Phase-separation immunoassays.

1987 ◽  
Vol 33 (9) ◽  
pp. 1509-1516 ◽  
Author(s):  
K Auditore-Hargreaves ◽  
R L Houghton ◽  
N Monji ◽  
J H Priest ◽  
A S Hoffman ◽  
...  
Keyword(s):  
Reaction Kinetics ◽  
Solid Phase ◽  
Specific Binding ◽  
Hepatitis B Surface Antigen ◽  
Solution Temperature ◽  
Critical Solution Temperature ◽  
Binding Reaction ◽  
Insoluble Polymer ◽  
Capture Antibody

Abstract Solid-phase-based immunoassays have traditionally been plagued by nonspecific binding to the solid phase and by slow reaction kinetics relative to reactants that are free to diffuse in solution. We have developed two novel immunoassays in which the solid phase is generated in situ after the specific binding reaction has occurred, thereby enhancing reaction kinetics and minimizing the opportunities for non-specific binding. In the first system, the capture antibody is conjugated to an organic monomer, polymerization of which to form insoluble polymer particles is initiated by a reaction involving free radicals. The amount of signal-labeled antibody incorporated into the resulting particles is directly proportional to the concentration of antigen. The principle is illustrated for the simultaneous assay of IgG and IgM in a single sample. In the second system, capture antibody is conjugated to a polymer, the solubility of which is a function of temperature. Specific binding is conducted below the critical solution temperature of the polymer, which is then separated from solution by increasing the temperature above the critical temperature. The incorporation of signal-labeled antibody into the precipitated polymer is directly proportional to the concentration of antigen. This principle is illustrated for the assay of hepatitis B surface antigen and Chlamydia trachomatis.

N-isopropylacrylamide-based Copolymers with Time-dependent LCST for a Bioresorbable Carrier

2004 ◽  
Vol 845 ◽  
Author(s):  
Bae Hoon Lee ◽  
Brent Vernon
Keyword(s):  
Body Temperature ◽  
Time Dependent ◽  
Solution Temperature ◽  
Gelation Temperature ◽  
Critical Solution Temperature ◽  
New Class ◽  
Polymeric Biomaterials ◽  
Localized Delivery ◽  
Hydrolysis Of

ABSTRACTTo develop a new class of in situ-forming, injectable, and biodegradable polymeric biomaterials based on time-dependent lower critical solution temperature (LCST) properties for localized delivery, copolymers of N-isopropylacrylamide (NIPAAm), 2-hydroxyethyl methacryl lactate (HEMA-lactate) and acrylic acid (AAc) were prepared with varying mole ratios of monomers. The copolymers showed LCST and gelation properties below body temperature in 0.1 N PBS solution of pH 7.4. The LCST and gelation temperature of the copolymers decreased as the HEMA-lactate content of the copolymers was increased. The copolymers also showed time-dependent LCST and gelation properties in 0.1 N PBS solution of pH 7.4 owing to hydrolysis of HEMA-lactate. Hydrolysis of HEMA-lactate caused the polymers to be more hydrophilic, resulting in an increase in LCST and gelation temperature. All the polymers with about 6 mol % AAc exhibited LCST and gelation temperature above body temperature after complete hydrolysis of HEMA-lactate.

Probing the thermal collapse of PNIPAM grafts by quantitative in situ ellipsometry

2013 ◽  
Vol 1544 ◽  
Author(s):  
E.S. Kooij ◽  
X.F. Sui ◽  
M.A. Hempenius ◽  
H.J.W. Zandvliet ◽  
G.J. Vancso
Keyword(s):  
Density Profile ◽  
Spectroscopic Ellipsometry ◽  
Quantitative Description ◽  
Solution Temperature ◽  
Thickness Variation ◽  
Dense Layer ◽  
Chain Segment ◽  
Critical Solution Temperature ◽  
Gradient Density

ABSTRACTWe demonstrate the potential of spectroscopic ellipsometry for the investigation of the chain segment density profile and layer thickness during the temperature-induced, reversible collapse−expansion transition of poly(N-isopropylacrylamide) (PNIPAM) grafted layers with variable grafting densities in aqueous systems. To obtain a quantitative description of the thickness of our swollen PNIPAM layers, various models were implemented to fit the ellipsometric data. From the ellipsometry results, the density and thickness variation accompanying the collapse transition across the lower critical solution temperature (LCST) was characterized. The collapse can be adequately explained by considering the PNIPAM film to consist of two layers: (i) a dense layer near the surface and (ii) a more diluted layer with a gradient density profile on the side of the film exposed to the solvent.

scholarly journals Mass-sensing, multianalyte microarray immunoassay with imaging detection

1998 ◽  
Vol 44 (9) ◽  
pp. 2036-2043 ◽  
Author(s):  
John W Silzel ◽  
Bibijana Cercek ◽  
Charles Dodson ◽  
Tsong Tsay ◽  
Robert J Obremski
Keyword(s):  
Near Infrared ◽  
Solid Phase ◽  
Binding Assays ◽  
Mass Sensing ◽  
Reagent Consumption ◽  
Microarray Immunoassay ◽  
Imaging Detection ◽  
Capture Antibody ◽  
Near Infrared Dye

Abstract Miniaturization of ligand binding assays may reduce costs by decreasing reagent consumption, but it is less apparent that miniaturized assays can simultaneously exceed the sensitivity of macroscopic techniques by analyte “harvesting” to exploit the total analyte mass available in a sample. Capture reagents (avidin or antibodies) immobilized in 200-μm diameter zones are shown to substantially deplete analyte from a liquid sample during a 1–3-h incubation, and the assays that result sense the total analyte mass in a sample rather than its concentration. Detection of as few as 105 molecules of analyte per zone is possible by fluorescence imaging in situ on the solid phase using a near-infrared dye label. Single and multianalyte mass-sensing sandwich array assays of the IgG subclasses show the sensitivity and specificity of ELISA methods but use less than 1/100 the capture antibody required by the 96-well plate format.

scholarly journals Thermo-responsive super porous p(NIPAM) cryogels affords enhanced thermal stability and activity for ɑ-Glucosidase enzyme by entrapping in situ

2021 ◽  
Author(s):  
Sahin Demirci ◽  
Nurettin Sahiner
Keyword(s):  
Enzyme Immobilization ◽  
Storage Stability ◽  
Storage Conditions ◽  
Solution Temperature ◽  
Critical Solution Temperature ◽  
Enzyme Functionality ◽  
And Storage ◽  
Enzyme Entrapment ◽  
Temperature Storage

The concept of using a thermo-responsive p(NIPAM) polymer matrix for enzyme immobilization with lower critical solution temperature (LCST) value is rationalized by availability of the compartmental milieu to enzymes to operate within super porous 3-D matrix with special environmental conditions. Therefore, the enzyme immobilization within a support material will be carried out under the storage conditions of enzymes, generally ~-20 oC to afford unnecessarily loss of enzyme functionality in comparison to the other enzyme entrapment methods. Thus, here ɑ-Glucosidase as a model enzyme was entrapped within thermo-responsive super porous p(NIPAM) cryogels (ɑ-Glu@p(NIPAM) during the synthesis that uses cryogenic condition, ~-20 oC. The LSCT value for the prepared p(NIPAM) based cryogels were determined as 34.6±1.2 oC. The immobilization yield, immobilization efficiency, and activity recovery% values were calculated as 89.4±3.1, 66.2±3.3, and 74.0±3.3%, respectively at pH 6.8 and 37 oC for ɑ-Glu@p(NIPAM) cryogel system. Interestingly, the optimum working conditions were achieved as 25 oC and pH 6.8 with higher activity, 98.4±0.2% for the prepared ɑ-Glu@p(NIPAM) cryogel system. The operational and storage stability studies revealed that the prepared ɑ-Glu@p(NIPAM) cryogel system possessed much better operational and storage stability than free ɑ-Glu enzyme e.g., more than 50% activity after 10th usage and 10-day room temperature storage time. Moreover, the kinetic parameters such as Km and Vmax of free-Glu enzyme and ɑ-Glu@p(NIPAM) cryogel system were calculated by non-linear Michaelis-Menten equation.

Polydopamine Nanoparticle for Poly(N-Isopropylacrylamide)-Based Nanocomposite Hydrogel with Good Free-Radical-Scavenging Property

2016 ◽  
Vol 848 ◽  
pp. 94-98 ◽  
Author(s):  
Shu Qiang Xiong ◽  
Yan Wang ◽  
Jing Zhu ◽  
Zu Ming Hu ◽  
Jun Rong Yu
Keyword(s):  
Free Radical ◽  
Radical Scavenging ◽  
Antioxidant Property ◽  
Solution Temperature ◽  
Scavenge Activity ◽  
Initial Reaction ◽  
Critical Solution Temperature ◽  
Nanocomposite Hydrogels ◽  
Isopropyl Acrylamide

Nanocomposite hydrogels (NC gels) consisting of poly (N-Isopropyl acrylamide) (pNIPAM)/polydopamine nanoparticles (PDAPs) were prepared by in-situ-free-radical polymerization of N-isopropyl acrylamide in the presence of modified PDAP in aqueous solution. The composition of the NC gels could be controlled directly by altering the composition of the initial reaction mixture. The lower critical solution temperature (LCST) of the NC gels were studied by DSC, and the LCST of both of pure pNIPAM hydrogel and NC gels was at 34°C. Besides, the NC gels showed superior antioxidant property, and the ability to scavenge activity of NC gel was up to 70% with the addition of 6 wt% modified PDAP into pNIPAM.

Systematic Study on the Remote Triggering of Thermo-Responsive Hydrogels Using RF Heating of Fe3O4 Nanoparticles

2015 ◽  
Vol 1718 ◽  
pp. 35-40 ◽  
Author(s):  
Daniel Denmark ◽  
Devajyoti Mukherjee ◽  
Janae Bradley ◽  
Sarath Witanachchi ◽  
Pritish Mukherjee
Keyword(s):  
Systematic Study ◽  
Ion Concentration ◽  
Solution Temperature ◽  
Rf Heating ◽  
Critical Solution Temperature ◽  
Responsive Polymers ◽  
Transmission Electron ◽  
Remote Triggering ◽  
Responsive Hydrogels

ABSTRACTIn this work, a systematic study on the factors that influence the lower critical solution temperature (LCST) of poly(N-isopropylacrylamide) (PNIPAM) solutions during remote radiofrequency (RF) heating, using Fe3O4 magnetic nanoparticles (MNPs) is reported. A series of PNIPAM solutions with varying concentrations of Fe3O4 MNPs were prepared and characterized using transmission electron microscopy and Raman spectroscopy. Preliminary studies showed the highest specific absorption rate (SAR) for 15 nm sized Fe3O4 MNPs, which monotonically decreased as the MNP sizes increased to 20-30 nm. In-situ transmission measurements were used to determine the LCST of PNIPAM under various aqueous concentrations with dispersed Fe3O4 MNPs. A systematic decrease in the LCST from 34 °C to 31 °C was observed as the concentration of PNIPAM was increased from 0.3 wt. % to 1.0 wt. %, keeping the concentration of Fe3O4 MNPs constant. On the other hand, varying the concentrations of the MNPs did not drastically affect the LCSTs of PNIPAM solutions. However, varying the ion concentration of the PNIPAM solutions by adding adjusted KOH pellets, showed a pronounced lowering of the LCST by 2-3 °C at all PNIPAM concentrations. The remote triggering of phase transitions in PNIPAM solutions by raising the temperature above the LCST using Fe3O4 MNPs as reported here is important in targeted drug-delivery applications using thermo-responsive polymers.

Dynamic studies of silicide-mediated crystallization of amorphous silicon

1992 ◽  
Vol 50 (2) ◽  
pp. 1352-1353
Author(s):  
C. Hayzelden ◽  
J. L. Batstone
Keyword(s):  
Ion Implantation ◽  
Solid Phase ◽  
Metallic Phase ◽  
Single Crystal Substrate ◽  
Free Electrons ◽  
Melt Temperature ◽  
Transmission Electron ◽  
Crystal Substrate ◽  
High Resolution Tem

Epitaxial reordering of amorphous Si(a-Si) on an underlying single-crystal substrate occurs well below the melt temperature by the process of solid phase epitaxial growth (SPEG). Growth of crystalline Si(c-Si) is known to be enhanced by the presence of small amounts of a metallic phase, presumably due to an interaction of the free electrons of the metal with the covalent Si bonds near the growing interface. Ion implantation of Ni was shown to lower the crystallization temperature of an a-Si thin film by approximately 200°C. Using in situ transmission electron microscopy (TEM), precipitates of NiSi2 formed within the a-Si film during annealing, were observed to migrate, leaving a trail of epitaxial c-Si. High resolution TEM revealed an epitaxial NiSi2/Si(l11) interface which was Type A. We discuss here the enhanced nucleation of c-Si and subsequent silicide-mediated SPEG of Ni-implanted a-Si.Thin films of a-Si, 950 Å thick, were deposited onto Si(100) wafers capped with 1000Å of a-SiO2. Ion implantation produced sharply peaked Ni concentrations of 4×l020 and 2×l021 ions cm−3, in the center of the films.

Solid Phase Excitation-Emission Matrix Spectroscopy for In-Situ Chemical Analysis of Combustion Aerosols

2020 ◽  
Author(s):  
Gaurav Mahamuni ◽  
Jiayang He ◽  
Jay Rutherford ◽  
Byron Ockerman ◽  
Edmund Seto ◽  
...  
Keyword(s):  
Chemical Analysis ◽  
Cigarette Smoke ◽  
Forest Fires ◽  
Solid Phase ◽  
Incident Angle ◽  
Personal Exposure ◽  
Traffic Emission ◽  
Excitation Emission Matrix ◽  
Internal Excitation

<p>Exposure to combustion generated aerosols such as PM from residential woodburning, forest fires, cigarette smoke, and traffic emission have been linked to adverse health outcomes. It is important to assess the chemical composition of PM to examine personal exposure. Excitation-emission matrix (EEM) spectroscopy has been shown as a sensitive and cost-effective technique for evaluation of combustion PM composition and as a source apportionment tool. However, EEM measurements are hindered by a solvent extraction step and a need for benchtop instrumentation. Here, we present a methodology that eliminates this labor-intensive sample preparation and allows to automate and miniaturize the detection platform. A miniature electrostatic collector deposits PM sample onto transparent polydimethylsiloxane (PDMS) coated substrate, where PAH components are extracted into solid-phase (SP) solvent and analyzed using EEM spectroscopy in-situ. We evaluated external and internal excitation schemes to optimized signal to noise ratio. Analysis of woodsmoke and cigarette smoke samples showed good agreement with liquid extraction EEM spectra. Internal excitation is hindered by fluorescent interference from PDMS at λ<250nm. The external excitation EEM spectra are dependent on the incident angle; ranges of 30-40⁰ and 55-65⁰ showed the best results. The proposed SP-EEM technique can be used for development of miniaturized sensors for chemical analysis of combustion generated PM. </p>

Synthesis of hepatitis B surface antigen pre-S2 region fragments and studies on their immunogenicity

1988 ◽  
Vol 53 (11) ◽  
pp. 2952-2956 ◽  
Author(s):  
Bernard Lammek ◽  
Zbigniew Maćkiewicz ◽  
Izabela Derdowska ◽  
Hanna Świderska ◽  
Adam Nowosławski ◽  
...  
Keyword(s):  
Hepatitis B ◽  
Surface Antigen ◽  
Solid Phase ◽  
Hepatitis B Surface Antigen ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Antigenic Determinants ◽  
Phase Method ◽  
Peptide Fragments ◽  
Humoral Immune

Two peptide fragments of hepatitis B surface antigen pre-S2 region were synthesized by the solid phase method. The peptides were purified by gel filtration or ion-exchange chromatography on Sephadex SP-C-25. Both peptides induced a cellular and humoral immune response in rabbits. The results showed that fragment 14-22 of pre-S2 region contains one of the antigenic determinants.

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