Last revised December 27, 2001 
View a Printer-Friendly Version of this Web Page!

It could be argued that the rate-limiting factor in biology at the moment is not the speed of assays but devising the assays themselves; that is, establishing new and imaginative ways of measuring biological activity in vivo or in vitro and then using genetics or biochemistry to use the players- Kim Nasmyth ["Opinions on the potential of yeast biochemical genomics" in "The awesome power of yeast biochemical genomics" Trends in Genetics 16 (2): 49-51 Feb. 2000]

Related glossaries are Applications Drug Discovery & Development, Sequencing Technologies Gene Amplification & PCR,  Imaging, Mass spectrometry, Microarrays, Miniaturization  Additional definitions appear in the In-depth glossary, after the Bibliography.

amplification: See signal amplification, target amplification (and gene amplification).

analysis, molecular - innovative: Molecular analysis technologies of interest [to the National Cancer Institute] include those that are entirely novel, or emerging but not currently in broad scale use, or technologies currently in use for one application or set of applications, that are being evaluated for utility for alternative applications. ... Technologies suited for this solicitation, include those that enable the: detection of alterations and instabilities of genomic DNA; measurement of expression of genes and gene products; analysis and detection of gene and or cellular products including differential expression, quantitation, post translational modification, and function of proteins; identification of exogenous infectious agents in cancer; assaying the function or major signal transduction networks involved in cancer. Additionally, technologies that will support molecular analysis in vitro, in situ, or in vivo (by imaging or other methods) are suitable. [NCI, Innovative Technologies for the Molecular Analysis of Cancer, 2001] http://otir.nci.nih.gov/tech/imat_ini.html#inno

The growing momentum of genomics and molecular analysis has enormous promise for earlier and non (or less) invasive diagnostics, novel therapeutics and more precise prognoses in cancer, cardiovascular and infectious diseases. Improved patient stratification holds the allure of faster, smaller clinical trials and fewer adverse effects for patients. Advances in gene sequencing and analysis have made these processes higher throughput, more scaleable and reproducible. Microarrays are revolutionizing basic research. Proteomics and protein chips further broaden the range of possibilities.  Molecular Analysis for Research and Diagnostics: Feb. 13-14  2002 • San Diego, California

analyte specific reagents: A new class of regulated product: analyte specific reagents. These products are usually a singular reagent, such as an antibody, which can be used toward developing a test by third parties, such as another company or a hospital. The manufacturer of this product is not planning to sell it as part of a kit, but only as an independent reagent. Therefore, it is of low risk to the user, it can be exempt from 501(k) [device]  requirements, and by definition must be used "for identification and quantification of an individual chemical substance or ligand in biological substances."  [Joseph Hackett, in CHI Summit Pharmacogenomics Report]

assay A set of operations having the object of determining the value of a quantity. In analytical chemistry, this term is synonymous with measurement. [IUPAC Compendium]

Generically a bioassay where biological activity is derived; associated with a bioactive effector molecule. Within the screening discipline, an assay will probably be robust enough and have the capacity to enable testing of up to 10,000 samples, generally with limited chemical diversity. [The precise definition of the following terms varies widely between drug discovery companies. The meanings given here are aligned with the use of the terms within the lead discovery function at GlaxoWellcome.  Martin J. Valler,  Darren Green  "Diversity screening versus focussed screening in drug discovery " Drug Discovery Today 5(7): July 2000]  http://162.105.138.23/pdl/web_course/swjs/diversity%20screening%20versus%20focussed%20screening%20in%20drug%20discovery.pdf

The drive of genomic and proteomic research and diagnostics towards greater performance is resulting in demands for improved approaches to labels and other methods for detection of biological molecules. Achieving greater speed, sensitivity and accuracy are being pushed to the limits, all while moving towards greater automation and lower costs. Innovative techniques, including nanolabels, extended multiplexing and direct detection, are being used to achieve some of these goals. Challenging applications, such as assays of living cells or single molecule detection, help push technology - which can also be beneficial for many other applications. 

Assays, Molecular Labels, Signaling  & Detection: Profiling PCR & Proteins  June 27-28, 2002, Washington DC

Narrower terms: In-depth Enzyme-Linked Immunosorbent Assay ELISA, primary assays, secondary and tertiary assays; In-depth: bioassay, cell assays, homogeneous assays, immunoassay, sandwich assay; In-depth primary assays, secondary assays

attomole: 10^-18 mole. One quintillionth.

competitive hybridization: Gene amplification & PCR glossary

dendrimers: Miniaturization glossary

detection: The Oxford English Dictionary points out that detecting involves finding what is otherwise apt to elude notice, particularly that which is "artfully concealed".

The drive of genomic and proteomic research and diagnostics towards greater performance is resulting in demands for improved approaches to labels and other methods for detection of biological molecules. Achieving greater speed, sensitivity and accuracy are being pushed to the limits, all while moving towards greater automation and lower costs. Innovative techniques, including nanolabels, extended multiplexing and direct detection, are being used to achieve some of these goals. Challenging applications, such as assays of living cells or single molecule detection, help push technology - which can also be beneficial for many other applications.  Advances in Assays, Molecular Labels, Signaling  & Detection: Profiling PCR  June 27-28, 2002, Washington DC  Narrower term: single molecule detection

detector technologies: Include direct detection, electrochemical, fluorescence, fluorescence polarization, colorimetry, mass spectrometry, luminescence, optical, primer extension and minisequencing. [Michael Phillips, CHI Nucleic Acids Technologies conference, June 7-9, 2000] Detector instrumentation includes CCD cameras and lasers.

colorimetry: The methods used to measure color and to define the results of the measurements. [Photonics]

diagnostics: Clinical genomics glossary

direct detection: Direct detection systems attach the reporter group (e.g. fluorescein) directly to the primer or probe. Compare with "indirect systems" , the primary label (e.g. biotin) is detected through interaction with a secondary system containing a detectable reporter group. [Synthetic Genetics "Synthetic DNA"] http://www.syntheticgenetics.com/historyDNA.html

dyes: See labels, reagents.

ELISA: SEE Enzyme-Linked Immunosorbent Assay 

electrochemistry: The study of chemical changes resulting from electrical action and electrical activity resulting from chemical changes. [MeSH]

Related terms: In- depth electrochemiluminescence, Electrogenerated Luminescence ECL, electroluminescence

Enzyme-Linked Immunosorbent Assay ELISA: An immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme- antibody- antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed. [MeSH]

excitation: Narrower terms: In- depth chemiexcitation, photoexcitation Related terms: quencher, quenching, scintillation

FISH Fluorescence In Situ Hybridization:  Gene Amplification & PCR

femtomole: 10^-15 mole. One quadrillionth. 

From the Norwegian for fifteen, approved by the International Committee [on Weights and Measures] in 1961. [OED]

fluorescence: Luminescence which occurs essentially only during the irradiation of a substance by electromagnetic radiation. [IUPAC Compendium]

The property of emitting radiation while being irradiated. The radiation emitted is usually of longer wavelength than that incident or absorbed, e.g., a substance can be irradiated with invisible radiation and emit visible light. [MeSH]

Narrower terms: FISH Fluorescence In Situ Hybridization, Fluorescence Resonance Energy Transfer FRET, green fluorescent protein GFP; In-depth FRAP Fluorescence Recovery After Photobleaching, fluorescence polarization, LIF Laser Induced Fluorescence Related terms fluorophore

Fluorescence In Situ Hybridization: See FISH.

Fluorescence Resonance Energy Transfer FRET: A distance- dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon. FRET is dependent on the inverse sixth power of the intermolecular separation, making it useful over distances comparable with the dimensions of biological macromolecules. Thus, FRET is an important technique for investigating a variety of biological phenomena that produce changes in molecular proximity. [Molecular Probes website, Handbook, 2001] http://www.probes.com/handbook/sections/0002.html

Has been used for many years to make spectroscopic distance measurements on ensembles of molecules. Recent advances in new fluorescent dyes and optical methods have increased the spatial resolution, distance range, and sensitivity of this method so that it continues to be one of the few tools available for measuring nanometer-scale distances in biological molecules. In FRET, energy is transferred from a donor fluorophore to an acceptor fluorophore over a range of 20-100 Å. ... Dynamic events, such as the relative motion between donor and acceptor molecules, however, cannot be detected by conventional FRET methods due to the lack of synchronized events in a population of molecules.  [NIGMS,  Single Molecule Detection and Manipulation Workshop "Single Molecule Fluorescence of Biomolecules and Complexes Protein Folding April 17-18, 2000] http://www.nigms.nih.gov/news/reports/single_molecules.html#examples  Narrower term single-pair FRET spFRET

fluorophore: The categories of greatest need [in single molecule studies] are improving the photophysical properties of fluorophores used for single molecule spectroscopy There is a need for synthesis of probes with desirable spectral and luminescent characteristics, such as small size, high quantum yield, high extinction, reduced photobleaching, blinking, and photoisomerization. The best probes will be compatible with conditions inside the cell and will move freely in the cell. Emerging technologies have made use of silicon and lanthanide nanocrystals (Quantum dots), which emit enough photons to be detected at very low concentrations, plasmon and Raman probes, and G/C/Y/R- fluorescent proteins, but there is still much to be done to optimize these probes. High throughput screening and combinatorial approaches need to be applied to this problem.   [NIGMS, Single Molecule Detection and Manipulation Workshop "Single Molecule Fluorescence of Biomolecules and Complexes Protein Folding April 17-18, 2000] http://www.nigms.nih.gov/news/reports/single_molecules.html#examples

May refer to either the fluorescent label or marker, or to the atoms which make the label fluorescent.

green fluorescent protein GFP: As a label for reporting cellular events in situ has been explored by a large number of laboratories. GFP and its mutants offer a powerful advantage as clonable markers for use in living tissue. However, photoisomerization and flickering of the emission signal ('blinking') create a challenge in single molecule experiments for both types of probe. Studies are in progress by W.E. Moerner and others (for example, see 6,7) to understand the basis for the long- lived dark states that lead to fluctuations in the emission spectra from these molecules, and to develop improved probes with reduced photoisomerization and blinking. [NIGMS  Single Molecule Detection and Manipulation Workshop" Single Molecule Fluorescence of Biomolecules and Complexes Protein Folding April 17-18, 2000] http://www.nigms.nih.gov/news/reports/single_molecules.html#examples 

GFP from the jellyfish Aequorea victoria is a revolutionary reporter molecule for monitoring gene expression and protein localization in vivo, in situ, and in real time. GFP fluoresces bright green upon mere exposure to UV or blue light — unlike other bioluminescent reporters which require additional proteins, substrates, or cofactors to emit light. [Clontech website] http://www.clontech.com/products/catalog/GeneExpression/GFPintro.html

High Throughput Screening HTS: Drug Discovery & Development glossary      http://www.whatman.plc.uk/

ISH: See in situ hybridization.

immunometric assay: See In-depth sandwich assay.

in situ hybridization ISH: Gene amplification & PCR

isothermal: Isothermal conditions are important in immunoassays, immunohistochemistry, in situ amplification and in cell- based assays, in which one wants to retain the morphology and viability of the cells. The relative simplicity of an isothermal reaction also indicates greater utility for point of care diagnostic applications. [CHI Summit Proteomics]

Having a constant temperature.

label: A marker, tag or indicator distinguishable by the observer but not by the system and used to identify a tracer. [IUPAC Radioanalytical, Pure & Applied Chemistry: 2514-2576, 1994]

There is a need to develop better methods for inserting site- specific labels in the samples for detection, as well as mechanical handles for manipulation. Site- directed mutagenesis, approaches using chimeras, clonable tags, reporter genes, protection/ deprotection protocols, and protein modification using derivatized amines and thiols, such as His tags, are currently used, but flexibility in the placement of chemical handles in the sample remains a limitation. [NIGMS, Single Molecule Detection and Manipulation Workshop "Single Molecule Fluorescence of Biomolecules and Complexes Protein Folding, April 17-18, 2000] http://www.nigms.nih.gov/news/reports/single_molecules.html#examples 

A chemical or radiochemical which can later be used for detection. Non- radioactive labels are increasingly in demand.  Increased sensitivity, specificity and other improvements are also desirable -- but cost- effectiveness, ease of use and robustness are as well.  Different purposes have varying degrees of tolerance (and need) for each of the many variables. Advances in Assays, Molecular Labels, Signaling  & Detection: Profiling PCR  June 27-28, 2002, Washington DC.  

Narrower terms nanolabels, In-depth biotin, differential labelling, ICAT, optical tagging, streptavidin, target labelling  Related terms: assays, dyes, reagents; In-depth target labelling, tracer 

luminescence: The property of giving off light without emitting a corresponding degree of heat. It includes the luminescence of inorganic matter or the bioluminescence of human matter, invertebrates and other living organisms. [MeSH]

Spontaneous emission of radiation from an electronically or vibrationally excited species not in thermal equilibrium with its environment. [IUPAC Compendium]

Narrower terms: In-depth bioluminescence, chemiluminescence, electrochemiluminescence, Electrogenerated Luminescence ECL. 

massively parallel: Many (assays or other procedures) at once.

Related term: Gene amplification & PCR glossary multiplexing

microsequencing: Sequencing glossary  In-depth

minisequencing: Sequencing glossary  In-depth

mole, mol: The mole is the amount of substance of a system which contains as many elementary entities as there are atoms in 0.012 kilogram of carbon 12. 2. When the mole is used, the elementary entities must be specified and may be atoms, molecules, ions, electrons, other particles, or specified groups of such particles. [Bureau International de poids et mesures, SI base units, Système International d'Unités, (International System of Units)] http://www.bipm.fr/enus/3_SI/base_units.html

molecular distillation: Imaging glossary

nanobarcodes particles: Nanobarcodes^TM particles -- a proprietary, nano- scale technology allowing multiplexed bioanalytical measurements at an unprecedented level on micro- volume samples with a common instrumentation platform. Cylindrical metal nanoparticles in which the composition along the particle length can be varied in a stripe- like fashion.  Because the number of stripes, the width of stripes, the identity of the metals, and the overall particle shape can be varied, trillions of unique "flavors" can theoretically be produced.  Analogous to conventional barcodes, which are read using the differential contrast of black and white stripes, Nanobarcodes particles can be identified using conventional optical microscopy, based on the pattern of differential reflectivity of adjacent metal stripes. [SurroMed. Inc. website "Technology" 2001] http://www.surromed.com/Technology.html

nanocrystals: Miniaturization glossary Broader term: labels

nanolabels: DNA ‘barcodes’, nanoscopic data molecules on the basis of DNA with a storage capacity of at least 32bit. They can be used as invisible markers for labeling of industrial products like paints, oils, lubricants etc., paper- based materials and documents, ink, pharmaceuticals, medical samples and organic materials. The molecules can even be used for labeling of single genes, thus, for the first time, allow labeling of genetically engineered products and food. Also, for the first time, the molecules can be encrypted to guarantee full fake- security of the labeled products. [Informium AG, Germany, Aug. 12, 2001] http://www.informium.com/article/articleview/8/1/6/

nanomole: 10 ^-9 mole.  One billionth. 

^{From the Greek for dwarf. One thousand- millionth. Adopted by the Union International [OED]}

nanoparticles: Advances in Assays, Molecular Labels, Signaling  & Detection: Profiling PCR  June 27-28, 2002, Washington DC Washington DC will have a session on Nanoparticles and other novel labels.  

Related terms: nanocrystals, quantum dots, others?

phosphorimagers: Microarrays glossary

photobleaching: Photobleaching by laser light has been a useful tool for biologists and biochemists in determining cellular structure and extracellular transport mechanisms in living things. [Rachel Kinghorn, "Laser Photobleaching" EE 488 Dept. of Electrical Engineering, Lasers and Optoelectronics, Oregon State. Univ. US Autumn 1994] http://www.ece.orst.edu/~ece482/lasers/ee48894/rachel/rachprop.htm

photon, photonics: Imaging glossary

picomole: 10 ^–12 mole. One trillionth.

From the Spanish pico beak, peak, (in phrases) little bit. [OED]

primary assay: Assays of drugs done on a single target or small groups of targets. [CHI Breaking Bottlenecks]

primer:  Gene Amplification & PCR

probe:  Gene Amplification & PCR

propidium iodide: [Available from] (Molecular Probes, Eugene, OR) is a membrane- impermeant dye that stains by intercalating into nucleic acid molecules. It binds both DNA and RNA, and is nonspecific with respect to base sequence. When bound to DNA, propidium iodide excites at 536 nm and fluoresces at 617 nm. It is useful in confocal microscopy studies of chromosome topography, DNA structure, and membrane integrity. It can also be used in double-experiments to detect DNA and a fluorescein- labeled antigen simultaneously. [Protocol Online, Cell Biology>Cell Cycle>Cytometry "Flow Cytometry sample preparation" DoubleTwist, 2001] http://www.protocol-online.net/cellbio/cell_cycle/cytometry.htm

quantum dot: An important strategy for nonisotopic labeling of single molecules is the use of highly luminescent semiconductor nanocrystals, or 'quantum dots,' that can be covalently linked to biological molecules. This class of detectors, which range in size from 1-5 nm, have been exploited for biological labeling by a number of laboratories, particularly those of Shimon Weiss, Paul Alivisatos and Shuming Nie (4, 5). Quantum dots offer several advantages over organic dyes, including increased brightness, stability against photobleaching, a broad continuous excitation spectrum, and a narrow, tunable, symmetric emission spectrum. Because quantum dots are nontoxic and can be made to dissolve in water, efforts are underway to explore their use in labeling single molecules in living cells.  [NIGMS  Single Molecule Detection and Manipulation Workshop" Single Molecule Fluorescence of Biomolecules and Complexes Protein Folding April 17-18, 2000] http://www.nigms.nih.gov/news/reports/single_molecules.html#examples 

reagents:  Substances used for the detection, identification, analysis, etc. of chemical, biological, or pathologic processes or conditions. .. Reagents are substances used for the detection or determination of another substance by chemical or microscopical means, especially analysis. Types of reagents are precipitants, solvents, oxidizers, reducers, fluxes, and colorimetric reagents. [MeSH ‘indicators and reagents’]   Related term reactant.

SPR Surface Plasmon Resonance: A biosensing technique in which biomolecules capable of binding to specific analytes or ligands are first immobilized on one side of a metallic film. Light is then focused on the opposite side of the film to excite the surface plasmons, that is, the oscillations of free electrons propagating along the film's surface. The refractive index of light reflecting off this surface is measured. When the immobilized biomolecules are bound by their ligands, an alteration in surface plasmons on the opposite side of the film is created which is directly proportional to the change in bound, or adsorbed, mass. Binding is measured by changes in the refractive index. The technique is used to study biomolecular interactions, such as antigen - antibody binding. [MeSH]  Narrower term Surface Plasmon Resonance microscopy Imaging glossary

scanning technology: Imaging glossary

screening: Drug Discovery & Development glossary

secondary assays (and tertiary assays): Undertaken after primary screening has identified "hit" compounds against targets, are more complicated - and time-consuming - tests of a drug and include ADME/Tox (absorption, distribution, metabolism, excretion/toxicology) studies (e.g., done on mice or rats). Test a drug against more than one target, complicated and time- consuming, so they have not been considered practical for use in very early drug development.... . The secondary and tertiary assays tell you more biology. ...Typically, secondary and tertiary assays are more comprehensive, but they also take longer, and they are more complex and less reproducible. [CHI Breaking Bottlenecks]

selectivity: The extent to which a compound hits the intended drug target. [CHI Breaking Bottlenecks] See also IUPAC note Clinical genomics glossary  about selectivity and specificity often being used interchangeably.

sensitivity: 100% sensitivity = 100% true positives, 0% false positives.

sensors: An equipment which detects, and may indicate, and/or record objects and activities by means of energy or particles emitted, reflected, or modified by objects. [Dept. of Defense US, Dictionary of Military Terms] http://www.dtic.mil/doctrine/jel/doddict/    

Narrower terms In-depth biosensor, electronic biosensor, electronic nose, sensor web.

signal amplification: Increasing the amount of signal per unit of target for better detection.

Because of the minute scale of hybridization, and the small amounts of target present, it is desirable to increase the signal from an array. This signal- boosting can be accomplished either by increasing the amount of target (target amplification) or increasing the amount of signal per unit of target (signal amplification). [CHI Microarrays]

Narrower term: Tyramide Signal Amplification TSA

signal to noise: Ratio which can interfere with detection. Can also refer to data analysis. Biological data is often very "noisy". This is particularly seen when trying to look at low abundance biomolecules.

signals: Produced by dyes, fluorescence or radioactivity. (Non-radioactive materials, because of disposal and other problems, and improvements in other technologies) are increasingly in demand.. 

Related terms detection, reagents In-depth actuators

single cell detection and manipulation: [Viola Vogel, director of the Center of Nanotechnology at the University of Washington] indicated that single cell detection is even more nascent. "If you want to make a single molecule measurement in a cell system, you need to develop a lot of the technology that does not exist today," remarked Jay Trautman, CEO of Praelux Inc., Lawrenceville, N.J. That technology should allow researchers to target individual molecules within the cell, track where they are going, and record changes--all without significantly interfering with cellular physiology. [Dave Amber " Researchers Seek Basics Of Nano Scale" Scientist 14 (16): 1, Aug. 21, 2000] http://www.the-scientist.com/yr2000/aug/amber_p1_000821.html

single cell studies: Cell biology glossary

single molecule detection: Recent advances in optical imaging and biomechanical techniques have demonstrated that it is possible to make observations on the dynamic behavior of single molecules, to determine mechanisms of action at the level of an individual molecule, and to explore heterogeneity among different molecules within a population. These studies have the potential to provide fundamentally new information about biological processes and are critical for a better understanding of cellular function. Current high- resolution methods, such as X- ray crystallography and NMR, have provided a vast array of structural detail for biological molecules, yet the output of these methods is limited by its static molecular view and ensemble averaging. Single molecule methods provide an alternative set of approaches that will lead to a more direct view of the action of individual molecules without the need to infer process or function from static structures. Real- time measurements on the spatial and temporal fluctuations of single molecules in living cells, which are not possible using other methods, are a major goal of this initiative. Despite the promise of single molecule methods, there are a number of technical challenges that must be met to optimize these studies.

Development of the collateral chemistry and instrumentation required to carry out single molecule studies is essential for progress. New tools and strategies, as well as refinement of current methods, are also needed. Single molecule methods are likely to lead to significant advances in understanding molecular movement, dynamics, and function. ... There is an urgent need to bring chemists into this field; many of the problems in single molecule chemistry may be easily addressed by well- trained chemists. Testing new chemistry in a biological system while carrying out parallel experiments to optimize it is essential and will require ongoing interaction and collaboration. Attracting chemists to be interested in these problems is considered to be a significant barrier to progress in the field. ... 

The participants agreed that the most sophisticated methods to study single molecules will depend, to a large extent, on instruments developed by physicists thinking about biological problems. Traditionally, technical advances that have led to radical changes in the spectroscopic methods have come from physicists supported by agencies other than the NIH. For this field to move forward, it is essential to attract physicists into biology laboratories. In addition to collaborations with physicists, the NIH should focus on new, and creative ways to train physicists to think about single molecule experiments  [NIGMS, NICDC, NHGRI, Single Molecule Detection and Manipulation, Feb. 12, 2001] http://grants.nih.gov/grants/guide/pa-files/PA-01-049.html

The question of how to develop such a [nanotechnology] research infrastructure took center stage at a June 25-26 [2000] conference, "Nanoscience and Nanotechnology: Shaping Biomedical Research," held in Bethesda, Md. The National Institutes of Health Bioengineering Consortium, or BECON, sponsored the meeting that conference cochair Lynn Jelinski called a "watershed for the development of biomedical nanotechnology." Scientists from a grab bag of disciplines outlined current nanotechnology research and developed some recommendations for NIH's future involvement. Many of the more than 600 participants left voicing specific needs, such as more rigorous interdisciplinary basic research-- especially in toolmaking -  and further understanding of single molecule and cell biology. [Dave Amber " Researchers Seek Basics Of Nano Scale" Scientist 14 (16): 1, Aug. 21, 2000] http://www.the-scientist.com/yr2000/aug/amber_p1_000821.html

Advances in Assays, Molecular Labels, Signaling  & Detection: Profiling PCR  June 27-28, 2002, Washington DC Washington DC  has a session on single molecule detection

solution arrays: Functionalized particle sets for multiplexed bioanalysis in solution. [Michael Natan, SurroMed, Inc.  personal communication Dec. 2001]

specificity: In the five disciplines that eventually contributed to the formation of molecular biology, ideas of specificity had widely different standing and character. In microbiology, the relevance of specificity in the present day sense awaited resolution of the long, piecemeal shift of opinions about what sort of creatures micro- organisms were....[Oswald Avery, Chargaff, Lwoff, Luria Delbruck, Jacques Monod, Alice Audureau]  Mendelian genetics itself had always been highly specific, of course, and from very early the genetic specificity - the map - was understood to form a strictly linear array  [Thomas Hunt Morgan, Boris Ephrussi, George Beadle, Edward Tatum]... In physical chemistry, specificity was not abstract and not linear, but concretely physical and three- dimensional [Pauling] ... Crystallography, in its way, was also permeated with specificity [Edward Tyson Reichert, Amos Peaslee Brown, Felix Haurowitz ... Specificity in the present- day sense of unique molecular structures was never a surprise to the crystallographers ... The fifth discipline in the synthesis that formed molecular biology was biochemistry. Yet the standard view of the rise of molecular biology has somewhat taken for granted, for example, the radical sharpening of ideas of specificity represented by Fritz Lipmann's elucidation of the way energy is supplied to the steps of cellular reactions.  And it grievously undervalues the work of the two biochemists who proved decisive in changing the way people thought about specificity.  The man who released the present day understanding of molecular specificity in living processes was Frederick Sanger ... the most general and profound [result of his methods] was that protein s are entirely and uniquely specified.  [HF Judson Eighth Day if Creation, Cold Spring Harbor Laboratory Press, 1996 pp. 583- 585]

100% specificity = 100% true negatives, 0% false negatives. See also Clinical genomics glossary (analytical and clinical sensitivity and specificity).

Stokes shift: Microarrays glossary

tag: See label; In-depth capture tag, Isotope Coded Affinity Tags ICAT, optical tagging

target amplification: Gene amplification & PCR

transducer: <physics> A device that transforms one type of energy to another. [OMD 17 Mar 1998] 

tunable lasers: Imaging glossary

ultrasensitive: Ever lower quantities are becoming detectable. Single molecule and even single atom detection have been reported. Related terms zeptomole, yoctomole.

yoctomole: 10 ^-24  mole. One septillionth.

One yoctomole is less than one molecule which is not a useful concept for most molecular biologists. [Fralin Biotechnology Center, Virginia Tech, US] http://www.biotech.vt.edu/outreach/biotech-times/9_98/page7.html

zeptomole: 10 ^–21 mole. One-sextillionth.

Thirty years ago the limit of detection for peptides was in the order of micromoles (10^-6 moles) or about 1017 molecules. ..Although micromoles are pretty small, scientists are closing in on the absolute limit of detection, a single molecule. In recent years the limit of detection has dramatically decreased from the micromole range (10^-6 moles or 1017 molecules) to nanomole (10^-9 moles), picomole (10^-12 moles), femtomole (10^-15 moles), attomole (10^-18 moles), and now zeptomole (10^-21 moles). If one mole contains Avogadro's number of molecules then a zeptomole contains about 600 molecules. [Fralin Biotechnology Center, Virginia Tech, US] http://www.biotech.vt.edu/outreach/biotech-times/9_98/page7.html

Bibliography

Alpha glossary index

IUPAC definitions are reprinted with the permission of the International Union of Pure and Applied Chemistry.

In-depth Assays, labels, signaling & detection glossary

actuator: A  peripheral [output] device which translates electrical signals into mechanical actions; e.g., a stepper motor which acts on an electrical signal received from a computer instructing it to turn its shaft a certain number of degrees or a certain number of rotations. See: servomechanism. [FDA, US Glossary of Computerized System and Software Development Terminology 1998] http://www.fda.gov/ora/inspect_ref/igs/gloss.html  

Related term: biosensor Broader term: sensors.

avidin: Protein found in raw egg white which binds to biotin (etymology is from its avidity to biotin).

bioassay: A procedure for determining the concentration or biological activity of a substance (e.g. vitamin, hormone, plant growth factor, antibiotic, enzyme) by measuring its effect on an organism or tissue compared with a standard preparation. [IUPAC Medicinal Chemistry]

bioluminescence: Luminescence produced by living systems. [IUPAC Photo]

biotin: A vitamin of the B complex. It is a co-enzyme for various enzymes that catalyse the incorporation of carbon dioxide into various compounds. It is essential for the metabolism of fats. Biotin is attached to pyruvate carboxylase by a long, flexible chain like that of lipoamide in the pyruvate dehydrogenase complex. Adequate amounts are normally produced by the intestinal bacteria in animals. a.k.a. vitamin H (in USA). [FAO Glossary]

Narrower terms: biotin labelling, biotinylated- DNA, biotinylation Related term: streptavidin

biotin labelling  1. The attachment of biotin to another molecule. 2. The incorporation of a biotin- containing nucleotide into a DNA molecule.  [FAO Glossary]

biotinylated-DNA  A DNA molecule labelled with biotin by incorporation of biotinylated -dUTP into a DNA molecule. It is used as a non- radioactive probe in hybridization experiments, such as Southern transfer. The detection of the labelled DNA is achieved by complexing it with streptavidin (an antibiotic with a high affinity for biotin) to which is attached a colour- generating agent such as horseradish peroxidase that gives a fluorescent green colour upon reaction with various organic reagents. [FAO Glossary]

biosensor: A device that uses specific biochemical reactions mediated by isolated enzymes, immunosystems, tissues, organelles or whole cells to detect chemical compounds, usually by electrical, thermal or optical signals. [IUPAC Bioinorganic] 

Narrower term: electrochemical biosensor. Related term: sensor web

biotinylation: To label a probe with biotin.

Catalyzed Reporter Deposition CARD: See Tyramide Signal Amplification TSA.

cell assays, cellular assays: Cell biology is also looking less traditional these days. Companies such as Automated Cell and Cellomics have developed live cell assays that fully automate sample handling and quantify cellular characteristics such as motility, proliferation and morphology. The ability to track the behavior of individual cells over time permits data gathering on functional behavior not available in any other kind of assay. This functional assay technology is amenable to high throughput analysis, and therefore can occupy a niche complementary to many proteomic technologies focused on identification of potential therapeutic targets. [CHI  Summit Proteomics]

Can be used for drug screening ... some companies are using such assays to gain insights about target function.... assays [can also be used] to get detailed functional information [CHI Breaking Bottlenecks]

chemiexcitation: Generation, by a chemical reaction, of electronically excited molecular entities from reactants in their ground electronic states. [IUPAC Photo]

chemiluminescence:  Emission of light as a result of a chemical reaction without an apparent change in temperature. [MeSH]

Luminescence arising from chemiexcitation. [IUPAC Photo]

chromophore, chromophore assisted inactivation: Pharmaceutical biology glossary

competitive immunoassays: Rely on the competition between a labeled and unlabeled antigen for a limited number of antibody binding sites. [B. Weigl et al “Novel Immunoassay formats for integrated microfluidic circuits” SPIE  BIOS 2000]  http://www.micronics.net/spiebios2000/spie2000novelIAformats.htm

A single antibody is bound to a small molecular weight antigen of less than 10,000 kD. The antibody, at a very low concentration, binds the antigen in  the sample. Then a known concentration of antigen is labelled with a detector … All  remaining antibody sites bind the labelled antigen.  The amount of either the bound or free- labeled antigen added to the  reaction is measured at the end of the immunological binding reaction. The  percentage bound is inversely proportional to the amount of unlabeled  antigen. The antibody bound enzyme- labeled antigen is separated at the  end of the immunological binding reaction using a secondary antibody coated  microplate that specifically binds the primary antibody. The resulting signal is  inversely proportional to the amount of antigen in the sample. [Whatman Polyfiltronics, Technical Support, Archives  “An introduction to assays”] http://www.whatman.plc.uk/

Broader term immunoassay Related term: competitive PCR

differential labeling: When comparing the proteomes of two cell states (e.g. diseased vs. normal), gel- to- gel variability in spot position and protein yield often places the results of such experiments in question. Differential labeling  enables one to analyze both states on a single gel, thus enabling direct comparison of protein levels. In this method, cells are treated with normal media, or media enriched in ¹⁵N. Corresponding proteins from each state will migrate to the same location on the gel, but analysis by mass spectrometry will distinguish the metabolically labeled peptides and thus quantify the two sets of proteins separately. This can have significant impact on reproducibility when comparing experiments. 

An analogous differential labeling technique uses isotope coded affinity tags (ICAT) that chemically modify peptide cysteines with a normal- or deuterium- labeled biotin reagent. Samples are pooled, purified by avidin chromatography and quantified as described above, but there is no need for metabolic labeling. Both differential labeling techniques permit combined samples to be pre- fractionated prior to separation, without losing information on their relative quantities. [CHI Summit Proteomics]

electrochemical biosensor: A self- contained integrated device, which is capable of providing specific quantitative or semi- quantitative analytical information using a biological recognition element (biochemical receptor) which is retained in direct spatial contact with an electrochemical transduction element. Because of their ability to be repeatedly calibrated, we recommend that a biosensor should be clearly distinguished from a bioanalytical system, which requires additional processing steps, such as reagent addition. A device which is both disposable after one measurement, i.e., single use, and unable to monitor the analyte concentration continuously or after rapid and reproducible regeneration should be designated a single use biosensor. [IUPAC Commission on Electroanalytical Chemistry, Electrochemical Biosensors: Recommended Definitions and Classification, 1999]  http://www.iupac.org/reports/1999/7112thevenot/  Broader term biosensor

electrochemiluminescence: See Electrogenerated Luminescence ECL. [IUPAC Photo]

Electrogenerated Luminescence ECL: Luminescence produced by electrode reactions. Also called electroluminescence or electrochemiluminescence. [IUPAC Photo]

electroluminescence: See electrogenerated chemiluminescence.  [IUPAC Photo]

electronic nose: An emerging technology with potential application across a wide range of application areas, including bacteriological and environmental monitoring, explosive detection, process monitoring and control, product quality control, and fraud detection. … An instrument which comprises an array of electronic chemical sensors with partial specificity and an appropriate pattern recognition system, capable of recognising simple or complex odours (and other gaseous mixtures)1. The ability of an electronic nose to rapidly discriminate between slight variations in complex mixtures makes the techniques ideal for on- line process diagnostics and screening across a wide range of application areas. [Quantitative characterisation of electronic noses, Competitive and Sustainable Growth Programme, European Union, Oct. 2000] http://ncp.stp.lt/docs/GROWTH/00.10.13/c_topic_4_10_200003.pdf

fluorescence polarization: Fluorescence polarization is based on the observation that emission signals from small fluorescent molecules are relatively depolarized, while binding to a larger molecule reduces the tumbling rate of the fluorescer, resulting in a relatively polarized emission signal. [CHI SNPs Update]

First described in 1926 (Perrin) and has been a powerful tool in the study of  molecular interactions. When fluorescent molecules are excited with plane polarized light, they emit light in  the same polarized plane, provided that the molecule remains stationary throughout the excited state (4  nanoseconds in the case of fluorescein). However, if the excited molecule rotates or tumbles out of the  plane  of polarized light during the excited state, then light is emitted in a different plane from that of the initial  excitation. If vertically polarized light is used to excite the fluorophore, the emission light intensity can be  monitored in both the original vertical plane and also the horizontal plane. The degree to which the emission  intensity moves from the vertical to horizontal plane is related to the mobility of the fluorescently labeled  molecule. If fluorescently labeled molecules are very large, they move very little during the excited state  interval, and the emitted light remains highly polarized with respect to the excitation plane. If fluorescently  labeled molecules are small, they rotate or tumble faster, and the resulting emitted light is depolarized  relative to the excitation plane. [PanVera website] http://www.panvera.com/ls/fpabout.html

Fluorescence Recovery After Photobleaching FRAP: Based on the principal of observing the rate of recovery of fluorescence due to the movement of a fluorescent marker into an area of the membrane which contains this same marker but which has been rendered non-fluorescent via an intense photobleaching pulse of laser light. The two- dimensional diffusion coefficient (D) of the fluorophore is related to both its rate and extent of recovery. FRAP has proved to be a popular means to assess the structure of artificial and biological membranes. [Cell & Developmental Biology, Univ. of North Carolina Chapel Hill School of Medicine] http://www-cellbio.med.unc.edu/facilities/frap.htm

homogeneous assay: These assays require no separation steps. Pipette, incubate, and measure  are the only steps required. The reactions occur completely in solution  generally without beads or solid phase attachments to interfere with low  affinity interactions. Homogeneous assay methods are essential for the throughputs required in drug discovery and for assay miniaturisation. In any homogeneous assay, all the components of the assay are present during  measurement. The elimination of separation steps is the major advantage of  these assays, but this presents difficulties because of non- specific  measurement of the assay constituents. [Whatman Polyfiltronics. Technical Support, Archives  “An introduction to assays”] 

immunoassay: A ligand- binding assay that uses a specific antigen or antibody, capable of binding to the analyte, to identify and quantify substances. The antibody can be linked to a radiosotope (radioimmunoassay, RIA), or to an enzyme which catalyses an easily monitored reaction (enzyme- linked immunosorbent assay, ELISA), or to a highly fluorescent compound by which the location of an antigen can be visualized (immunofluorescence). [IUPAC Compendium] 

Related terms ELISA, Pharmaceutical Biology

Only method possible for  small molecular weight antigens, such as steroids, drugs, lipids, and  peptides. There are three basic components in any immunoassay, the  antigen to be detected and/or quantified, a specific antibody to this antigen,  and a system to measure the amount of the antigen in the sample. The  separation at the end of the immunological reaction uses a microplate. [Whatman Polyfiltronics. Archives “An introduction to assays”] http://www.whatman.plc.uk/

Narrower term competitive immunoassay

immunohistochemistry: Histochemical localization of immunoreactive substances using labeled antibodies as reagents. [MeSH]

Immunohistochemistry involves using antibodies (typically visualized via an enzyme- linked antibody assay) that specifically bind to proteins of interest. This method allows one not only to assess levels of a protein but also to localize the protein within cells in the tissue sample. [CHI Target Validation]

LIF Laser Induced Fluorescence: The optical emission from molecules that have been excited to higher energy levels by absorption of electromagnetic radiation. The main advantage of fluorescence detection compared to absorption measurements is the greater sensitivity achievable because the fluorescence signal has a very low background. For molecules that can be resonant excitated, LIF provides selective excitation of the analyte to avoid interferences. LIF is useful to study the electronic structure of molecules and to make quantitative measurements of analyte concentrations. Analytical applications include monitoring gas-phase concentrations in the atmosphere, flames, and plasmas; and remote sensing using light detection and ranging (LIDAR). [Brian Tissue, "LIF" Chemistry Hypermedia Project, Chemistry Dept. Virginia Tech, Virginia Polytechnic Institute, US, 2000 ] http://www.chem.vt.edu/chem-ed/spec/laser/lif.html

Related term: Imaging glossary  fluorescence spectroscopy- single molecule

luminometer: Instrument that measures light. [Celsis website “Hygiene monitoring glossary”] http://www.celsis.com/hygen-glossary.cfm

Optical tagging OT: See under LIF Laser Induced Fluorescence.

photoexcitation: Luminescence arising from photoexcitation. [IUPAC Photo]

quencher: A molecular entity that deactivates (quenches) an excited state of another molecular entity, either by energy transfer, electron transfer, or by a chemical mechanism. [IUPAC Photo]

quenching: 1. Arresting the course of a chemical reaction by chemical or physical means. (in photochemistry) 2. The deactivation of an excited molecular entity intermolecularly by an external environmental influence (such as a quencher) or intramolecularly by a substituent through a nonradiative process. 3. (in radiation chemistry) The process of inhibiting continuous or multiple discharges following a single event in certain types of radiation detectors. [IUPAC Compendium]

reactant: A  substance that is consumed in the course of a chemical reaction.  It is sometimes known, especially in the older literature, as a reagent, but this term is better used in a more specialized sense as a test substance that is added to a system in order to bring about a reaction or to see whether a reaction occurs (e.g. an analytical reagent). [IUPAC Compendium] Related term reagents.

sandwich assay: The antigen is "sandwiched"  between the two antibodies, one is attached to the solid phase, and the  other is labelled with an enzyme. The amount of solid phase antibody and  enzyme conjugated antibody are in a higher proportion than the amount of  antigen in the sample. The result is an assay that produces a signal that is  proportional to the amount of antigen in solution. [Whatman Polyfiltronics. Technical Support, Archives  “An introduction to assays”] http://www.whatman.plc.uk/

sensor web: An independent network of wireless, intra- communicating sensor pods, deployed to monitor and explore a limitless range of environments. This adaptable instrument can be tailored to whatever conditions it is sent to observe. [Jet Propulsion Lab, NASA "Sensor Web Project"]  http://sensorwebs.jpl.nasa.gov/

scintillation: Burst of luminescence of short duration caused by an individual energetic particle. [IUPAC Radioanalytical]

single-pair FRET spFRET: Designed to overcome the averaging effects of ensemble studies because measurements are made on single molecules freely diffusing in solution. This method limits the observation period to the diffusion time of each molecule through the focal spot of a laser on the order of a few hundred milliseconds, but it permits the rapid gathering of data at single-molecule resolution on a large number of molecules in a short time period. SpFRET can be used to study intramolecular conformational changes by placing the donor and acceptor fluorescent tags on two different sites of the same macromolecule, or alternatively, intermolecular interactions can be studied by attaching the donor and acceptor tags to two different macromolecules.  [NIGMS, Single Molecule Detection and Manipulation Workshop "Single Molecule Fluorescence of Biomolecules and Complexes Protein Folding April 17-18, 2000] http://www.nigms.nih.gov/news/reports/single_molecules.html#examples 

streptavidin:  A 60 kD extracellular protein of Streptomyces avidinii with four high-affinity biotin binding sites. Unlike AVIDIN,  streptavidin has a near neutral isoelectric point and is free of carbohydrate side chains. [Metathesaurus/MeSH]

Tyramide Signal Amplification TSA: TSA - also known as Catalyzed Reporter Deposition (CARD) - is a signal- amplification technology designed to enhance detection sensitivity in DNA arrays, in situ hybridization (ISH) assays, and other applications. It is being commercialized by NEN Life Science Products. With TSA, the enzyme horseradish peroxidase is used to activate and deposit a reactive tyramide- labeled tag (e.g., biotin or fluorescent dyes). This amplifies the signal, which can then be detected by routine means. NEN reports that in some applications, TSA increases detection limits up to 100-fold, enabling very low levels of nucleic acids or target proteins to be discerned. Researchers in the Netherlands used biotinylated tyramine- based CARD to amplify ISH signals and reported that the detection limit was "highly increased" and that CARD is especially suitable for visualizing very weak ISH signals. [Kerstens HM, Poddighe PJ, Hanselaar AG. "A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine." Journal of Histochemistry & Cytochemistry. 1995. 43:347-352.]

target labelling: Targets for arrays are labelled representations of cellular mRNA pools. Typically reverse transcription from an oligo-dT primer is used … Frequently total RNA pools (rather than mRNA selected on oligo-dT) are labelled, to maximize the amount of message that can be obtained from a given amount of tissue.  [DJ Duggan et al “Expression profiling using cDNA microarrays” Nature Genetics 21(1s): 10-14, Jan 1999]

tracer: Labelled members of a population used to measure certain properties of that population.  [IUPAC Radioanalytical]