Biosensor

Details Biosensor Biosensor

1998-06-18

2004-02-17

Smith, Lynette R. F. (Department: 1645)

Chemical apparatus and process disinfecting, deodorizing, preser

Analyzer, structured indicator, or manipulative laboratory...

C422S067000, C422S068100, C422S082050, C422S052000, C422S082080, C422S083000, C422S051000, C435S252300, C435S254110, C435S257200, C435S410000, C435S817000, C435S007310, C435S004000, C435S007230, C435S006120, C435S070100, C435S325000, C435S007200, C435S007210, C435S007800, C435S007100, C435S029000, C435S034000, C435S091500, C436S501000, C204S192130, C204S403060

Reexamination Certificate

active

06692696

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods and apparatus for detecting one or more ligands in candidate substances. More specifically, the invention is directed to methods and apparatus using biosensors incorporating G-Protein Coupled Receptors (GPCRs) for detecting specific substances or ligands, or for detecting specific objects or people associated with specific substances or ligands.
2. Description of Related Art
Biosensors have been in use for more than 30 years. In the past 7 to 8 years, however, the rate of development of biosensor technology has rapidly increased.
Biosensors are composed of a biological sensing element in intimate contact with some form of a physical transducer. Together, these two elements relate the concentration of a target analyte or ligand with some measurable signal. The earliest biosensors were simply enzymes immobilized on solid surfaces of oxygen electrodes which measured the consumption of oxygen in response to the enzymatic breakdown of substrate.
Enzyme-based biosensors have found their greatest use in the medical field for body fluid analyses (e.g. urine sugar content, blood sugar levels, serum cholesterol, lactate, and acetylcholine). Recently, a variety of other oxidative and reductive enzymes have been immobilized and coupled to colorimetric changes in product or to potentiometric or electrochemical changes in response to enzyme activities. These enzyme-based systems have been used most extensively for environmental monitoring (contaminants, pesticides, herbicides, and organic solvents) and in some cases for process stream monitoring. The sensors are suitable for multiple use and continuous monitoring up to certain limits. In general, end-product build-up leads to inhibition of the enzymatic activity, enzyme inactivation can occur as well as enzyme degradation all of which can lead to deterioration of the sensor function. Typical sensor lifetimes are only a few days.
Two additional types of biosensors have been recently developed and field-evaluated. One type exploits the highly selective recognition between antibodies and their antigens, while the second type exploits whole bacterial cells that report the presence of the target ligand through the production of light, or through a change in metabolic function.
An antibody sensor in an advanced state of development is that developed by the Naval Research Laboratory by Ligler (Naval Research Reviews, vol. 14, 1994). These biosensors use either monoclonal antibodies, which recognize a single epitope or site on the antigen or polyclonal antibodies, which recognize many epitopes on the antigen. The NRL biosensor can exploit two different types of immunological assay conditions. The response times for antibody sensors are in the range of minutes and sensitivities are nanograms per liter.
In one case, a sandwich immunological assay is used. In this assay type, antibodies for the target analyte or ligand are immobilized on an optical fiber. A solution containing the same antibodies which are labeled with a fluorophore is added with the experimental sample of ligand. The ligand binds to both sets of antibodies creating links between the immobilized antibodies on the fiber and the labeled antibodies. Thus the antigen is ‘sandwiched’ between the immobilized antibodies on the fiber and the labeled antibodies. The excitation of the fluorophore occurs through the fiber and only those fluorophores in very close proximity (ca. 150 nm) to the fiber (those bound in the sandwich) create an optical field on the surface of the fiber called the evanescent wave. This is the signal that is measured and it is proportional to the amount of ligand (and, consequently, fluorophore) bound to the fiber.
This type of antibody sensor requires sufficiently large antigens on the order of the size of small peptides (10,000 Daltons) to the size of large proteins (hundreds of thousands of Daltons), bacterial spores or viruses to ensure that a ‘sandwich’ is generated.
The second type of antibody sensor is based upon the competition between fluorescently labeled antigen and unlabeled antigen for binding sites on the fiber-immobilized antibodies. The same basic sensor design is used. The measurement is based on the reduction in fluorescence on the fiber due to binding of the unlabeled ligand which competes for sites where the labeled antigen was present in the initial calibration. The competitive immunoassay is highly suitable for small molecular species (e.g. less than 2000 Daltons). Other types of antibody sensors have been developed that exploit a variety of optical transduction schemes, including, resonance energy transfer between the chromophore bound on the antibody, which then transfers energy to chromophores bound to the ligand.
Cell-based sensors have also been developed. Among the most common to date, are genetically transformed bacterial cells are used in which the genes for bioluminescence (bacterial genes for bioluminescence are called lux genes) have been inserted to provide a means of reporting, through the generation of light, the presence of an analyte that is ‘recognized’ by the bacteria. For example, bacteria that possess the genes to bind intracellular copper (Cu
++
) were transformed to possess the lux genes as well. When Cu
++
enters the cell, the genes encoding the copper-binding proteins are ‘turned on’ or expressed. Concomitant with the activation of the genes for Cu
++
binding is expression of the lux or light-producing genes. This coupling of gene expression for these two different functions is achieved by placing the lux genes under the influence of the promoter of the genes encoding the Cu
++
binding proteins. Consequently, when the cells take up Cu
++
, they generate light thereby reporting the presence of intracellular Cu
++
. The light emission can be detected with a CCD (Charged Coupled Device) camera, or if the cells are immobilized either on a fiber optic or a surface, the light production can be quantified using a CCD chip.
Since Cu
++
is the bioactive form of copper, the cell reports and can quantify the bioavailable copper in a sample making this biosensor more useful for many ecological and environmental studies than analytical techniques that quantify total copper. The current sensitivity of the copper biosensor is in the micromole range. In a similar fashion, investigators have linked lux genes to genes encoding degrading enzymes for a variety of organic toxicants such as naphalene and toluene and have achieved nano- to picomole sensitivities in cell-based sensors with high specificity.
Because these systems require gene expression in response to the presence of a target ligand to report the presence of the ligand, they do not possess rapid response times—generally 15-30 minutes for a detectable response and 60 minutes for maximal light production. However, they can be used remotely and can have significantly long operational lifetimes. More recently, bacterial metabolic functions have been exploited as biosensors. Intracellular degradation or conversion of target ligands are “reported” through the electrochemical detection of intermediates or end-products that are suitable for detection modes when the target ligand is not. Lastly, bacterial cell biosensors have been generated by the expression of non-endogenous enzymes on the cell surface that convert target ligands that are not normally taken up by the cells into chemical species that can be readily metabolized and reported as described above.
Tissue and organ based biosensors have also been developed. These often exploit insect antennae or excitatory tissues (neurons). Such biosensors are often coupled to electrical transduction and recording schemes. A major shortcoming to these technologies is the short (generally hours) operational lifetimes of the tissues or organs in the biosensor format.
The current technologies for ligand or substance detection in the vapor or aqueous phase of the environment and in me

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