change in cantilever mass, and thus the observed changes are due to the attachment of spores on the sensor surface. Selectivity of the antibody-functionalized sensor was determined with samples of BA (3x10(6)/mL) mixed with Bacillus thuringiensis (BT; 1.5x10(9)/mL) in various volume ratios that yielded BA:BT ratios of 1:0, 1:125, 1:250, 1:500 and 0:1. The corresponding resonance frequency decreases were, respectively, 2345, 1980, 1310, 704 and 10 Hz. Sample containing 100% BT spores (1.5x10(9)/mL and no BA) gave a steady state frequency decrease of 10 Hz, which is within noise level of the sensor, indicating excellent selectivity. The observed binding rate constant for the pure BA and BT-containing samples ranged from 0.105 to 0.043 min-1 in the spore concentration range 300 to 3x10(6)/mL. These results show that detection of B. anthracis spore at a very low concentration (300 spores/mL) and with high selectivity in presence of another Bacillus spore (BT) can be accomplished using piezoelectric-excited millimeter-sized cantilever sensors.
Asunto(s)
Bacillus anthracis/aislamiento & purificación , Técnicas Biosensibles/instrumentación , Recuento de Colonia Microbiana/instrumentación , Electroquímica/instrumentación , Transductores , Técnicas Biosensibles/métodos , Recuento de Colonia Microbiana/métodos , Electroquímica/métodos , Diseño de Equipo , Análisis de Falla de Equipo , Inmunoensayo/instrumentación , Inmunoensayo/métodos , Miniaturización , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Esporas Bacterianas/aislamiento & purificaciónRESUMEN
In this paper, we describe a new method of measuring alkanethiol monolayer formation on a gold surface. A gold-coated millimeter-sized rectangular-shaped lead zirconate titanate (PZT) cantilever of dimensions 3.5 x 2 x 0.05 mm, previously shown to detect a picogram level of mass change, was used to measure the adsorption kinetics of 1-hexadecanethiol in ethanol over six orders of concentration range (1 nM to 10 mM) in real time. The flexural mode of cantilever vibration, 45.5 +/- 0.01 kHz, was monitored during the self-assembly. The total resonant frequency change obtained for the 1 nM, 10 nM, 100 nM, 1 microM, 4 mM, 8 mM, and 10 mM thiol concentrations were 116 +/- 2 (n = 2), 225 (n = 1), 270 +/- 10 (n = 2), 440 +/- 10 (n = 2), 900 +/- 10 (n = 2), 900 +/- 10 (n = 2), and 900 +/- 10 (n = 2) Hz, respectively. These results compare favorably to literature results in that the rate of the monolayer formation is concentration-dependent and the exponential change during adsorption follows the reversible first-order Langmuir kinetic model. The rate constants of adsorption and desorption were 0.061 M(-1) s(-1) and 3.61 x 10(-4) s(-1), respectively. The significance of the results is that millimeter-sized PZT cantilevers can be used in real-time for characterizing self-assembly of monolayer formation at nanomolar concentration levels. In addition, at 1 nM, the adsorption was found not to be diffusion limited.
Asunto(s)
Oro , Adsorción , CinéticaRESUMEN
A composite self-excited PZT-glass cantilever (4mm in length and 2mm wide) was fabricated and used to measure the binding and unbinding of model proteins. A key feature of the cantilever is that its resonant frequency is dependent on its mass. The fabricated cantilever has mass change sensitivity in liquid of 7.2 x 10(-11)g/Hz. Resonant frequency change was measured as protein reacted or bound with the sensing glass cantilever surface. Protein concentrations, 0.1 and 1.0mg/mL, which resulted in nanogram mass change were successfully detected. The mass change sensitivity gave a total mass change of 54+/-0.45 ng for the binding of anti-rabbit IgG (biotin conjugated) to rabbit IgG immobilized cantilever and the subsequent binding of captavidin. The unbinding of anti-rabbit IgG and captavidin gave a total mass change of 54+/-1.70 ng. Fluorescence based assays showed the combined mass of both proteins in the released samples was 54+/-2.24 ng. The binding kinetics of the model proteins is modeled as first order. The initial binding rate constant of anti-rabbit IgG to rabbit IgG was 1.36+/-0.02(min(mg/mL))(-1). The initial binding rate constant of captavidin to biotinylated anti-rabbit IgG was (2.57 x 10(-1))+/-0.003(min(mg/mL))(-1). The significance of the results we report here is that millimeter-sized PZT-actuated glass cantilevers have the sensitivity to measure in real-time protein-protein binding, and the binding rate constant.
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Electroquímica/instrumentación , Plomo/química , Análisis por Matrices de Proteínas/instrumentación , Mapeo de Interacción de Proteínas/instrumentación , Proteínas/análisis , Proteínas/química , Titanio/química , Transductores , Circonio/química , Electroquímica/métodos , Diseño de Equipo , Análisis de Falla de Equipo , Vidrio/química , Miniaturización , Análisis por Matrices de Proteínas/métodos , Mapeo de Interacción de Proteínas/métodosRESUMEN
Composite self-excited PZT-glass cantilevers (5 and 3 mm in length, 1.8 and 2.0 mm wide) were fabricated and their resonance characteristics were determined in air and at 1 mm liquid immersion. In air, resonance occurred at 65.8 and 63.4 kHz for the two cantilevers used in this paper. Monoclonal antibody (MAb) specific to the pathogen Escherichia coli (E. coli) O157:H7 was immobilized at the cantilever glass tip, and then exposed to pathogen in the concentration range of 7x10(2) to 7x10(7)bacteria/mL. Resonance of the second mode decreased due to pathogen attachment in accordance with a proposed kinetic model. The specific attachment rate constant was found to be 3x10(-9) to 5x10(-9) min-1 (cell/mL)-1. Exposure to a mixed population containing both a pathogenic and non-pathogenic strain showed that the antibody-immobilized cantilever is highly selective, thus demonstrating its usefulness for detecting water-borne pathogens.
Asunto(s)
Técnicas Biosensibles/instrumentación , Recuento de Colonia Microbiana/instrumentación , Electroquímica/instrumentación , Escherichia coli O157/aislamiento & purificación , Inmunoensayo/métodos , Plomo/química , Titanio/química , Transductores , Circonio/química , Anticuerpos/inmunología , Técnicas Biosensibles/métodos , Materiales Biocompatibles Revestidos/química , Recuento de Colonia Microbiana/métodos , Electroquímica/métodos , Diseño de Equipo , Análisis de Falla de Equipo , Escherichia coli O157/inmunología , Vidrio/químicaRESUMEN
A composite self-excited millimeter-sized lead zirconate titanate (PZT) glass cantilever (2 mm x 1.8 mm; sensing area of 6 mm2) was fabricated for the detection of Escherichia coli (E. coli) O157:H7. The fundamental and second mode resonance in air was 10.95 +/- 0.05 kHz and 43.45 +/- 0.05 kHz, respectively. Affinity purified monoclonal antibody (anti-E. coli O157:H7) specific to the pathogen E. coli O157:H7 was immobilized at the cantilever glass tip, and then immersed in liquid containing the pathogen (70 to 7 x 10(7) cells/mL). The resonant frequency showed a reduction and reached a steady state shift of 0 +/- 5, 46 +/- 5, 260 +/- 5, and 1010 +/- 5 Hz corresponding to 0, 700, 7000, and 7 x 10(7) cells/mL. From the experiments conducted, the detection limit of the sensor was 700 cells/mL.