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New developments in particle-based immunoassays: Introduction
Pure and Applied Chemistry
Abstract
There have been many innovations in diagnostics since white latex particles or microspheres were first used in medical diagnostic applications as "latex" agglutination tests (LAT) in the late 1950's. These innovations include colored particles permitting multivalent (or simultaneous) analyses, and special devices for simplifying test execu- tion and result interpretation. Dyed agglutinated particles caught on filters form the ba- sis of another class of tests. Sensitive particle-enhanced turbidimetric assays are in common use and are read with clinical chemistry analyzers via spectrophotometric or nephelometric methods. Particle capture ELISA tests and assays are in common use. The popular new strip tests for pregnancy, ovulation, drugs of abuse in urine, and many other tests all use dyed microspheres (and some use wo types of microspheres). Solid phase assays and tests use particles as a solid phase for positive or negative cap- ture of a wide variety of analytes. Solid-liquid separations can be made by centrifugal density separation, or filtration, or via magnetic separation of superparamagnetic parti- cles. Single microsphere (and perhaps single molecule sensitivity?) assays are now possible in flow cytometers.
... The DPPs are exploited in various bioassays: from identification of biomarkers that correspond to diseases and infectious organ- isms [2], monitoring of metabolic syndrome [3], separation and purification of biological objects [4] to genomic technologies [5]. This demonstrates great abilities of DPPs for effective, specific assays that can be realized with high sensitivity, selectivity, repro- ducibility in small sample volume at a relatively low price. ...
... A high T g of polystyrene imparts robustness to particles under stress avoiding coalescence, which provides their application in microfluidic analysis [23]. The hydrophobic surface properties are suitable for the biomolecule adsorption allowing biomolecule attachments [2]. The polymer properties of the particles can be varied by copolymerization of two or more monomers or during seed polymerization that affect surface functionality, diameter, morphology, T g value, etc. ...
... The particle size of DPPs and its narrow size distribution are very important parameters that need to be monitored depending on the further bioassay format involving DPPs. As such as, particle aggluti- nation reaction in plate with naked eye detection requires particles with diameter more 1.5 m, whereas agglutination reaction with turbidimetric detection involves particles, which diameter <0.3 m [2]. The control of sizes and their distribution is usually carried out during the synthesis by choosing a heterophase polymeriza- tion technique. ...
... [44] Such colorimetric chemosensors are finding increased use in various fields such as medical diagnostics (blood glucose monitoring, pregnancy test, etc.), biological and environmental monitoring. [45,46] Generally, colorimetric sensors work in the visible region of electromagnetic radiations and analyte recognition achieved by utilizing various photoactive moieties such as pyrene, BODIPY, anthracene, rhodamine, fluorescein, nitrophenyls, julolidine, 1,8-naphthalimide, Near-Infrared (NIR) absorbing dyes (squarines, hemocyanins), coumarin, nitrobenzoxazole (NBD), etc. [47] and it was soon recognized that most of them contain a system of conjugated bonds thus, it was critical to determine the color of dye by the energy difference between the HOMO and the LUMO. However, it was well-established that, smaller the energy differences between ground and excited state of molecule more bathochromic (red) shift of absorption band. ...
Development of chemosensors for the biologically and chemically important ionic species has been an active area of research of supramolecular chemistry and received great attention because of their high sensitivity, selectivity, rapid response time and versatility. Moreover, exploration of new sensing mechanisms of interaction between recognition and signal reporting unit is of current interest. This minireview provides a detailed overview about the recent advances and developments in the field of colorimetric and fluorescent chemosensors, summarized types of optical chemosensors with their design principles and discuss various sensing mechanisms and photophysical processes like metal‐ligand charge transfer (MLCT), ligand‐metal charge transfer (LMCT), photoinduced electron transfer (PET), intramolecular charge transfer (ICT), paramagnetic fluorescence quenching, fluorescence resonance energy transfer (FRET), excited state intramolecular proton transfer (ESIPT), excimer/ exciplex formation that emerged in the past few years. Moreover, the mechanism of action of cation and anion chemosensors also summarizes in this review.
... In the first and second regions, an analyte-explicit counter acting agent responds with the analyte in the example and is chromatographically exchanged to the nitrocellulose film. The immune response will undoubtedly shaded latex particles as a label [8] . If the example contains the analyte, it responds with the named counter acting agent. ...
... They are also used in the Carter-Wallace home pregnancy's "First Response" kit. During its use, when the latex micro-and gold NPs derivatized with a hormone released by pregnant women were mixed, the micro-and nanoparticles coagulated, forming clumps of pink color (Bangs 1996). Bio-detection sensitivity obtained from spherical NPs was not strong enough to trace the interaction of biomolecules (Orendorff et al. 2005). ...
In the recent years, gold nanoparticles (NPs) have been extensively studied for their widespread uses, particularly in biology, medicine and agriculture. Of the physical, chemical and biological methods of their fabrication, the last ones are simple, cost-effective, eco-friendly and may be easily scaled up for high yields and/or production. Plants and their extracts contain metabolites such as amines, amino acids, aldehydes, ketones, carboxylic acids, phenols, proteins, flavonoids, saponins, steroids, alkaloids, tannins and different nutritional compounds that have been frequently and successfully used in green synthesis of gold NPs. Many phytochemicals are used as the reducing agents for producing NPs from metal salts. Their production rate and size are controlled by monitoring the concentration, pH and temperature of plant extracts and that of the gold salt. The plant-mediated synthesized NPs have done better in various applications. This chapter provides an overview of plant-mediated fabrication of gold NPs, their characterization by UV-vis spectroscopy, thermogravimetric analysis, X-ray diffractometry, and SEM/TEM, among others, and their application in various cutting-edge areas.
... They are also used in the Carter-Wallace home pregnancy's "First Response" kit. During its use, when the latex micro-and gold NPs derivatized with a hormone released by pregnant women were mixed, the micro-and nanoparticles coagulated, forming clumps of pink color (Bangs 1996). Bio-detection sensitivity obtained from spherical NPs was not strong enough to trace the interaction of biomolecules (Orendorff et al. 2005). ...
In the recent years, gold nanoparticles (NPs) have been extensively studied for their widespread uses, particularly in biology, medicine, and agriculture. Of the physical, chemical, and biological methods of their fabrication, the last ones are simple, cost-effective, and eco-friendly and may be easily scaled up for high yields and/or production. Plants and their extracts contain metabolites such as amines, amino acids, aldehydes, ketones, carboxylic acids, phenols, proteins, flavonoids, saponins, steroids, alkaloids, and tannins and different nutritional compounds that have been frequently and successfully used in green synthesis of gold NPs. Many phytochemicals are used as the reducing agents for producing NPs from metal salts. Their production rate and size are controlled by monitoring the concentration, pH, and temperature of plant extracts and that of the gold salt. The plant-mediated synthesized NPs have done better in various applications. This chapter provides an overview of plant-mediated fabrication of gold NPs; their characterization by UV-vis spectroscopy, thermogravimetric analysis, X-ray diffractometry, and SEM/TEM, among others; and their application in various cutting-edge areas.
... The magnetic nanoparticles (MNPs) that illustrated superparamagnetic behavior are preferred for biomedical applications, for example, antibacterial, biosensors and drug delivery [16][17][18][19]. They should have essential features to their extensive use in the biomedical field including 1-The MNPs size should match well with the cellular level of the human body because diseases occur at cellular levels. ...
Magnetic nanoparticles that preferred for biomedical applications are required to be biocompatible, nanosized and superparamagnetic. In this research, ultra-small superparamagnetic Fe 3 O 4 nanoparticles and novel super-paramagnetic Fe 3 O 4 @ZnS core/shell nanocomposites were fabricated using biocompatible ethylenediaminete-traacetic acid (EDTA) as a capping agent by a facile refluxing assisted co-precipitation method at optimum condition. The Fe 3 O 4 and Fe 3 O 4 @ZnS nanoparticles were investigated using X-ray diffraction (XRD), scanning and transmission electron microscopy (SEM and TEM), UV-Vis spectroscopy, vibrating sample magnetometer (VSM) and Dynamic Light Scattering (DLS). The VSM results indicated that all of the samples have super-paramagnetic behavior. The particle size of the Fe 3 O 4 and Fe 3 O 4 @ZnS nanoparticles were obtained at about 10 and 22 nm, respectively.
... However, the coating of antigen or antibody on plates and electrochemical sensor surface make them expensive. Nowadays, magnetic beads (MBs) have been increasingly applied in the development of immunoassays for food analysis (Llamas et al. 2007;Gessler et al. 2006) and clinical diagnosis (Haukanes and Kvam 1993;Bangs 1996) due to their large surface area for convenient functional groups attachment, efficient magnetic force, slow sedimentation rate during incubation, which can be easily manipulated by an external magnetic field (Centi et al. 2007;Hayat et al. 2012). These advantages of MBs can highly shorten washing time, decrease matrix effect, and simplify the fabrication processes. ...
Okadaic acid (OA), a representative diarrhetic shellfish poisoning toxin, mainly produced by toxigenic dinoflagellates, has significant hazard to public health. Traditional methods for detection of OA can not give the consideration to the need of rapid, high sensitive, quantitative and in-situ detection at the same time. Herein, a new effective detection method of OA was developed based on fluorescence immunosensor and flow cytometry (FCM). In this assay, Streptavidin-coated magnetic beads (MBs) were used as the supporter to immobilize the biotinylated OA. Modified MBs competed with the free OA in the sample solution to bind with the anti-OA monoclonal antibody (OA-MAb). The R-phycoerythrin (R-PE) dye labeled IgG was served as a secondary antibody to perform fluorescence detection. A portable flow cytometry was applied for the in-situ fluorescence quantification. The results showed that the OA concentration was inversely proportional to the R-PE fluorescence intensity. The detection method took within 50 min with a limit of detection (LOD) was 0.05 μg/L and range from 0.2 to 20 μg/L for OA detection. Moreover, the matrix effect and the recovery rate were assessed during real sample measurement, showing a high recovery. Performance features such as high sensitivity, low LOD, speediness and simplicity of the analysis protocol, shows this biosensing-systems as a promising tool for routine use.
... We require an intact 1.25-µL droplet to be able to quantify the assay; therefore, plug stability is necessary. Using a nonionic surfactant additionally acts as a blocker of the hydrophobic surfaces of the microbeads, which reduces nonspecific agglutination of the microbeads [7,73]. ...
In this paper, we present a two-phase microfluidic system capable of incubating and quantifying microbead-based agglutination assays. The microfluidic system is based on a simple fabrication solution, which requires only laboratory tubing filled with carrier oil, driven by negative pressure using a syringe pump. We provide a user-friendly interface, in which a pipette is used to insert single droplets of a 1.25-µL volume into a system that is continuously running and therefore works entirely on demand without the need for stopping, resetting or washing the system. These assays are incubated by highly efficient passive mixing with a sample-to-answer time of 2.5 min, a 5–10-fold improvement over traditional agglutination assays. We study system parameters such as channel length, incubation time and flow speed to select optimal assay conditions, using the streptavidin-biotin interaction as a model analyte quantified using optical image processing. We then investigate the effect of changing the concentration of both analyte and microbead concentrations, with a minimum detection limit of 100 ng/mL. The system can be both low- and high-throughput, depending on the rate at which assays are inserted. In our experiments, we were able to easily produce throughputs of 360 assays per hour by simple manual pipetting, which could be increased even further by automation and parallelization. Agglutination assays are a versatile tool, capable of detecting an ever-growing catalog of infectious diseases, proteins and metabolites. A system such as this one is a step towards being able to produce high-throughput microfluidic diagnostic solutions with widespread adoption. The development of analytical techniques in the microfluidic format, such as the one presented in this work, is an important step in being able to continuously monitor the performance and microfluidic outputs of organ-on-chip devices.
... The complementary modules iii) and iv) have now to be added and the whole system assayed. Compared to fluorescent output, the revelation system involving black latex beads ensures an optimal reading condition for blood samples [22] and is not affected by the paper self-fluorescence. ...
Nowadays, STIs constitute a major public health issue. Indeed, treatments are often started too late because of belated diagnosis resulting in health problems, such as sterility. If prevention is probably the most effective action one can take to prevent the spread of STIs, early detection could help limit their deleterious effects. In this work, a new diagnosis approach based on aptamers is presented. Bound to paper, they allow the detection of HIV and Hepatitis B biomarkers from a blood sample. The associated device is composed of an anchor, the streptavidin protein, allowing the fixation of the aptamer to the paper via biotin (see graphical abstract). With this system, the HIV-1 Reverse Transcriptase (BBa_K1934060 and BBa_K1934061: protein subunits p51 and p66) and HBsAg (surface antigen of Hepatitis B) are specifically targeted. Then, the biomarker/aptamer complex is detected by two methods. The first one is based on fluorescence. As a proof of concept, a paired ATP/aptamer was used and enabled to successfully detect ATP up to 10 µmol.L-1. However, the signal was not detectable with naked eyes or with a cell phone equipped with blue and green filters either. Therefore, a lateral flow assay with nano-sized latex black beads was tested. This second technique showed that a protein biomarker, such as thrombin, could be complexed with latex beads coated with aptamers, in liquid. Finally, the ultimate step, migration of the latex beads inside paper, needs further optimization. Moreover, to easily handle several STI-tests on a single paper strip, an innovative bio-sourced PLA casing was designed and 3D printed to offer an additional intuitive user-interface.
... Immobilized proteins are extensively used for preparation of affinity chromatography resins for antigen and antibody purification, isolation and expansion of cells using antibody-coated particles, production of immobilized enzymes for industrial applications etc. [2,3]. Though different types of magnetic materials based on metals such as Cu, Co, Fe and Ni with diverse physical and biochemical properties are available, iron oxide (Fe3O4) nanoparticles have demonstrated greater potential for research applications [4][5][6]. Due to properties such as superparamagnetism, low toxicity, high surface area, biocompatibility and easy separation under external magnetic field, iron oxide nanoparticles have attracted much attention in the past decade [2]. ...
A method for immobilization of functional proteins by chemical cross-linking of the protein of interest and uncoated iron oxide nanoparticles in the presence of Epichlorohydrin is described. As a result of the cross-linking, the proteins form a matrix in which the particles get entrapped. The optimum concentration of Epichlorohydrin that facilitates immobilization of protein without affecting the functional properties of the protein was determined. This method was used to immobilize several functional proteins and the development and functional activity of Protein A-magnetic nanoparticles (MNPs) is described here in detail. The Protein A-MNPs possess high binding capacity due to the increased surface area of uncoated nanoparticles and robust magnetic separation due to the absence of polymeric coating materials. Protein A-MNPs were successfully used for purification of antibodies and also for immunoprecipitation. We also immobilized enzymes such as horse radish peroxidase and esterase and found that by providing the optimum incubation time, temperature and protein to nanoparticle ratio, we can retain the activity and improve the stability of the enzyme. This study is the first demonstration that Epichlorohydrin can be used to entrap nanoparticles in a cross-linked matrix of protein without impairing the activity of immobilized protein.
... Utilising the aggregation tendency of AuNPs, the tests combined micrometre sized latex particles and AuNPs functionalised with different epitopes of human chorionic gonadotropin antibodies. When the gonadotropin hormone, present in the urine of woman during pregnancy, contacts AuNPs within the sensor, the particles agglomerate forming visible pink aggregates [348]. Working via a similar principle, ImmunoCAP (Phadia, Inc.) is a commercially available allergy test which uses whole blood samples to create an immunoglobulin E (IgE) profile [349]. ...
Colloidal gold is undoubtedly one of the most extensively studied nanomaterials, with 1000s of different protocols currently available to synthesise gold nanoparticles (AuNPs). While developments in the synthesis of AuNPs have progressed rapidly in recent years, our understanding of their biological impact, with particular respect to the effect of shape, size, surface characteristics and aggregation states, has struggled to keep pace. It is generally agreed that when AuNPs are exposed to biological systems, these parameters directly influence their pharmacokinetic and pharmacodynamic properties by influencing AuNPs distribution, circulation time, metabolism and excretion in biological systems. However, the rules governing these properties, and the science behind them, are poorly understood. Therefore, a systematic understanding of the implications of these variables at the nano-bio interface has recently become a topic of major interest. This Review Article attempts to ignite a discussion around the influence of different physico-chemical parameters on biological activity of AuNPs, while focussing on critical aspects of cellular interactions, uptake and cytotoxicity. The review also discusses emerging trends in AuNP uptake and toxicity that are leading to technological advances through AuNP-based therapy, diagnostics and imaging.
... Iron oxide nanoparticles (NPs) have extensively been studied in living systems as the carriers of therapeutic agent and in humans for clinical purposes [1]. Owing to their magnetic characteristics, small diameter, water solubility and low toxicity, these NPs are useful in life sciences such as in bio-imaging, bio-sensing and drug delivery systems [2]. ...
Present study was conducted to study the in vivo biocompatibility of cobalt iron oxide magnetic nano-particles (CoFe2O4 MNPs) in rabbits. CoFe2O4 MNPs were synthesized by the conventional micro emulsion technique in crystallite size range of 30 to 50 nm. The lattice constant (a) and cell volume were found to be 8.386 Å and 589.75 Å3, respectively, revealed by XRD. Subject animals were divided in three groups—low dose, high dose and control group without nanoparticles implantation for biocompatibility evaluation. CoFe2O4 was intraperitoneally implanted in rabbits: low dose (1mg CoFe2O4/Kg body weight) and high dose (10mg CoFe2O4/Kg body weight). Blood, serum and histological study of vital organs (liver, heart, kidney and spleen) were carried out in seven days of time protocol after sacrificing of animals. Results indicated that CoFe2O4 had drastically affected the blood chemistry in a dose-dependent manner as RDWa (P=0.01), Platelet (P<0.001) and Plateletcrit (P<0.001) concentrations reduced significantly in low dose and high dose CoFe2O4 treatments as compared to sham treated control group. Histological analysis revealed that CoFe2O4 exposure resulted in disordered and abnormal histology of liver, kidney and that of muscles at surgical site. It is concluded that CoFe2O4 has low biocompatibility and higher toxicity levels in living system at the applied doses.
... For instance, in memory storage applications, the storage material is desired to be made up of particles that are stable and possess a switchable magnetic state which is compatible to represent the information stored in the form of bits and these should not be readily affected by the fluctuations in temperature. Similarly for biomedical applications, the use of such materials is preferred which possess superparamagnetism at room temperature which is chiefly characterized by virtually nil residual magnetism for the rapidly fluctuating values of the applied magnetic field131415. Therefore, we can say that magnetic nanomaterials behave in a highly different manner as compared to their conventional counterparts. ...
Nanomaterials have been the topic of intense industrial research for the past several years. They have unique physical, chemical, optical and magnetic properties as compared to those of their bulky counterparts. Intense research has been done on reducing the size of memory devices in the electronics industry. This requirement has made it extremely urgent to explore the materials with larger scope of functionalities on their surfaces. In this respect the conventional silicon based materials have been engineered for better and better control and modification of their surfaces but unfortunately this conventional route does not provide us with both higher functionalities as well as high speeds. In this regard, magnetic nanomaterials have been researched for interesting and far sighted effects. These have been the hot destinations for their sensitive responses in the biomedical and diagnostic applications. Magnetism at nanoscale is also significantly different from the one observed at bulk scale. Magnetic nanoparticles are normally made up of materials which have sensitive magnetic properties arising from the unpaired electrons in their d-orbitals and their coupling effect with their nuclear spins. This review explores the synthesis methods of the magnetic nanomaterials and their possible implementation in making memory based electronic storage devices. This also highlights the significant benefits and aspects that compel the use for investigating memory based potential for magnetic nanomaterials.
... Turbidity and nephelometry are two widely used methods to determine the extent of particle aggregation in medical diagnostic and environmental monitoring for biomolecules, microbes, heavy metal ions, and so on. 10 In these two assays, the intensity of light scattered from or transmitted through an assay solution is recorded and correlated to analyte concentration. Nevertheless, these two assays are mostly used for high concentration analyte analysis because of their low sensitivity. ...
Homogeneous assays possess important advantages that no washing or physical separation is required, contributing to robust protocols and easy implementation which ensures potential point-of-care applications. Optimizing the detection strategy to reduce the number of reagents used and simplify the detection device is desirable. A method of homogeneous bead-agglutination assay based on micro-chip sheathless flow cytometry has been developed. The detection processes include mixing the capture-probe conjugated beads with an analyte containing sample, followed by flowing the reaction mixtures through the micro-chip sheathless flow cytometric device. The analyte concentrations were detected by counting the proportion of monomers in the reaction mixtures. Streptavidin-coated magnetic beads and biotinylated bovine serum albumin (bBSA) were used as a model system to verify the method, and detection limits of 0.15 pM and 1.5 pM for bBSA were achieved, using commercial Calibur and the developed micro-chip sheathless flow cytometric device, respectively. The setup of the micro-chip sheathless flow cytometric device is significantly simple; meanwhile, the system maintains relatively high sensitivity, which mainly benefits from the application of forward scattering to distinguish aggregates from monomers. The micro-chip sheathless flow cytometric device for bead agglutination detection provides us with a promising method for versatile immunoassays on microfluidic platforms.
... In the immunoassay field, the streptavidin-biotin system is widely employed to improve the sensitivity and specificity of an immunoassay due to its ultra-high affinity (the streptavidin-biotin affinity constant in solution is approximately 10 15 M −1 [18,19]) and signal amplification effect (four biotins could be combined per streptavidin). Most clinical immunoassay technologies such as enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay (CLIA) rely on the recognition and capture of specific analytes by a solid-phase carrier such as plastic plates, latexes and magnetic particles (MPs) [20,21]. In contrast to homogeneous analysis technologies, the reaction thermodynamics and kinetics of solid phase-based immunoassay are dramatically affected by the solid-phase carrier [22][23][24]. ...
... It is agreed that for many of the expected applications tailoring the composition, size, morphology of the iron oxide nanoparticles and their surface by various processes is of key importance [3][4][5][6]. For instance, in data storage devices, the particles need to have a stable, switchable magnetic state to signify bits of information [7]. For biomedical applications, it is preferred to use particles that possess superparamagnetic behavior at room temperature [8]. ...
... Magnetic nanoparticles, widely used as an antibody carrier in clinical laboratories, are standard in high-throughput assays (Bangs 1996). These materials offer several benefits, including easy separation and localization of the analyte in the reaction mixture by an external magnet, extremely high surface/volume ratio to enhance the amounts of loaded biomolecules, fast reaction between antigen and antibody, and easy miniaturization (Hsing et al. 2007). ...
This paper reports a novel enzyme-free electrochemical immunoassay for Escherichia coli (ATCC 25922) that involves the formation of an immunocomplex between antibodies immobilized on the surface of a platinum electrode and bacteria-labelled magnetite nanoparticles. Magnetite nanoparticles modified with chitosan were used as the signal label in the immunoassay. The main novelty is the collection of an electrochemical signal from a magnetic nanolabel included within the immunocomplex on the electrode surface. The developed immunosensor is highly specific, enabling the determination of E. coli from 10 to 105 colony-forming units (CFU)/mL with the relative standard deviation below 10%. The limit of detection for the immunosensor was 9.3 CFU/mL. Bacteria were determined in model mixtures and real samples. The recovery was between 95 and 99%, comparable with results from enzyme-linked immunosorbent assays.
... Magnetite nanoparticles (NPs) have attracted considerable interest in the recent years because of their fundamental importance and potential applications in various fields [1][2][3]. These magnetic particles can be synthesized so small that each particle becomes a single domain, showing unusual magnetic properties, known as superparamagnetism [4,5]. ...
... Au NPs are also of interest for agglomeration-based biosensing if the NPs surfaces are engineered to drive agglomeration in the presence of an analyte. The color change that accompanies agglomeration is often discernible by eye, and agglomeration of Au NPs is the mechanism of the color change employed in some pregnancy test kits (Bangs, 1996). In some instances, the SPR shift can also be calibrated to measure the distance between NPs, which is known as a plasmon ruler (Jain et al., 2007;Sönnichsen et al., 2006). ...
Here we report our efforts to teach the first multidisciplinary undergraduate nanotechnology laboratory course in the College of Engineering at North Carolina State University (NCSU). The course was designed to provide undergraduate students with hands-on experience in nanoscience and
nanotechnology. The theme of this laboratory course is the integration of nanotechnology with microsystem technology, i.e., bottom-up synthesis meeting top-down fabrication. This course consists of seven carefully designed lab modules that bridge the major "pillars" of nanotechnology–nanomaterials,
nanofabrication, nanoscale characterization, and nanodevices. Final projects provide students opportunities to conduct nanotechnology research through problem-based learning and to improve their communication and presentation skills for educating the public about nanotechnology. A pedagogical
approach that features problem-based learning, group learning, visual/tactile assistance and interdisciplinary interaction was employed during the offering of this course.
... Their high surface-tovolume ratio and variety of surface functionalities makes them ideal as solid supports for a range of (bio)chemical reactions. Microfluidic applications utilising microparticles have included immunoassays, DNA hybridisation, affinity assays, and a number of protein, DNA and enzymological studies (Bangs, 1996). ...
We demonstrate a versatile microfluidic system for performing rapid, consecutive (bio)chemical reactions in continuous flow. Surface-functionalised magnetic microparticles are introduced into a chamber and pulled, via a magnet, across a series of laminar flow streams containing different reagents, thus performing multiple sequential reactions on the particles' surface. Such a continuous flow method eliminates many of the inefficiencies associated with batch techniques, such as the time-consuming, laborious sequential reaction and washing steps, to yield a system that can perform analyses far more rapidly and with less reagent volume than conventional methods. This innovative device has been applied to a two-reaction step mouse IgG sandwich immunoassay and one-and two-reaction step DNA hybridisation assays, all of which were completed within one minute. These results pave the way for a multi-purpose microreactor that can perform a variety of analytical and synthetic processes.
... However, nonspecific adsorption causes lots of problems, because proteins adsorb on almost all surfaces. One might assert that the saturation by ligands could solve this problem, but this saturation causes the socalled prozone phenomenon, which lowers the efficiency of affinity ligands (5,15). (The general scheme of optimal ligand coupling is shown in Fig. 2.) Therefore, we need a precise control mechanism to prevent this nonspecific adsorption. ...
... Among the various known immobilization techniques are 128 In this case, the last possibility was taken. In order to covalently link biomolecules to the carboxy-modified PAN-COOH/OH nanospheres for the avidin assay, a two-step reaction was preferred over the one-step coupling reaction due to limitations that may be expected when coupling larger molecules [129][130][131][132][133][134][135][136][137][138][139] . A few drops of 1 M hydrochloric acid were added to 50 mL of the nanosphere suspension described above until the nanospheres were ...
Biocatalysis is the peculiar feature of microbial enzymes nowadays, which primarily belongs to the consumption of natural catalysts for novel purposes. The current demands of the world’s biotechnological industries are better enzyme efficiency and perfection of novel techniques for boosting the shelf life of biocatalysts. With the introduction of nanotechnology, the unique physicochemical properties of the nanomaterials constitute novel and interesting matrices for enzyme immobilization. Enzyme-related industry involving concepts and application(s) of biocatalysis and biotransformation has also been uplifted by the utilization of nanoparticles (NPs). These particles are often functionalized prior to immobilization to offer essential groups, which aid in covalent attachment of enzyme molecule onto nanosurfaces. These particles have turned out as influential tools in bringing enzyme technology on the forefront for biocatalysis. As opposite and inert enzyme carriers, they offer an extensive variety of applications in improving the success and efficiencies of the bioprocess. The present article undoubtedly depicts the understanding of the role of NPs-based immobilization strategies, which provide a new avenue for better biocatalysis.
Resumen El presente trabajo muestra datos objetivos sobre la exposición a drogas ilícitas en una sub-población no considerada de alto riesgo. En un primer estudio se analizó una muestra aleatoria de orinas provenientes de 119 pacientes masculinos con edades comprendidas entre los 18 y los 45 años que por cualquier motivo, no asociado con hechos de violencia, acudieron a la consulta de triaje del Servicio de Emergencia del Hospital Universitario de Caracas, y a quienes por razones médicas se les indicó la realización de un examen de orina de rutina. Una vez realizado dicho examen de orina, alícuotas de las muestras fueron refrigeradas, centrifugadas y almacenadas bajo congelación a-20 ºC hasta el momento del análisis para detectar la presencia de drogas ilícitas. Del libro de registro de pacientes consultantes se tomaron datos de edad, procedencia y motivo de consulta, omitiendo deliberadamente la identidad de los pacientes emisores de las muestras, con el fin de preservar su anonimato. Las mismas fueron sometidas a análisis cualitativo mediante dispositivos inmuno-cromatográficos tipo tira reactiva para detectar la presencia visual de metabolitos de marihuana (mTHC, 7 casos positivos, 6%) y metabolitos de cocaína (mCOC, 15 casos positivos, 13%). En un segundo estudio realizado para detectar mCOC mediante extracción alcohólica e inmunocromatografía, se analizó un total de 34 muestras de cabello donadas voluntariamente por un grupo de pacientes de características similares a las del primer estudio, encontrándose nueve (09) casos positivos, esto es 26%. Se discuten estos resultados con las cifras de prevalencia general del consumo de sustancias adictivas en nuestro medio. Abstract ESTIMATION OF ILLICIT DRUGS SELFADMINISTRATION PREVALENCE IN VENEZUELA.EPIDEMIOLOGICAL SURVEILLANCE AT THE UNIVERSITY HOSPITAL OF CARACAS The present work shows objective data about the exposure to illicit drugs in a low-risk sub-population of hospital-patients. In a first study we analyzed 119 samples of urine taken at random among male patients with ages 18 to 45 years who attended a first level clinic for medical reasons unrelated to violence and who had indication for a routine urine test. After such a test was completed, the samples were centrifuged, decanted into clean glass-tubes, and stored frozen at-20ºC until they were analyzed to detect the presence of illicit drugs. Data of age, patient-address area and consultation motive, were also recorded on each aliquot-label, patient identification was deliberately omitted to ensure the anonymous nature of the study. Samples were analyzed using immuno-chromatographic rapid-test strips to visually detect the presence of marihuana metabolites (mTHC, 7 positive cases, 6%) and cocaine metabolites (mCOC, 15 positive cases, 13%). A second study was performed using scull-hair samples obtained by informed consent from 34 male patients with similar inclusion-criteria to those described for the first study. Alcoholic extracts of the hair-samples were dried, resuspended in water and processed as urine samples, in order to detect the presence of mCOC alone. Nine hair-samples were positive for mCOC (26%). We discuss these results in relation to generally accepted prevalence figures. Introducción ebido al perjuicio social que ocasiona el consumo de drogas ilícitas y la corre spondiente adicción, todos los países in tentan desarrollar programas de prevención y control para el uso de estas sustancias. Cualquier programa de este tipo requiere la utilización de metodologías adecuadas para la estimación absoluta o relativa de D la prevalencia del consumo de estas drogas en la población. Entre estos métodos se encuentran las encuestas de hogares, y las encuestas escolares, 1-4 las cuales deberían ser realizadas periódicamente en cada país. Sin embargo, se considera que la sensi bilidad de estas encuestas es generalmente muy baja, lo cual conduce lógicamente a la subestimación del problema, o al reconocimiento de un alto grado de incertidumbre en la apreciación de los resultados
Compositional micro-grained immune-magnetic absorbents (IMA) were obtained on the basis of silica - aluminocilicate, modified by polyglucin and sodium secondary alkylsulphate. IMA were shown to possess high adsorption activity due to their developed surface and implanted specific ligand, standard structural features, mechanical reliability, chemical and microbiological stability. The existence of magnetic material provided facilitation and convenience of operations with absorbents in analysis performance. Applying of IMA allowed to release of any mixtures with their adverse effect on reaction during samples preparing by means of rinsing the absorbent with fixed infected agent on it; concentrating the unknown pathogen to the extent possible increased the specificity and sensitivity of express-analysis methods (EIA and PCR), the time spending for analysis reduced substantially (up to 1-3hours).
Methods for the preparation of multifunctional dispersions of polymer particles (DPP) in the course of free radical heterophase polymerization, which provides careful control of both macromolecular and colloidal properties, are considered. Approaches that, according to the polymerization technique, make it possible to control the particle diameter and shape, particle size distribution, colloidal stability, morphology, surface chemistry and functionality, as well as the formation of organo-inorganic hybrid particles, are demonstrated. The possibilities of DPP application in homogeneous and heterogeneous assays and cell labelling are reviewed. The use of DPP leads to decrease the reaction scales and costs and to increase the speed, sensitivity, selectivity, and reproducibility of assays.
Principal possibility of using a smart polymer, poly-N-vinylcaprolactam, for immobilization of biopharmaceuticals (antibodies and antigens) in the course of phase transition of a soluble coil into an insoluble globule was demonstrated. A procedure for preparing a hydrosol stable at room temperature in the presence of a stabilizer was examined. The hydrosol obtained, in which the collapsed polymer with the captured antigen acts as a solid support, can be used for agglutination with a complementary antibody, i.e., the immunochemical reaction can be evaluated visually.
Magnetic nanoparticles have been investigated for use as biomedical purposes for several years. For biomedical applications the use of particles that present superparamagnetic behavior at room temperature is preferred [1-4]. To control the magnetic materials by magnetic field is essential locate particle to the suitable destination on feeding by injection. In order to use them properly, the particles should be nano size. However there are many difficulties in applications, because there is lack of identifications in nano magnetic properties. In our studies, structural and magnetic properties of iron oxide nanoparticles were investigated by XRD, VSM, TEM, and Mossbauer spectroscopy. At 13 K, hyperfine fields of {\gamma}-Fe_2O_3 were 516 kOe and 490 kOe, that of Fe_3O_4 were 517 kOe and 482 kOe. The saturation magnetizations were 21.42 emu/g and 39.42 emu/g. The particle size of powders is 5?19 nm.
Dynamic surface tension of non-ionic surfactants pluronic F-168 and poly(dimethylsiloxan) bis(hydroxyalkyl) terminated (PDS) were studied using the drop profile analysis tensiometry. The comparison of these data with those from styrene heterophase polymerization studies show that only in the PDS:pluronic mixing ratio range between 1:0.2 and 1:0.8 the contribution of each surfactant component manifests its effect and provides appropriate conditions for the synthesis of polymer particles with required properties.
We recently defined a magnetic bead-based assay that exploited an agglutination-like response for DNA and applied it to DNA-containing cell enumeration using inexpensive benchtop hardware [ J. Am. Chem. Soc. 2012 , 134 ( 12 ), 5689 - 96 ]. Although cost-efficient, the open-well format assay required numerous manual steps, and the magnetic field actuation scheme was not readily adaptable for integration. Here, we demonstrate a low-cost (<$2 in-lab), higher-throughput "pinwheel assay" platform that relies on a combination of a disposable rotation-driven microdisc (RDM), and a simple bidirectional rotating magnetic field (bi-RMF). The assay was transformed into an integrated microfluidic system using a multilayered polyester microfluidic disc created through laser print, cut and laminate fabrication, with fluid flow controlled by rotation speed without any mechanical valves. The RDM accepts four samples that undergo on-chip dilution to five different concentrations that cover the effective concentration range needed for downstream cell counting by pinwheel assay. We show that a bi-RMF is effective for the simultaneous actuation of pinwheel assays in 20 detection chambers. The optimization of the bi-RMF frequencies allows the RDM-based pinwheel assay detect human genomic DNA down to a mass of human genomic DNA (5.5 picograms) that is roughly equal to the mass in a single cell. For proof of principle, enumeration of the white blood cells in human blood samples on the RDM provided data correlating well (C.V. of 10%) with those obtained in a clinical lab. Fusing the cost-effective RDM with a simple bi-RMF provides a promising strategy for automation and multiplexing of magnetic particle-based agglutination assays.
The methods for preparation of polyacrolein-based dispersions of microspheres containing organic dyes and luminescent inorganic nanocrystals capable of light excitation in both the ultraviolet and the infrared spectral regions are considered. Microspheres of dispersions act as carriers of bioligands in immunochemical analysis with the visual and instrumental detection of results during labeling of cell receptors and as visualizing agents for in vivo studies. The methods for obtaining dispersions of polyacrolein-containing microspheres with pH- and thermosensitive fluorescence and the peculiarities of the application of these dispersions in bioanalysis are discussed. This review deals with studies that were performed at the Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry.
We describe the synthesis and properties of Au-decorated Fe2O3 nanoparticle catalysts, Fe2O3-Au, where the supporting Fe2O3 nanoparticles are of different shapes: spheres, rings, and tubes. The decoration procedure of the Fe2O3-Au nanoparticles is identical for each of the shapes, and is also analogous to the synthesis of pure Au nanoparticles (AuNPs). These similarities allows for the direct comparison between the different shapes and between the pure AuNPs. The morphological, structural, optical, and magnetic characterizations reveal that the Fe2O3-Au nanoparticles are hybrid structures exhibiting both plasmonic and magnetic properties. The catalytic properties of the different shape Fe2O3-Au nanoparticles are evaluated for a model catalytic reaction, specifically the reduction of 4-nitrophenol, and these are compared to those of the pure AuNPs fabricated in an identical manner. Remarkably, it is found that the Fe2O3-Au nanoparticles can be more efficient catalysts than the AuNPs, even with a much lower loading of gold, which is the catalytically-active material. Taking into account the Au-loadings, the Fe2O3 rings and tubes are superior to the Fe2O3 spheres as catalytic supports due to their γ-Fe2O3 crystal phase. It is also shown that the Fe2O3-Au nanoparticles have the additional benefit for catalysis in that they can be recovered and reused using magnetic collection. Furthermore, the Fe2O3-Au nanoparticles and AuNPs are found to efficiently transduce heat from light through plasmonic absorbance, and this phenomenon is exploited to demonstrate the photothermal catalytic reduction of 4-nitrophenol.
The scientific and technical history of polymerization can be divided into three periods, which will be illustrated for emulsion polymerization. The first period was when emulsion polymers were originally produced, and was developed as an attempt to copy natural rubber latex. Indeed, the natural process is quite different from the synthetic process of emulsion polymerization, which in fact does not even need an emulsion to be present: the term is a misnomer! The results were functional but limited. In the second period, the first theories appeared, and a huge range of products was made for surface coatings, adhesives, commodity polymers such as SBR, neoprene, etc. The work of the outstanding pioneers was based on limited types of experimental data, and some suppositions are now seen to be incorrect. Nevertheless, many excellent products were made and have evolved to many materials currently in everyday use. The third period of emulsion polymerization is now dawning. The scientific efforts of many teams over previous decades, aided by the advent of new physical techniques for investigation, have resulted in better understanding of the fundamentals of emulsion polymerizations. Some examples from the author's group involve creating novel materials using controlled seeded emulsion polymerization from natural rubber latex and other polyenes. Latex topology and controlled free-radical chemistry can be combined to produce a) a comb polymer with hydrophobic backbone and hydrophilic "teeth", or b) with sufficient in situ compatibilizer between two otherwise incompatible polymers to yield a spatially uniform material down to the nanostructure level, and c) to produce controlled nanostructures.
Mixed monolayers composed of α-(carboxyethyl)-ω-(trimethylsiloxy)polydimethylsiloxane (PDS), styrene, polystyrene, and sodium alkylsulfonate (E-30) in various combinations modeling the interfacial adsorption layer of latex particles were studied. A change in the composition of monolayers is accompanied by changes in their qualitative and quantitative characteristics (an increase or decrease in the area per molecule of PDS and the absence of the transition plateau between the liquid-expanded and liquid-condensed monolayer states). The obtained results made it possible to explain the observed nonmonotonic dependence of the polymerization rate on the ratio of surfactant components and to show that their mutual contribution to the maintenance of the stability of the interfacial absorption layer of polymer-monomer particles is realized only at a certain ratio between PDS and E-30.
The sections in this article are
Introduction
Self‐Assembly
Polypeptide‐Based Nanomaterials
De Novo‐Designed Helix Loop Helix Polypeptides
Polypeptides with Controllable Folding Properties
Polypeptide Scaffolds for Protein Recognition
Polypeptide‐Based Nanofibers
Biomolecular Fibers for the Assembly of Nanowires
Self‐Assembly of Hybrid Nanomaterials
Gold Nanoparticles
Synthesis of Gold Nanoparticles
Optical Properties of Gold Nanoparticles
Surface‐Functionalization of Gold Nanoparticles
Biomimetic Self‐Assembly of Gold Nanoparticles
Nanoparticle Assembly in Biodiagnostics
Conclusions and Outlook
Acknowledgments
Abbreviations
The present work shows objective data about the exposure to illicit drugs in a low-risk sub-population of hospital-patients. In a first study we analyzed 119 samples of urine taken at random among male patients with ages 18 to 45 years who attended a first level clinic for medical reasons unrelated to violence and who had indication for a routine urine test. After such a test was completed, the samples were centrifuged, decanted into clean glass-tubes, and stored frozen at -20°C until they were analyzed to detect the presence of illicit drugs. Data of age, patientaddress area and consultation motive, were also recorded on each aliquot-label, patient identification was deliberately omitted to ensure the anonymous nature of the study. Samples were analyzed using immuno-chromatographic rapid-test strips to visually detect the presence of marihuana metabolites (mTHC, 7 positive cases, 6%) and cocaine metabolites (mCOC, 15 positive cases, 13%). A second study was performed using scull-hair samples obtained by informed consent from 34 male patients with similar inclusion-criteria to those described for the first study. Alcoholic extracts of the hair-samples were dried, resuspended in water and processed as urine samples, in order to detect the presence of mCOC alone. Nine hair-samples were positive for mCOC (26%). We discuss these results in relation to generally accepted prevalence figures.
A highly robust enzyme immobilization platform has been fabricated for electrochemical biosensors by synergistically using bio-mimetic dopamine polymer film and nanogold particles. Taking horseradish peroxidase (HRP) as an enzyme model, this presented platform has been successfully exploited to develop a novel electrochemical enzyme sensor for the detection of H2O2. Experimental results show that the nanocomposite matrix can allow HRP to be immobilized with high loading amount and well-retained bioactivity. Moreover, it can achieve rapid electron transferring between the enzyme's active sites and the electrode surface, facilitating quick electroanalysis of H2O2. In addition, compared with other polymer materials (i.e., chitosan), the as-proposed biosensor design using the bio-mimetic dopamine polymer can show much better analytical performances. The developed enzyme sensor can allow for the detection of H2O2 with linear concentration range of 4.0×10-7~4.5×10-4 mol·L-1, detection limit of 3.7×10-7 mol·L-1 and sensitivity of 100.2 μA·L·mmoL-1. High detection reproducibility, storage stability and good interference-against ability can be additionally expected.
This review describes new polymer materials for biomedical applications developed in the Polymers for Biology Laboratory of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences. These include composite rigid sorbents for biochromatography, polymer dispersions for immunoassay, polymer hydrogels for immobilization of enzymes and cells, and polymer ultra thin films as biomembrane models and materials for biosensors. Some general and specific properties of these new materials and models as well as examples of their applications are discussed.
The influence of surface curvature on the adsorption of bovine serum albumin (BSA) was evaluated through the combination of two fairly simple techniques: electrophoretic light scattering and UV/vis spectroscopy. Measurements were carried out for a range of protein concentrations (0–320 μg/ml) at pH 3.5, 4.5 and 7 using hydrophobic polystyrene nanospheres of 38.8, 82 and 220 nm in diameter. The results obtained demonstrate that the charge of the BSA molecules in solution dictates the pH-dependent behavior of the protein-coated nanospheres, indicating in all cases a significant adsorption of BSA molecules. At a fixed pH, however, it is the zeta potential that characterizes the uncoated nanospheres normalized by their surface area that primarily controls protein adsorption. In particular, it is found that the rate at which BSA interact with the different nanospheres increases as their negative zeta potential per unit area (or diameter) increases (decreases) regardless of the pH. Moreover, provided that adsorption occurs away from the isoelectric point of the protein, highly curved surfaces are found to stabilize the native-like conformation of BSA upon adsorption by likely reducing lateral interactions between adsorbed molecules.
Our method utilizes the specific interaction between antigen and antibody to minimize time-to detection and maximize accuracy and precision of phenylalanine (Phe) measurement in urine and
blood at home.
The report deals with the unique properties of nanoparticles in bioanalysis. Recent advances in deoxyribonucleic acid detection, immunoassay, bioseparations and sensors are described. 66 references are cited.
Diagnostic dry reagent strips are common products used in clinical settings for urinalysis and blood testing, particularly glucose monitoring. Results are obtained instrumentally or visually as thresholds and quantitative outputs. Dry reagents are applied in the construction of strips in a variety of ways. The mechanism used to make strips operational, as well as practices used by manufacturers to establish performance, are reviewed. The limitations and benefits of reagent strips are assessed. After reading this article the reader will be able to identify the types of technology used in reagent strip products
Die Konkurrenz zwischen Tartrat und einem gängigen Indikator (1) um einen synthetischen Wirt (2) wurde genutzt, um Tartrat in Getränken aus Trauben quantitativ zu bestimmen. Der Ansatz demonstriert eine allgemeine Methode zur Entwicklung colorimetrischer Assays für hochkompetitive Medien.
Inorganic nanoparticles are non-toxic, hydrophilic, biocompatible and highly stable compared to organic materials. Drug delivery systems, designed for enhanced drug efficacy and reduced adverse effects, have evolved accompanied by the development of novel materials. Biomedical applications of nanotechnology are mainly suited for diagnostic techniques, nano drugs and delivery systems, and biomedical implants. Nano-enabled drug delivery has been projected as the single largest market opportunity. Recent advancement in nanotechnology led to the introduction of various other inorganic nanoparticles as excellent drug delivery matrices beside calcium phosphates. Nanoparticles are having highly advanced chemical properties and many inorganic nanoparticles have been used as drug carriers. This chapter reviews some of the recent developments and applications of calcium phosphate nanoparticles, gold nanoparticles and iron oxide nanoparticles in drug delivery and tissue engineering.
nanoparticles have been prepared by a sol-gel method. The structural and magnetic properties have been investigated by DTA/TGA, XRD, SEM, and spectroscopy, VSM. powder that was annealed at has spinel structure and behaved superparamagnetically. The estimated size of superparammagnetic Ni-Zn ferrite nanoparticle is around 10 nm. The hyperfine fields at 13 K for the A and B patterns were found to be 533 and 507 kOe, respectively. The blocking temperature () of superparammagnetic nanoparticle is about 250 K. The magnetic anisotropy constant and relaxation time constant of nanoparticle were calculated to be and s, respectively. Also, Temperature increased up to within 10 minutes under AC magnetic field of 7 MHz. It is considered that powder that was annealed at is available for biomedicine application such as hyperthermia, drug delivery system and contrast agents in MRI.
Nanotechnology can be defined as a research for the design, synthesis and manipulation of structure of particles with dimension smaller than 100 nm. The nanoparticles are of great scientific interest as they bridge the gap between bulk materials and atomic or molecular structures. Metallic nanoparticles have possible applications in diverse areas such as electronics, cosmetics, coatings, packaging and biotechnology. Although chemical and physical methods may successfully produce pure, well defined nanoparticles, these are quite expensive and potentially dangerous to the environment. Use of biological materials could be an alternative to chemical and physical methods for the production of nanoparticles in an eco-friendly manner. A promising approach to achieve this objective is to exploit the array of biological resources in nature. Over the past several years, plants, algae, fungi, bacteria, viruses and enzymes have been used for the production of low-cost, energy –efficient and non toxic metallic nanoparticles. The integration of biological molecules and materials at the nanoscale has the potential to revolutionize many fields of science and technology. The nanometer size scale is particularly relevant in biology, because the dimensions of large molecules such as proteins and DNA, as well as those of many important sub cellular structures, fall in the 1-1000 nm range.
In this chapter, we provide an overview of various methods of synthesis of metallic nanoparticles by biological means.
We have used a magnetic cell sorter (MACS) technique designed for animal cells to produce large samples of potato somatic hybrids. Selected biotinylated lectins are used to mediate, via streptavidin, the binding of superparamagnetic microbeads to the protoplasts of one fusion partner. The other partner contains the selectable marker neomycinphosphotransferase II (NPT II). After fusion, the protoplast mixture is sorted by MACS, which retains protoplasts labeled with magnetic microbeads. This fraction is incubated on kanamycin-containing medium, where only hybrid cells grow into calli. We have optimized the protoplast staining with lectins, the protoplast separation with MACS and the regeneration of hybrid calli into plants. With MACS treatment, among regenerated plants the hybrids between two clones of Solatium tuberosum increased from 8 to 36 percent. In the interspecific somatic hybridization S. tuberosum x S. bulbocastanum the increase was from 28 to 82 percent. Hybrid plants were identified by DNA fingerprinting and had the morphology and cytology expected for fusion products. The large number of plants which can be obtained should enable selection of hybrids with desired chromosome number, morphology and seed fertility.
There is need for integrated systems that provide rapid and sensitive diagnosis of Chlamydia trachomatis. Here we describe for the first time that specific antibodies to a C. trachomatis surface protein can be used for magnetic recovery of bacteria directly from crude urine samples. The combination of immunomagnetic separation (IMS), where the bacterial cells are both purified and concentrated, with a colorimetric PCR assay makes the method sensitive and suitable for routine clinical screening in microtiter format. No filtrations, centrifugations, extractions, or electrophoretic techniques are required throughout the whole procedure. Using magnetic beads as the solid phase, the colorimetric detection of immobilized amplified nucleic acids (DIANA) also allow direct DNA sequencing of positive samples for more detailed analysis. Of the 27 urine samples analyzed, eight were found to be positive using this method. Only five out of these eight were determined to be positive by cell culture.
Estimation of the extent and location of infarct is important for the prognosis and hence therapeutic strategy in patients with acute myocardial infarction (AMI). Because cardiac myosin is the major structural protein of the myocardium, and may thus reflect the extent of injured tissue, we established a new sensitive immunoradiometric assay, using a pair of monoclonal antibodies (Mabs) that specifically bind the myosin heavy chain fragments liberated from the myocyte into plasma after a heart attack. A first Mab is linked to a magnetic solid phase. A second Mab, radiolabeled with 125I, is used to detect myosin trapped on the solid phase by the first Mab during a 3-h incubation. This assay can detect 10 micro-units of myosin per liter and is highly reproducible.
During the last decade, immunomagnetic separation of cells has become popular due to the simplicity and speed of the technology. A variety of magnetic materials have been developed that have fundamentally different characteristics. Some of these act as true colloids and have the properties of solutions, while others are larger and are based on the polymerization of materials, such as styrene, in the presence of magnetite. In this article, the different magnetic matrices are discussed and reviewed with respect to their use for the separation of cells of the hematopoietic system. Additionally, a brief introduction into the field of immunomagnetic separation of nucleic acids is presented, as this technology may be used to perform investigations at the molecular level on hematopoietic cells.
A new method using traditional hybridization methodology, coupled with the new magnetic particle technology, has been developed for DNA purification, specifically for sequencing applications. The method is similar to the reverse hybridization blot system; however, a specific oligonucleotide probe was attached to the paramagnetic particle instead of a sheet membrane. The target DNA containing the complementary sequence of the probe hybridizes to the probe that is attached to the bead and is then magnetically removed from solution, washed and collected. This system eliminates the need of organic extractions and precipitation/concentration steps. The entire hybridization-purification system can be done in a 1.5-ml microcentrifuge tube making the method ideal for automation. M13 phage clones were purified with this method, both by manual means and by using the CATALYST 800 Molecular Biology LabStation fitted with a prototype magnetic station, and then sequenced. DNA sequencing results obtained with this system were reproducible and gave excellent length of read with low background.
We examined three enzyme-linked immunosorbent assay (ELISA) kits for human choriogonadotropin (hCG) (pregnancy tests) for use with urine and serum samples: the Tandem Icon II hCG Urine and Tandem Icon II hCG Serum, the NovoClone Target hCG Test, and the Abbott TestPacks hCG-urine and hCG-serum. Paired comparison of the results from each kit indicated that the NovoClone Target assay showed significantly lower diagnostic sensitivity (P less than 0.05) than did the Tandem Icon II or Abbott TestPack, both for urine and for serum samples. None of the products demonstrated any significant difference (P greater than 0.05) in diagnostic specificity, but the NovoClone Target kit showed several serious false-negative results with both urine and serum. Paired testing of urine kits vs serum kits also showed no significant differences (P greater than 0.05) in diagnostic sensitivity or specificity. We found the Abbott kits to be the most convenient to use and to read.
A rapid reversed passive latex agglutination method that uses high-density latex particles for the detection of staphylococcal enterotoxins (SE) A to E was developed. It took 3 h for incubation, much less than the 16 h needed with a customary latex agglutination test for SE detection such as a commercial test kit (SET-RPLA; Denka Seiken Co. Ltd., Tokyo, Japan). The rapid test was shown to be highly specific and sensitive for SE detection (detection limit, about 0.5 ng of SE per ml), comparable to the SET-RPLA test. The rapid test was also efficient in SE detection in foods and culture supernatants of staphylococcal strains, similar to the SET-RPLA test. This showed that a rapid test with high-density latex particles is fully reliable for use.
We describe the first homogeneous, nonradioactive, high-sensitivity assay for human thyrotropin (TSH). The assay is based on particle immunoassay techniques, wherein 800-nm particles form the basis for the immunochemistry, delivery, and the detection technologies, respectively. Our assay also is the first to involve the use of fragmented monoclonal antibodies (to eliminate serum interferences) covalently coupled to particles without loss of their binding properties. Assays are performed in a semiautomated mode with use of a new modular system (Multipact). Equilibrium is reached in less than 2 h. Precision profile, sensitivity, and clinical studies indicate that the assay is accurate, has good precision at low concentrations, and that detection-limit characteristics compare well with those of a leading commercial high-sensitivity immunoradiometric assay (IRMA) for TSH. Dilution characteristics were satisfactory down to the assay's detection limit for a range of clinical samples. Correlation studies vs a reference IRMA method yielded the regression equation, present method = 0.976 (IRMA) + 0.002 milli-int. unit/L (r = 0.98), for 223 samples with TSH concentrations in the range 0 to 30 milli-int. units/L. For 40 samples with TSH less than or equal to 1.0 milli-int. unit/L it was: present method = 0.94 (IRMA) + 0.005 milli-int. unit/L (r = 0.96).
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