Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1999-11-17
2002-07-30
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C204S400000, C204S403060, C422S051000, C422S051000, C422S082010, C422S082050, C422S082080, C422S082070, C435S287100, C435S287200, C435S288700, C435S808000, C436S517000, C436S528000, C436S536000, C436S149000, C436S805000, C436S806000, C530S391100
Reexamination Certificate
active
06426231
ABSTRACT:
BACKGROUND
1. Field of the Invention
This invention relates to the field of biosensing and, more particularly, to an improved method and system for analyte sensing mediated by adapter/carrier molecules which act as an adapter between the analyte and sensor element or deliver the analyte to the sensor element.
2. Description of the Background
Stochastic sensing is based on the detection of individual binding events between analyte molecules and a single receptor, which acts as a biosensor element. The read-out depends on a property of the receptor, usually a protein, that is altered when the binding site is occupied. In its simplest manifestation, stochastic sensing provides a binary signal (occupied/unoccupied) comprising fluctuations in, e.g. electrical current, fluorescence, or force. The frequency of occurrence of the binding events is determined by the concentration of the analyte. The nature of the binding events, e.g. the magnitude and duration of the associated signal, is determined by the properties of the analyte. The ability to identify an analyte by its characteristic signature is a distinctive feature of stochastic sensing.
The ability to observe changes in the state of single protein molecules has been available with respect to ion channels for over twenty years. The electrical currents generated by the large ion fluxes through these molecules (e.g., 10
8
s
−1
) can be monitored by single channel recording. More recently, structural changes in single protein molecules have been detected by fluorescence techniques and by force measurements (Doleman, B. J., et al.,
Proc. Natl. Acad. Sci. USA
95:5442-5447, 1998; Hellinga, H. W., et al.,
Trends Biotechnol.
16:183-189, 1998; Czarnik, A. W.,
Nature
394:417-418, 1998).
Genetically engineered versions of the bacterial pore forming protein &agr;-Hemolysin (&agr;HL) have been used as sensor elements. Current stochastic schemes are limited in that the type of analytes that can be sensed are restricted to those which interact with the pore. Current schemes cannot be used, for example, to analyze organic molecules, molecules insoluble in aqueous media and certain mixtures of analytes.
Better sensors would be useful in many situations. For example, in medicine, improved means for detecting physiological markers and therapeutic agents are needed; in environmental protection, various pollutants and effluents from factories must be monitored more thoroughly; for defense, new ways to detect explosives and chemical and biological agents are urgently required. In only a few cases are the available technologies optimal for the task at hand. Better devices are needed with improved sensitivity and rapid “real time” response.
There is therefore a need for analyte sensors, including stochastic biosensors, that can detect the presence and concentration of a wider variety of analytes as well as samples containing mixtures of analytes.
SUMMARY OF THE INVENTION
The present invention overcomes the problems and disadvantages associated with current strategies and designs and provides analyte sensing systems and methods which extend the categories of analytes that give a response and improve the dynamic range of response. A host molecule is used to serve as an adapter between the analyte and sensor element, or to deliver the analyte directly to the sensor element, producing a unique signal that indicates both the concentration and identity of the analyte. The invention is particularly useful in detecting organic molecules.
The present invention is a substantial improvement over stochastic sensing using pore proteins as sensor elements without the benefit of a host molecule. The host molecule of the present invention allows interactions between an analyte and a sensor element that would not normally occur. The host molecule of the present invention can concentrate analyte, even from the vapor phase. The host molecule may mediate the analysis of organic molecules that are normally insoluble in aqueous media. Two or more analyte molecules that interact with a host molecule can be analyzed simultaneously with a single sensor element. The complex signal can be resolved to reveal the concentrations of multiple components in a mixture. In addition, more than one host molecule can be used in combination with a single sensor element.
Accordingly, one embodiment of the invention is directed to a system for sensing at least one analyte comprising a sensor element and a host molecule. The sensor element has a receptor site. The host molecule, which acts as a carrier or adapter, is configured to interact with both the receptor site of the sensor element and the analyte to produce a detectable signal unique for the analyte.
Sensing may comprise identifying or quantitating/determining the concentration of the analyte, or both. The host molecule is preferably a cyclodextrin, such as &bgr;-cyclodextrin (&bgr;CD), and the sensor element is preferably an &agr;-Hemolysin (&agr;HL) pore.
Systems of the invention may be highly combinatorial. For example, another embodiment of the invention is directed to a system for sensing a plurality of different analytes comprising a plurality of different sensor elements, each sensor element comprising a pore and having a receptor site, and a plurality of different host molecules. The host molecules are each configured to interact with a receptor site of one of the plurality of different sensor elements and one of the different analytes to produce a detectable and unique signal.
Another embodiment of the invention is directed to a biosensor for detecting an analyte in a sample comprising a bilayer, which separates the biosensor into a first compartment and a second compartment, and a sensor element disposed in the first compartment so that it forms a channel in the bilayer. The biosensor further comprises a host molecule which is designed or configured to interact with a receptor site on the sensor element and the analyte to produce a detectable signal. The host molecule may be disposed in the first, second or both compartments.
Another embodiment of the invention is directed to a method for sensing at least one analyte in a sample comprising the steps of providing a biosensor, the biosensor comprising a sensor element having a receptor site and a host molecule, the host molecule configured to interact with the receptor site of the sensor element and the analyte to produce a detectable signal, allowing the sample to interact with the biosensor to produce a signal, and detecting the signal.
Still another embodiment of the invention is directed to a method of making a biosensor for detecting an analyte in a sample comprising providing a bilayer apparatus, the bilayer apparatus comprising a bilayer separating the apparatus into a first compartment and a second compartment, adding a sensor element to the first compartment and allowing it to form a channel in the bilayer, and providing a host molecule, the host molecule being configured to interact with a receptor site on the sensor element and the analyte to produce a detectable signal. The method further comprises the step of adding the host molecule to the first or second compartments or both. The host molecule may be added substantially simultaneously with the addition of the sample.
Another embodiment is directed to a method for modifying an interactive property of a protein with a second molecule comprising modifying the interactive property of the protein by contacting the protein with a third molecule, said third molecule comprising a non-covalent molecular adapter.
Other embodiments and advantages of the invention are set forth in part in the description which follows, and in part, will be obvious from this description, or may be learned from the practice of the invention.
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patent: 5252743 (1993-10-01), Barrett et al.
patent: 5620850 (1997-04-01), Bamdad et al.
patent: 5744305 (1998-04-01), Fodor et al.
patent: 5817771 (1998-10-01), Bayley et al.
patent: 5824776 (1998-10-01), Bayley et al.
patent: 6083763 (2000-07-01), Balch
patent: WO 99/05167 (19
Bayley Hagan
Braha Orit
Gu LiQun
Baker & Botts L.L.P.
Chin Christopher L.
The Texas A&M University System
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