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At least 37 records · Page 2Linked to original sources
PubMed1999

Single Molecule Detection Technologies in Miniaturized High Throughput Screening: Fluorescence Correlation Spectroscopy.

Fluorescence assay technologies used for miniaturized high throughput screening are broadly divided into two classes. Macroscopic fluorescence techniques (encompassing conventional fluorescence intensity, anisotropy [also often referred to as fluorescence polarization] and energy transfer) monitor the assay volume- and time-averaged fluorescence output from the ensemble of emitting fluorophores. In contrast, single-molecule detection (SMD) techniques and related approaches, such as fluorescence correlation spectroscopy (FCS), stochastically sample the fluorescence properties of individual constituent molecules and only then average many such detection events to define the properties of the assay system as a whole. Analysis of single molecular events is accomplished using confocal optics with an illumination/detection volume of approximately 1 fl (10(-15) L) such that the signal is insensitive to miniaturization of HTS assays to 1 µl or below. In this report we demonstrate the general applicability of one SMD technique (FCS) to assay configuration for target classes typically encountered in HTS and confirm the equivalence of the rate/equilibrium constants determined by FCS and by macroscopic techniques. Advantages and limitations of the current FCS technology, as applied here, and potential solutions, particularly involving alternative SMD detection techniques, are also discussed.

Journal Article↗
PubMed2000

A thousand points of light: the application of fluorescence detection technologies to two-dimensional gel electrophoresis and proteomics.

As proteomics evolves into a high-throughput technology for the study of global protein regulation, new demands are continually being placed upon protein visualization and quantitation methods. Chief among these are increased detection sensitivity, broad linear dynamic range and compatibility with modern methods of microchemical analyses. The limitations of conventional protein staining techniques are increasingly being encountered as high sensitivity electrophoresis methods are interfaced with automated gel stainers, image analysis workstations, robotic spot excision instruments, protein digestion work stations, and mass spectrometers. Three approaches to fluorescence detection of proteins in two-dimensional (2-D) gels are currently practiced: covalent derivatization of proteins with fluorophores, intercalation of fluorophores into the sodium dodecyl sulfate (SDS) micelle, and direct electrostatic interaction with proteins by a Coomassie Brilliant Blue-type mechanism. This review discusses problems encountered in the analysis of proteins visualized with conventional stains and addresses advances in fluorescence protein detection, including immunoblotting, as well as the use of charge-coupled device (CCD) camera-based and laser-scanner-based image acquisition devices in proteomics.

Animals↗
PubMed2003

An improved mechanically durable electrophoresis gel matrix that is fully compatible with fluorescence-based protein detection technologies.

Unfortunately, conventional large-format polyacrylamide gels are mechanically fragile, often tearing during the subsequent manipulations required for visualization of the proteins. This problem is compounded when large-format two-dimensional gels are subjected to multiple staining procedures in order to detect different classes of proteins, such as total protein, phosphoproteins, and glycoproteins. A mechanically durable liquid polyacrylamide-based matrix has been developed that, upon polymerization, facilitates the handling of one-dimensional and two-dimensional gels. The matrix, referred to as Rhinohide liquid acrylamide, is stable as a refrigerated solution for up to one year, and forms a polymer-reinforced polyacrylamide gel suitable for electrophoresis, upon addition of catalysts. The matrix is superior to previously reported durable gel matrices in that it does not cause distortion of high-molecular-weight bands and does not suffer from other spot morphology artifacts, such as doubling of protein spots in the molecular weight dimension. The matrix is particularly valuable for the analysis of proteins applying multiple applications of fluorescent dyes, as required with serial staining of proteins for phosphorylation, glycosylation, and total protein expression, using Pro-Q Diamond phosphoprotein stain, Pro-Q Emerald glycoprotein stain and SYPRO Ruby protein gel stain, respectively.

Acrylamide↗
PubMed2000

Arrayed primer extension: solid-phase four-color DNA resequencing and mutation detection technology.

The technology and application of arrayed primer extension (APEX) is presented. We describe an integrated system with DNA chip and template preparation, multiplex primer extension on the array, fluorescence imaging, and data analysis. The method is based upon an array of oligonucleotides, immobilized via the 5' end on a glass surface. A patient DNA is amplified by PCR, digested enzymatically, and annealed to the immobilized primers, which promote sites for template-dependent DNA polymerase extension reactions using four unique fluorescently labeled dideoxy nucleotides. A mutation is detected by a change in the color code of the primer sites. The technology was applied to the analysis of 10 common beta-thalassemia mutations. Nine patient DNA samples, each of which carries a different mutation, and four wild-type DNA samples were correctly identified. The signal-to-noise ratio of this technology is, on the average, 40:1, which enables the identification of heterozygous mutations with a high confidence level. The APEX method can be applied to any DNA target for efficient analysis of mutations and polymorphisms.

DNA↗
PubMed2005

Enzymatic mutation detection technologies.

Mutation is as necessary for life as fidelity is in DNA replication. The study of mutations reveals the normal functions of genes, messages, proteins, the causes of many diseases, and the variability of responses among individuals. Indeed, recent mutations that have not yet become polymorphisms are often deleterious and pertinent to the disease history of afflicted individuals. This review discusses the principles behind a variety of methods for the detection of mutations and factors that should be considered in future methods design. One enzymatic approach in particular using orthologs of the CEL I nuclease that show high specificity for all mismatches, appears to be easy and robust. Further developments of this and other methods will allow mutation detection to become an integral component of individualized medicine.

Base Pair Mismatch↗
PubMed2006

Detection Technologies. Ambient mass spectrometry.

A recent innovation in mass spectrometry is the ability to record mass spectra on ordinary samples, in their native environment, without sample preparation or preseparation by creating ions outside the instrument. In desorption electrospray ionization (DESI), the principal method described here, electrically charged droplets are directed at the ambient object of interest; they release ions from the surface, which are then vacuumed through the air into a conventional mass spectrometer. Extremely rapid analysis is coupled with high sensitivity and high chemical specificity. These characteristics are advantageously applied to high-throughput metabolomics, explosives detection, natural products discovery, and biological tissue imaging, among other applications. Future possible uses of DESI for in vivo clinical analysis and its adaptation to portable mass spectrometers are described.

Animals↗
PubMed2005

Miniaturized detection technology in molecular diagnostics.

Miniaturization of genetic tests represents the convergence of molecular biology and engineering and is leading to a new class of small analyzers and test systems for genetic testing with improved analytical characteristics. Miniaturization initially focused on devices that contained micrometer-sized features designed for a particular analytical purpose (e.g., filters for cell isolation and chips for capillary electrophoresis). Now, the focus is shifting to analytical applications based on nano-sized objects such as nanotubes, nanochannels, nanoparticles, nanopores and nanocapacitors. These nanofabricated objects provide new tools for sequencing of nucleic acids and rapid, multiplexed, nucleic acid detection.

Animals↗
PubMed2004

The safety helmet detection technology and its application to the surveillance system.

The Automatic Teller Machine (ATM) plays an important role in the modem economy. It provides a fast and convenient way to process transactions between banks and their customers. Unfortunately, it also provides a convenient way for criminals to get illegal money or use stolen ATM cards to extract money from their victims' accounts. For safety reasons, each ATM has a surveillance system to record customer's face information. However, when criminals use an ATM to withdraw money illegally, they usually hide their faces with something (in Taiwan, criminals usually use safety helmets to block their faces) to avoid the surveillance system recording their face information, which decreases the efficiency of the surveillance system. In this paper, we propose a circle/circular arc detection method based upon the modified Hough transform, and apply it to the detection of safety helmets for the surveillance system of ATMs. Since the safety helmet location will be within the set of the obtainable circles/circular arcs (if any exist), we use geometric features to verify if any safety helmet exists in the set. The proposed method can be used to help the surveillance systems record a customer's face information more precisely. If customers wear safety helmets to block their faces, the system can send a message to remind them to take off their helmets. Besides this, the method can be applied to the surveillance systems of banks by providing an early warning safeguard when any "customer" or "intruder" uses a safety helmet to avoid his/her face information from being recorded by the surveillance system. This will make the surveillance system more useful. Real images are used to analyze the performance of the proposed method.

Algorithms↗
PubMed1996

Aluminosilicates enhance the infectivity of cytomegalovirus in urine using centrifugation-enhanced antigen detection technology.

Due to the inherent lability of CMV, necessary laboratory identification of this infectious agent is often compromised by a delay in specimen transport. Previous studies have addressed the phenomenon of infectivity enhancement/reduction in the rate of infectivity loss by the incorporation into various viral assay systems of trace concentrations of the adsorbents montmorillonite (bentonite [M]) or kaolinite (kaolin [K]). We extended these studies to the clinical setting to identify whether such aluminosilicates would effect an enhanced level of CMV infectivity. The shell vial assay-indirect immunofluorescent assay (SVA-IFA) was utilized in comparative testing throughout this study. The addition of trace concentrations of M or K to the SVA-IFA was found to enhance the infectivity of CMV in urine by 115 and 126%, respectively. The total CMV detection rate by SVA-IFA was 29% (30/105). Three of the 30 (10%) CMV positive specimens were detected only in shell vials which had been supplemented with K or M. Two specimens were isolation positive alone. The addition of K or M to shell vials immediately prior to the start of the SVA-IFA has the potential of (a), enhancing assay readability by increasing the number of fluorescent focus units per vial monolayer and (b), of detecting positive urine specimens with low viral titers which might otherwise not be identified using the conventional SVA-IFA procedure.

Antigens, Viral↗
PubMed2000

A review of molecular recognition technologies for detection of biological threat agents.

The present review summarizes the state of the art in molecular recognition of biowarfare agents and other pathogens and emphasizes the advantages of using particular types of reagents for a given target (e.g. detection of bacteria using antibodies versus nucleic acid probes). It is difficult to draw firm conclusions as to type of biorecognition molecule to use for a given analyte. However, the detection method and reagents are generally target-driven and the user must decide on what level (genetic versus phenotypic) the detection should be performed. In general, nucleic acid-based detection is more specific and sensitive than immunological-based detection, while the latter is faster and more robust. This review also points out the challenges faced by military and civilian defense components in the rapid and accurate detection and identification of harmful agents in the field. Although new and improved sensors will continue to be developed, the more crucial need in any biosensor may be the molecular recognition component (e.g. antibody, aptamer, enzyme, nucleic acid, receptor, etc.). Improvements in the affinity, specificity and mass production of the molecular recognition components may ultimately dictate the success or failure of detection technologies in both a technical and commercial sense. Achieving the ultimate goal of giving the individual soldier on the battlefield or civilian responders to an urban biological attack or epidemic, a miniature, sensitive and accurate biosensor may depend as much on molecular biology and molecular engineering as on hardware engineering. Fortunately, as this review illustrates, a great deal of scientific attention has and is currently being given to the area of molecular recognition components. Highly sensitive and specific detection of pathogenic bacteria and viruses has increased with the proliferation of nucleic acid and immuno-based detection technologies. If recent scientific progress is a fair indicator, the future promises remarkable new developments in molecular recognition elements for use in biosensors with a vast array of applications.

Biological Warfare↗
PubMed2003

Highly sensitive fluorescence detection technology currently available for HTS.

Homogeneous fluorescence methods are providing an important tool for HTS technologies. A wide range of different techniques have been established on the market, with read-outs ranging from total fluorescence intensity to statistical analysis of fluorescence fluctuations for biochemical assays or fluorescence imaging techniques for cellular systems. Each method has its own advantages and limitations, which have to be accounted for when designing a specific assay. Here, recently developed fluorescence techniques and some of their applications, with a particular focus on sensitivity, are summarized and their principles are presented.

Fluorescence Polarization↗
PubMed1999

Fluorescence-intensity distribution analysis and its application in biomolecular detection technology.

A methodology, fluorescence-intensity distribution analysis, has been developed for confocal microscopy studies in which the fluorescence intensity of a sample with a heterogeneous brightness profile is monitored. An adjustable formula, modeling the spatial brightness distribution, and the technique of generating functions for calculation of theoretical photon count number distributions serve as the two cornerstones of the methodology. The method permits the simultaneous determination of concentrations and specific brightness values of a number of individual fluorescent species in solution. Accordingly, we present an extremely sensitive tool to monitor the interaction of fluorescently labeled molecules or other microparticles with their respective biological counterparts that should find a wide application in life sciences, medicine, and drug discovery. Its potential is demonstrated by studying the hybridization of 5'-(6-carboxytetramethylrhodamine)-labeled and nonlabeled complementary oligonucleotides and the subsequent cleavage of the DNA hybrids by restriction enzymes.

DNA↗
PubMed2006

Detection technologies. Probing cellular chemistry in biological systems with microelectrodes.

Over the past 20 years, the technological impediments to fabricating electrodes of micrometer dimensions have been largely overcome. These small electrodes can be readily applied to probe chemical events at the surface of tissues or individual biological cells; they can even be used to monitor concentration changes within intact animals. These measurements can be made on rapid time scales and with minimal perturbation of the system under study. Several recent applications have provided important insights into chemical processes at cells and in tissues. Examples include molecular flux measurements at the surface of single cells and through skin-which can offer insights into oxidative stress, exocytosis, and drug delivery-and real-time brain neurotransmitter monitoring in living rats, which reveals correlations between behavior and molecular events in the brain. Such findings can promote interdisciplinary collaborations and may lead to a broader understanding of the chemical aspects of biology.

Animals↗
PubMed2001

Microspectrophotometric measurements of vertebrate photoreceptors using CCD-based detection technology.

We have developed a charge-coupled-device (CCD)-based microspectrophotometer (MSP) system and provide the first report on the successful employment of this technology to measure the spectral absorbance properties of vertebrate photoreceptors. The principal difference between the CCD-based MSP system and wavelength-scanning MSP systems, commonly used in vision biology, is that a short duration (800-1200 ms), broad-spectrum flash is employed rather than ascending and descending wavelength scanning. Data acquisition is thus significantly faster, with the added possible advantages of less variance due to movement of target photoreceptors during measurement, reduced spectral distortion due to photoproduct interference and an ability to measure fast, transient changes in absorbance as bleaching proceeds. Rainbow trout photoreceptors, previously measured with a wavelength-scanning MSP system, were again measured using the CCD-based MSP system. Our analysis of optical recordings from 102 photoreceptors corroborated data obtained previously with rainbow trout photoreceptors on lambda(max) (wavelength of maximum absorbance), A(max) (maximum absorbance) and half maximum bandwidth (HBW) of ultraviolet-, blue-, green- and red-sensitive cones and rods. There were slight differences in lambda(max) and half-maximum bandwidth of the ultraviolet-, blue- and green-sensitive cone classes, but this was most probably due to variation in the A(1):A(2) visual pigment ratio of the trout used in the two different studies. However, we were capable of resolving the A(1) and A(2) visual pigment spectra in the red-sensitive cones and the rods.

Animals↗
PubMed2004

Evolution of HLA antibody detection: technology emulating biology.

New technological advances in the field of histocompatibility have provided an approach to systematically address the specificity of positive lymphocyte crossmatches. These approaches can now confirm whether a positive crossmatch is (or is not) due to class I and/or class II antibodies directed against donor HLA antigens. The information gained from the application of these sensitive and specific technologies can be used to predict crossmatch results for highly sensitized patients. In summary, these emerging technologies have provided the tools to reliably determine the clinical relevance of a positive lymphocyte crossmatch.

Antibodies↗