AU762948B2 - In vivo biosensor apparatus and method of use - Google Patents

Description

WO 00/33065 PCT/US99/287-3

DESCRIPTION

IN ViVO BIOSENSOR APPARATUS AND METHOD OF USE 1.0 BACKGROUND OF THE INVENTION The present application is a continuing application that claims priority to United States Provisional Application Serial Number 60/110,684, filed December 2, 1998, the entire contents of which is specifically incorporated herein by reference in its entirety.

The United States government has certain rights in the present invention pursuant to grant number R21RR14169-01 from the National Institutes of Health.

1.1 FIELD OF THE INVENTION The invention generally relates to the field of implantable diagnostic devices (i.e.

devices deployed within the body of an animal) for monitoring one or more target substances, analytes, or metabolites in the animal. More particularly, the invention provides implantable biosensor devices for monitoring and regulating the level of analytes in the tissues and circulatory system of a human. In illustrative embodiments, the apparatus comprises a biosensor that is utilized to monitor the level of blood glucose in a diabetic or hypoglycemic patient. The disclosed sensors may also be used to control or regulate the delivery of a drug or other pharmaceutical agent from an external or an implantable drug delivery system. For example, the device may form part of an artificial pancreas to regulate insulin dosage in response to the level of glucose detected in situ.

1.2 DESCRIPTION OF RELATED ART 1.2.1 BIOSENSORS Biosensors are hybrid devices combining a biological component with an analytical measuring element. The biological component reacts and/or interacts with the analyte(s) of interest to produce a response measurable by an electronic, optical, or mechanical transducer. The most common configurations presently available utilize immobilized macromolecules such as enzymes or antibodies to form the biological component. Examples of analytes and immobilized macromolecules include: glucose and WO 00/33065 PCT/US99/28733 immobilized glucose oxidase Wilkins et al., 1995): nitrate and immobilized nitrate reductase (Wu et al., 1997); hydrogen peroxide and 2.3-dichlorophenoxyacetic acid and immobilized horseradish peroxidase (Rubtsova et ai.. 1998); and aspartate and immobilized L-aspartase (Campanella et al., 1995).

1.2.2 WHOLE-CELL BIOSENSORS A further refinement for biosensors has been developed in recent years that utilizes intact living cells, such as a microorganism, or an eukarvotic cell or cell culture as an alternative to immobilized enzymes. Microbial cells are especially well suited for biosensor technologies; they are physically robust, capable of existing under extremely harsh and widely fluctuating environmental conditions, they possess an extensive repertoire of responses to their environment, and they can be genetically engineered to generate reporter systems that are highly sensitive to these environmental responses.

Polynucleotide sequences that comprise specific promoter sequences are operably linked to a gene or a plurality of genes that encode the desired reporter enzyme(s) and then introduced into and maintained within the living cell. When the target analyte is present, the reporter genes are expressed, generating the enzyme(s) responsible for the production of the measured signal. Commonly used reporter systems have utilized either the pgalactosidase (lacZ) or catechol-2,3-dioxygenase (xylE) enzymes (Kricka, 1993).

Unfortunately, a limitation of these systems has been that following exposure to the target substance(s), the cells must be destructively lysed and the enzyme(s) isolated.

This lysis is then followed by the addition of one or more secondary metabolites to yield a colorimetric signal that is proportional to the concentration of enzyme(s) in solution, providing a means to quantif. the concentration of the original target substance.

A more recent improvement in such sensors utilizes green fluorescent protein as a reporter system, with the significant advantage that cells do not require destructive assay techniques to produce colorimetric signals. Because a substrate must be added to the green fluorescent protein constructs to first initiate the light response, however, these systems are quite complicated and offer little advantage for detection of analytes in situ (Prasher, 1995).

WO 00/33065 PCT/US99/28733 1.2.3 IN VIvo SENSORS The development of an integrated in vivo implantable glucose monitor was first reported by Wilkins and Atanasov (1995). This system utilizes glucose oxidase immobilized within a micro-bioreactor. This enzyme catalyzes the oxidation of 3-Dglucose by molecular oxygen to yield gluconolactone and hydrogen peroxide, with the concentration of glucose being proportional to the consumption of 02 or the production of

H

2 0 2 Unfortunately, the presence of a glucose oxidase inhibitor molecule in the human bloodstream tended to offset proportionality constants, and made the device unsatisfactorily inaccurate for precise glucose monitoring and control (Gough et al., 1997). Also limiting was the device's relatively large size x 7 cm), which negated its usefulness as an implantable device.

Although several smaller needle-type and microdialysis glucose sensors have since been developed to circumvent size limitations (Gough et al., 1997, Selam, 1997), their reliance on a glucose oxidase enzyme-based system limits their overall effectiveness and reliability.

Several nonspecific electrochemical sensors have also been investigated as potential in vivo glucose sensors Yao et al., 1994; Larger et al., 1994), but problems including limited sensitivity, instability, and limited long-term reliability have prevented their wide-spread utilization (Patzer et al., 1995). According to Atanasov et al. (1997), continuously functioning implantable glucose biosensors with long-term stability have yet to be achieved.

1.3 DEFICIENCIES IN THE PRIOR ART Despite a significant miniaturization of biosensors during the past decade, they are still relatively large and obtrusive to serve as ideal implantable devices. Current methodologies using mammalian bioluminescent reporter cells require cell lysis and addition of an exogenous substrate to generate a measurable response. Consequently, these cells cannot serve as continuous on-line monitoring devices.

Therefore, there remains a need for the development of a small implantable monolithic containing both biological and electrical components constructed on a single substrate layer) bioelectronic monitor that is durable, inexpensive, wireless, and 4 that can communicate remotely to a drug delivery system to provide the controlled delivery of a therapeutic agent such as insulin.

SUMMARY OF THE INVENTION Embodiments of the present invention overcome these and other inherent limitations in the prior art by providing implantable apparatus and methods for detecting and quantitating particular analytes in the body of an animal. In particular, embodiments of the invention provide devices for the in vivo detection and quantitation of metabolites, drugs, hormones, toxins, or microorganisms such as viruses in a human or animal. In illustrative embdoiments, the invention provides an implantable monolithic bioelectronic device useful for the detection of glucose in a human. Such devices provide for the first time an accurate on-line detector for glucose monitoring, ."and offer the ability to control the administration of pharmaceutical agents via an external or implantable drug 20 delivery system. Also disclosed are implantable monolithic bioelectronic devices for detecting the S"concentration of signature molecules proteins released from cancer cells, etc.), clotting factors, enzymes and the like, and other analytes present in the 25 bloodstream or interstitial fluid. In the area of oncology, the biosensor devices find utility in both initial and remission monitoring, on-line measurement of e ~.the effectiveness of chemotherapy, and stimulation/activity of the immune system. Likewise, the biosensor devices are useful in other areas of medicine, including on-line monitoring for enzymes associated with the occurrence of blood clots (strokes, heart attacks, etc.), detection and quantitation of clotting factors (maintain level), hormone replacement, continuous drug monitoring (testing for controlled substances in prisoners, military personnel, etc.), monitoring of soldiers exposure to sub-lethal exposure to nerve agents H,\janel\Keep\Speci\20408-0o.doc 11/04/02 4a and other debilitating agents, monitor levels of compounds affecting mental illness, and the like.

In one embodiment there is provided an implantable monolithic bioelectronic device for detecting analyte within the body of an animal, said device comprising: an integrated circuit including at least one transducer for generating an electrical signal in response to light incident thereon; and a bioreporter capable of metabolizing said analyte and emitting light when exposed to said analyte, said bioreporter positioned so that at least a portion of said emitted light reaches said transducer.

Preferably, the implantable monolithic bioelectronic device further comprises a biocompatible container in which said integrated circuit and said bioreporter are located, whereby said implantable monolithic bioelectronic device can be implanted in the body of said animal by implanting said biocompatible 20 container.

o oo* oo*o* H.\janel\Keep\Speci\20408-0O0doc 11/04/02 WO 00/33065 PCT/US99/28733 container that is implanted within the body of the animal in which the analyte detection is desired.

The biocompatible container may be comprised of silicon nitride, silicon oxide, or a suitable polymeric matrix, with exemplary matrices such as polyvinyl alcohol, poly-Llysine, and alginate being particularly preferred. The polymeric matrix may also further comprise a microporous, mesh-reinforced or a filter-supported hydrogel.

In certain embodiments, it may also be desirable to provide a transparent, biocompatible, bioresistant separator that is operably positioned between the phototransducer and the bioreporter.

The bioreporter preferably comprises a plurality of eukaryotic or prokaryotic cells that produce a bioluminescent reporter polypeptide in response to the presence of the target analyte. Prokaryotic cells such as one or more strains of bacteria, and eukaryotic cells such as mammalian cells are particularly preferred. Exemplary mammalian cells are human cells such as islet p-cells, immortal stem cells, or hepatic cells, with immortal stem cells being particularly preferred.

These cells preferably comprise one or more nucleic acid segments that encode a luciferase polypeptide or a green fluorescent protein that is produced by the cells in response to the presence of the analyte. Preferably the nucleic acid segment encodes an Aqueorea Victoria, Renilla reniformis, or a humanized green fluorescent protein, or more preferably, a bacterial Lux polypeptide, such as the LuxA. LuxB, LuxC, LuxD, or LuxE polypeptide, or the LuxAB or LuxCDE fused polypeptides described herein.

Exemplary bacterial lux gene sequences that may be employed to prepare the genetic constructs include the Vibrio fischerii or more preferably, the Xenorhabdus luminescens luxA, luxB, luxC. hlxD, luxE, luxAB, or luxCDE genes.

Exemplary lux gene sequences that may be employed for preparation of the genetic constructs as described herein include the gene sequences disclosed in SEQ ID NO:1. Exemplary Lux polypeptide sequences are disclosed in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

The Lux polypeptides preferably comprise at least a 10 contiguous amino acid sequence from one or more of the polypeptide sequences disclosed in SEQ ID NO:2 through SEQ ID NO:6. More preferably the Lux polypeptides comprise at least a contiguous amino acid sequence from one or more of the polypeptide sequences disclosed WO 00/33065 PCT/US99/28733 in SEQ ID NO:2 through SEQ ID NO:6, and more preferably still, comprise at least a contiguous amino acid sequence from one or more of the polypeptide sequences disclosed in SEQ ID NO:2 through SEQ ID NO:6.

Such polypeptides are preferably encoded by a nucleic acid sequence that comprises at least 20, at least 25, at least 30, at least 35. at least 40, or at least 45 or more contiguous nucleotides from SEQ ID NO:1.

The expression of the Lux-encoding nucleic acid segments is preferably regulated by a nucleic acid regulatory sequence operably linked to the Lux-encoding segment.

Preferably this regulatory sequence comprises a cis-acting element that is responsive to the presence of the target analyte. Exemplary cis-acting response elements are selected from the group consisting of an S14 gene sequence, a hepatic L-pyruvate kinase gene sequence, a hepatic 6-phosphofructo-2-kinase gene sequence, a p-islets insulin gene sequence, a mesangial transforming growth factor-p gene sequence, and an acetylcoenzyme-A carboxylase gene sequence.

In an illustrative embodiment, the cis-acting response element comprises a contiguous nucleotide sequence from a P-islets insulin gene sequence or a hepatic Lpyruvate kinase gene sequence. Expression of the nucleic acid sequence is preferably regulated by a promoter sequence such as the one derived from an L-pyruvate kinaseencoding gene described herein.

The device may further comprise a wireless transmitter, an antenna, and a source of nutrients capable of sustaining the bioreporter cells. Likewise the biocompatible container enclosing the bioreporter may further comprise a membrane that is permeable to the analyte but not to the bioreporter cells themselves. Such a semi-permeable membrane permits analytes to flow freely from the bodily fluid into the detector device, but restricts the migration of bioreporter cells from the device into the surrounding tissues or circulatory system of the body in which the device is implanted.

In one embodiment, the integrated circuit is a complementary metal oxide semiconductor (CMOS) integrated circuit. The integrated circuit may comprise one or more phototransducer, that themselves may be comprised of one or more photodiodes.

Likewise. the integrated circuit may also further comprise a photodiode, a current-tofrequency converter, a digital counter, and/or a transmitter that is capable of transmitting either digital or analog data.

7 The invention also provides an implantable controlled drug delivery system that comprises both the implantable monolithic bioelectronic device and an implantable drug delivery pump that is capable of being operably controlled by the implantable monolithic bioelectronic device and that is capable of delivering the drug to the body of the animal in response to controls by the device. The invention also concerns a method of providing a controlled supply of a drug to a patient in need thereof. The method generally involves implanting within the body of the patient the controlled drug delivery system.

The invention also provides a method of determining the amount of a drug required by a patient in need thereof, such as in the case of giving a diabetic patient an appropriate amount of insulin. The method generally involves implanting within the body of the diabetic patient one or more implantable monolithic bioelectronic devices that are responsive to either 20 glucose, glucagons, insulin, or another glucose oooo oo metabolite, and determining the amount of insulin required oo S"by the patient based upon the levels of the analyte detected in the body fluids by the device. When the device indicates that higher levels of insulin are .e required, the appropriate control signal can be sent to the drug delivery system and more insulin is injected into body. When the device indicates that lower levels of insulin are required, then the appropriate control signal can be sent to the drug delivery system and less insulin can be administered. Such "real-time" monitoring of glucose in the body of the animal permits for controlled release of insulin throughout the day, and obviates the need for daily or more frequent injections of insulin that may either be too much or too little for the particular time of administration. This affords a more costeffective administration of the drug, and also provides a more stable dosing of the insulin to the patient on an "as H \janel\Keep\Speci\20408-000doc 11/04/02 8 needed" basis.

The invention also provides a kit for the detection of an analyte, and such kits generally will include one or more of the disclosed implantable monolithic bioelectronic devices in combination with appropriate instructions for using the detection device.

Such kits may also routinely contain one or more standardized reference solutions for calibrating the device, and may also include suitable storage or nutrient medium for sustaining the bioreporter cells either during storage or during use once implanted within the body of the animal. In the case of therapeutic kits, such kits will also generally include one or more controlled delivery systems for administration of the drug to the body of the animal.

The invention also provides a method of regulating the blood glucose level of an animal in need thereof. This method generally comprises monitoring the level of glucose in the bloodstream or inerstitial fluid 20 of the patient using the implantable monolithic o bioelectronic device, and administering to the patient an 9 effective amount of an insulin composition sufficient to regulate the blood glucose level.

This new type of bioluminescence-based 25 bioreporter is capable of monitoring target substances without the disadvantageous requirement that cells be destroyed to produce the measurable signal. This allows 9 for monitoring to occur continuously, on-line and in realtime (Simpson et al., 1998a, 1998b). These cells rely on luciferase genes (designated lux in prokaryotes and luc in eukaryotes) for the reporter enzyme system. U.S. patent Appl. Ser. No. 08/978,439 and Intl. Pat Appl. Ser. No.

PCT/US98/25295 (each of which is specifically incorporated herein by reference in its entirety) disclose a selfcontained miniature bioluminescence bioreporter integrated circuit ("BBIC") that was designed to detect specific molecular targets ex situ or ex vivo.

H\janel\Keep\Speci\20408-00.doc 11/04/02 9 BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Illustrative embodiments of the present invention are depicted in the drawings, with like numerals being used to refer to like and corresponding parts of the various drawings.

FIG. 1 shows a perspective view of one illustrative embodiment of the invention.

FIG. 2 shows a side view of an illustrative embodiment of the present invention.

FIG. 3 shows a block diagram of an illustrative embodiment of the integrated circuit.

FIG. 4A shows a hig-quality photodetector that 20 can be made using a standard N-well CMOS process.

Hi\Janel\Keep\Speci\20408-OO.doc 11/04/02 is This page is intentionally blank.

H \janel\Keep\Speci\20408-OO.doc 11/04/02 WO 00/33065 PCT/US99/28733 FIG. 4B shows two photodetector structures fabricated in a silicon-on-insulator CMOS process: on the left, a lateral PIN detector; on the right, a device similar to left except that the junction is formed with a Schottky junction.

FIG. 5A shows a simple photodiode consisting of a P-diffusion layer, an N-well, and a P-substrate.

FIG. 5B shows a circuit using a large area photodiode for efficient light collection, and a small-area diode in a feedback loop to supply the forward bias current that cancels out the photocurrent.

FIG. 5C shows a circuit using correlated double sampling (CDS) to minimize the effects of low frequency (flicker) amplifier noise as well as time or temperature dependent variations in the amplifier offset voltage.

FIG. 6 shows the current-to-frequency converter architecture of the apparatus.

FIG. 7 shows a prototype BBIC biosensor.

FIG. 8 shows a minimum detectable concentration of toluene as a function of integration time for the prototype BBIC employing the bioreporter Pseudomonas putida TVA8.

FIG. 9 shows the schematic representation of a peritoneal glucose biosensor and insulin pump.

FIG. 10A shows a schematic representation of an implantable biosensor containing two separate photodetectors with the bioreporters responding to either an increase or decrease in glucose concentrations.

FIG. 10B shows a side view of biosensor showing silastic covering.

FIG. 10C shows a schematic representaion of the utilization of a selectably permeable membrane to protect bioreporters from the immune response.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS The luciferase system has been adapted for use in biosensors in vivo. In prokaryotes, the lux system consists of a luciferase composed of two subunits coded for by the genes luxA and luxB that oxidize a long chain fatty aldehyde to the corresponding fatty acid resulting in a blue-green light emission at an approximate wavelength of 490 nm (Tu and Mager, 1995). The system also contains a multienzyme fatty acid reductase consisting of three proteins, a reductase encoded by hluC. a transferase encoded by luxD, WO 00/33065 PCT/US99/28733 and a synthetase encoded by luxE that convert and recycle the fatty acid to the aldehyde substrate. The genes are contained on a single operon, allowing for the cloning of the complete lux gene cassette downstream from user-specific promoters for the utilization of bioluminescence to monitor gene expression. The majority of bioluminescent bioreporters consist of Gram-negative organisms engineered to detect and monitor critically important chemical and environmental stressors (Ramanathan et al., 1997, Steinberg et al., 1995). Luciferase fusions in Gram-positive bacteria, as well as in yeast cell lines, are also being successfully performed (Andrew and Roberts, 1993, Srikantha et al., 1996).

Eukaryotic luciferase genes cloned into bacterial reporters include the firefly luciferase (luc) producing light near 560 nm and the click beetle luciferase (lucOR) emitting light near 595 nm (Cebolla et al., 1995, Hastings, 1996). Eukaryotic bioreporters have been designed to monitor glucose concentrations in rat islet p-cells (Kennedy et al., 1997), steroid activity in HeLa cells (Gagne et al., 1994), ultraviolet light effects in mouse fibroblast cells (Filatov et al., 1996), toxicity effects in human liver cancer cells (Anderson et al., 1995), estrogenic and antiestrogenic compounds in breast cancer cell lines (Demirpence et al., 1995), and erythropoiten gene induction in human hepatoma cell lines (Gupta and Goldwasser, 1996). To date, most eukaryotic bioluminescent reporters require cell destruction and the addition of an exogenous substrate, usually luciferin, to generate a measurable luminescent response.

Green fluorescent protein is also routinely used as a reporter system, with the significant advantage that cells do not require destructive assay techniques to produce colorimetric signals (Hanakam et al., 1996; Grygorczyk et al., 1996; Siegel and Isacoff, 1997; Biondi et al., 1998). However, a substrate must be added to the GFP constructs to first initiate the light response (Prasher, 1995). Humanized GFP cDNA has been developed which is specifically adapted for high-level expression in mammalian cells, especially those of human origin (Zolotukhin 1996). Humanized GFP can be efficiently inserted into mammalian cells using viral vectors (Levy et al., 1996; Gram et al., 1998).

Detection of the bioluminescent signal from the reporter organisms is achieved through the use of optical transducers, including photomultiplier tubes, photodiodes, microchannel plates, photographic films, and charge-coupled devices. Light is collected and transferred to the transducer through lenses, fiber optic cables, or liquid light guides.

WO 00/33065 PCT/US99/28733 However, applications requiring small volumes, remote detection, or multiple parallel sensing necessitate a new type of instrumentation that is small and portable, yet maintains a high degree of sensitivity.

4.1 OVERVIEW OF THE SYSTEM The present invention describes an implantable BBIC that detects selected substances. The bioreporter is a genetically engineered cell line in which the nucleic acid sequence contains a cis-activating response element that is responsive to the selected substance. In preferred embodiments, the selected substance is glucose. Exposure of the bioreporter to the selected substances causes the response element to up-regulate a nucleic acid sequence that encodes one or more polypeptides that generate a luminescent response. In a preferred embodiment, the luminescent response is generated by a prokaryotic lux system.

The function of the IC portion of the BBIC is to detect, filter, amplify, digitize, and report the bioluminescent signal. In effect, the IC serves as a complete laboratory instrument-on-a-chip: a microluminometer.

Silicon-based ICs can detect optical signals in the near ultraviolet, visible, and near infrared regions using the PN junctions normally used to form transistors (Simpson et al., 1999a). Using an n-well/p-substrate photodiode in a 0.5-,m bulk CMOS IC process, an -66% quantum efficiency has been measured at the 490-nm bioluminescent wavelength (Simpson et al., 1999b). A variety of signal-processing schemes can be employed.

However, counting the pulses from a current-to-frequency converter circuit forms a long time-constant integrator and is the causal portion of the matched filter for a low-level bioluminescent signal in white noise. Using the photodiode mentioned above with this signal-processing scheme, an rms noise level of 175 electrons/second was measured for a 13-minute integration time. corresponding to a detection limit of -500 photons/second (Simpson et al., 1999b).

A prototype BBIC was constructed by placing the toluene sensitive bioreporter, P.

putida TVA8, above a custom integrated microluminometer. FIG. 6 shows the prototype BBIC (including the bioreporter enclosure) as used in the characterization studies (Simpson et al., 1998b: Simpson et al.. 1998c; Simpson et al., 1998d).

WO 00/33065 PCT/US99/28733 With no luminescent signal coming from the cells, multiple measurements were taken with the integration time set to 1-minute. Leakage currents produced a signal of -6 counts/minute with a standard deviation of 0.22 counts/minute. As expected, the a decreased with the square root of the integration time. Longer integration times were produced off-line by summing 1-minute measurements.

Bioluminescence was induced in the BBIC cells and a control sample of cells by exposure to toluene vapor. From the control sample measurements, we estimate that the toluene concentration was no more than 1 ppm. A signal of 12 counts/minute (6 counts/minute above background) was measured. From previous measurements, P. putida TVA8 is known to have a linear response to toluene concentration until saturating when the concentration reaches a level of approximately 10 ppm. The minimum detectable toluene concentration for this BBIC as a function of integration time is shown in FIG. 8.

In general, the minimum detectable concentration is also a function of the number of bioreporter cells and the area of the photodiode.

A naphthalene-sensitive BBIC was produced using the microluminometer described above and the bioreporter P. fluorescens 5RL. Using the same experimental procedure described above, this BBIC was exposed to naphthalene vapor with a concentration of approximately 10 ppm. A signal of 240 counts/minute was recorded.

To eliminate the need for the addition of exogenous substrate, cells must themselves supply the appropriate substrate for the luciferase. In the bacterial system the substrate is generated by a fatty acid reductase complex coded for by the luxCDE genes.

This enzyme complex reduces short chain fatty acids to the corresponding aldehyde. The luciferase then oxidizes the aldehyde to the corresponding fatty acid. The preferred fatty acid for this reaction is myristic acid, which is present in eukaryotic organisms (Rudnick et al., 1993). Myristic acid is usually involved in the myristoylation of the amino terminus that is associated with membrane attachment (Borgese et al., 1996, Brand et al., 1996).

In a preferred embodiment, the bioreporter for glucose monitoring will be a mammalian bioluminescent reporter cell line that has been genetically engineered to express luminescence in response to glucose concentrations on a continuous basis, without the need for cell destruction and exogenous substrate addition. Current methodologies using mammalian bioluminescent reporter cells require cell lysis and addition of an WO 00/33065 PCT/US99/28733 exogenous substrate to generate a measurable response. Consequently, these cells cannot serve as continuous on-line monitoring devices. In a preferred embodiment, this new cell line is constructed with a bioluminescent reporter utilizing the luxAB and luxCDE genes from X luminescens incorporated into a plasmid-based system designated p.LPK.Lucw which contains a eukaryotic luc gene able to respond to glucose concentrations.

Replacement of the luc gene with the luxAB gene will allow for bioluminescence measurements to occur in real-time with glucose concentrations, negating the requirement for cell destruction and substrate addition.

To form an implantable, glucose-monitoring BBIC, the bioreporters may be entrapped in a container behind a semi-permeable membrane that keeps them in place over the IC photodetector. Alternatively the bioreporter may be encased in a polymer matrix. The BBIC is enclosed in a biocompatible housing with a semi-permeable membrane covering the bioreporter region. This membrane allows glucose to pass to the bioreporters, yet stops the passage of larger molecules that could interfere with the glucose measurement. When the glucose reaches the bioreporter, it is metabolized and the cells emit visible light. The IC detects this light, amplifies and filters this signal, and then reports this measurement. This measurement could be reported to the patient to a wristwatch receiver) or could be reported to an insulin pump in a closed-loop system that functions much like the pancreas.

FIG. 1 shows a perspective view of the present invention. Glucose 10 that is being detected enters the BBIC 11 through the semi-permeable membrane 12 that covers the bioreporter.

FIG 2 shows a side view of the present invention. The BBIC is enclosed in a biocompatible housing 20 with a semi-permeable membrane 21 covering the bioreporter held in a container 22. The cells constituting the bioreporter may be in suspension or encapsulated in a polymer matrix. The bioreporter is separated from a photodetector 23 by a protective coating 24. A single substance 25 contains the photodetector as well as additional circuits 26 that process and transmits the signal.

FIG. 3 shows a block diagram of one embodiment of the integrated circuit The photodetector is a photodiode 33 connected to a current to frequency converter The photodiode responds to light by sinking a current. The current is converted to a series of pulses that are accumulated in a digital counter 31. The number of counts in the WO 00/33065 PCT/US99/28733 counter in a fixed amount of time is directly proportional to the amount of light collected by the photodiode, which is directly proportional to the concentration of glucose. Digital processing circuitry in the digital counter would determine the appropriate next step for an insulin pump based on the measured glucose levels. The measured concentration or next instruction for the insulin pump could be reported via the wireless transmitter 32. All these circuits (photodiode, signal processing, and wireless transmission) can be fabricated on one IC.

FIG. 4 shows the bioreporter being supplied with water and nutrients. A fluid and nutrient reservoir 141 is connected to a microfluidic pump 142 so that nutrient and fluid 144 may flow through the polymer matrix 143 enclosing the bioreporter. Each of these components can be constructed on a single substrate 140.

FIG. 5A shows a high-quality photodetector made using a standard N-well CMOS process. The photodetector consists of two reverse biased diodes in parallel. The top diode is formed between the P+ active layer 45 and the N-well 46, and the bottom diode is formed between the N-well 46 and the P-substrate 47. The top diode has good short wavelength light sensitivity (400 550 nm), while the bottom diode provides good long wavelength sensitivity (500 1100 nm). Thus, the complete diode is sensitive over the range from 400 to 1100 nm. The luminescent compound under test 41 is separated from the photodetector by a layer 40 of Si 3

N

4 and a layer 42 of SiO 2 FIG. 10A, FIG. 10B, and FIG. 10C show schematic representations of an implantable biosensor containing two separate photodetectors with the bioreporters responding to either an increase or decrease in glucose concentrations.

FIG. 10B shows a side view of biosensor showing silastic covering.

FIG. 10C shows a schematic representaion of the utilization of a selectably permeable membrane to protect bioreporters from the immune response.

4.2 PHOTODETECTOR The first element in the micro-luminometer signal processing chain is the photodetector. The key requirements of the photodetector are: Sensitivity to wavelength of light emitted by the bioluminescent or chemiluminescent compound under test; WO 00/33065 PCT/US99/2873 Low background signal leakage current) due to parasitic reverse biased diodes; Appropriate coating to prevent the materials in the semiconductor devices from interfering with the bioluminescent or chemiluminescent process under study and to prevent the process under study from degrading the performance of the micro-luminometer; and.

Compatibility with the fabrication process used to create the microluminometer circuitry.

Two photodetector configurations that satisfy these requirements are described below. It should be understood, however, that alternative methods of constructing such a photodetector can be used by one skilled in the art without departing from the spirit and scope of the invention as defined in the claims.

In the first embodiment, the photodetector is fabricated in a standard N-well CMOS process. Shown in FIG. 5A, this detector is formed by connecting the PN junction between the PMOS active region and the N-well in parallel with the PN junction between the N-well and the P-type substrate. The resulting detector is sensitive to light between approximately 400 nm and approximately 1100 nm, a range that encompasses the 450 to 600 nm emission range of most commonly used bioluminescent and chemiluminescent compounds or organisms. In order to meet the requirement that the device have a low background signal, the device is operated with a zero bias, setting the operating voltage of the diode equal to the substrate voltage. The photodiode coating may be formed with a deposited silicon nitride layer or other material compatible with semiconductor processing techniques.

In the second photodetector embodiment, the detector is fabricated in a silicon-oninsulator (SOI) CMOS process. The internal leakage current in an SOI process is two to three orders of magnitude lower than in standard CMOS due to the presence of a buried oxide insulating layer between the active layer and the substrate. Two photodetector structures are envisioned in the SOI process. The first structure, shown on the left of FIG.

consists of a lateral PIN detector where the P-layer is formed by the P+ contact layer, the I (intrinsic) region is formed by the lightly doped active layer, and the N region is formed by the N+ contact layer of the SOI CMOS process. The spectral sensitivity of this WO 00/33065 PCT/US99/28733 lateral detector is set by the thickness of the active layer, which may be tuned for specific bioluminescent and chemiluminescent compounds.

The second structure, shown on the right side of FIG. 3B, is similar to the first except that the junction is formed with a Schottky junction between a deposited cobalt silicide (CoSi,) or other appropriate material layer and the lightly doped active layer.

The inventors contemplate that other photodetector configurations may be envisioned in silicon or other semiconductor processes meeting the criteria set forth above.

4.3 Low NOISE ELECTRONICS The low noise electronics are the second element in the micro-luminometer signal processing chain. The requirements for the low noise electronics are: Sensitivity to very low signal levels provided by the photodetector; Immunity to or compensation for electronic noise in the signal processing chain; 0 Minimum sensitivity to variations in temperature; Minimum sensitivity to changes in power supply voltages (for battery powered applications); S For some applications the electronics must have sufficient linearity and dynamic range to accurately record the detected signal level; and, S In other applications the electronics must simply detect the presence of a signal even in the presence of electronic and environmental noise.

Three embodiments that satisfy these requirements are described below. It should be understood, however, that alternative methods of detecting small signals while satisfying these requirements may be used without departing from the spirit and scope of the invention as defined in the claims.

FIG. 6A schematically shows the first approach to the detection of very small signals. This device uses a P-diffusion/N-well photodiode. a structure compatible with standard CMOS IC processes, in the open circuit mode with a read-out amplifier (fabricated on the same IC with the photodiode). The luminescent signal generates electron-hole pairs in the P-diffusion and the N-well. The photo-generated electrons in the P-diffusion are injected into the N-well, while the photo-generated holes in the N-well WO 00/33065 PCT/US99/28733 are injected into the P-diffusion. The N-well is tied to ground potential so that no charge builds up in this region. However, since the P-diffusion is only attached to the input impedance of a CMOS amplifier (which approaches infinity at low frequencies), a positive charge collects in this region. Thus, the voltage on the P-diffusion node begins to rise.

As the P-diffusion voltage begins to rise, the P-diffusion/N-weill photodiode becomes forward biased, thereby producing a current in a direction opposite to the photogenerated current. The system reaches steady state when the voltage on the P-diffusion node creates a forward bias current exactly equal in magnitude (but opposite in polarity) to the photocurrent. If this PN junction has no deviations from the ideal diode equation, then the output voltage is given by the following equation: V, In(I/ (A (Eq. 1) where V, is the thermal voltage (approximately 26 mV at room temperature), I is the photo-current, A is the cross-sectional area of this PN junction, and I, is the reverse saturation current for a PN junction with unit cross-sectional area. The value of I, depends greatly on the IC process and material parameters.

Two major error currents are present in PN junctions operating at low current density: recombination current and generation current. Except at very low temperatures, free carriers are randomly created in the PN junction space charge region. Since this region has a high field, these thermally excited carriers are immediately swept across the junction and form a current component (generation current) in the same direction as the photocurrent. Carriers crossing the space-charge region also have a finite chance of recombining. This creates another current component (recombination current) in the opposite direction of the photocurrent. Therefore, taking into account these error currents, Eq. 1 becomes: V, ln((I, (Eq. 2) This output voltage is a function of parameters that are generally beyond the inventors' control. However, the inventors do have control over the junction area, A.

Unfortunately, to make the inventors' output signal larger, the inventors want a small A, while the inventors want a large A for a high quantum efficiency (QE).

WO 00/33065 PCT/US99/28733 FIG. 6B shows a second microluminometer embodiment that satisfies both of these needs. This circuit uses a large area photodiode for efficient light collection, but uses a small-area diode in a feedback loop to supply the forward bias current that cancels out the photocurrent. Once again, the amplifier and feedback diodes are fabricated on the same IC as the photodiode. For this circuit: Vo,, 3 V, In((Ip,+ I) (Eq. 3) where Af is the small cross-sectional area of the feedback diode. More than one diode is used in the feedback path to make the output signal large compared to the DC offset of any subsequent amplifier stages. This technique allows efficient collection of the light with a large-area photodiode, yet produces a large output voltage because of the smallarea diodes in the feedback path.

The feedback circuit of FIG. 6B maintains the photodiode at zero bias. With no applied potential, the recombination and generation currents should cancel. Eq. 3 becomes: Vo= 3 V, ln((I/ (Af 1) (Eq. 4) if the smaller recombination and generation currents in the smaller feedback diodes are neglected.

The principal advantages of the second micro-luminometer embodiment shown in FIG. 6B include: The SNR is totally determined by the photodiode; noise from the small diode and amplifier are negligible; Diodes can be added in the feedback path until the signal level at the output of the amplifier is significant compared to offset voltages (and offset voltage drift) of subsequent stages; This method is completely compatible with standard CMOS processes with no additional masks, materials, or fabrication steps; This detection scheme can be fabricated on the same IC with analog and digital signal processing circuits and RF communication circuits; and, Measurement can be made without power applied to the circuit. Power must be applied before the measurement can be read, but the measurement can be obtained with no power.

WO 00/33065 PCT/US99/28733 A third microluminometer implementation shown in FIG. 6C uses correlated double sampling (CDS) to minimize the effects of low frequency (flicker) amplifier noise as well as time or temperature dependent variations in the amplifier offset voltage. As shown in FIG. 6C. a photodiode with capacitance C, and noise power spectral density S, is connected to an integrating preamplifier with feedback capacitance C and input noise power spectral density S, through a set of switches that are controlled by the logical level of a flip-flop output. When the flip-flop output is low, the switches are positioned so that the photocurrent flows out of the preamplifier, causing the output voltage of the integrator to increase. When the low-pass filtered integrator output voltage exceeds a threshold, the upper comparator "fires" setting the flip-flop and causing its output to go high. The detector switches change positions, causing current to flow into the integrating amplifier, which in turn causes the amplifier output voltage to decrease. When the integrator output goes below a second threshold, VO, the lower comparator "fires" resetting the flip-flop and causing the output to go low again. The process repeats itself as long as a photocurrent is present.

The average period of the output pulse, At, is given by the following equation: 2Cf (VHI

VLO)

A/ (Eq. where VHI and VIo are the threshold voltages of the comparators and I, is the diode photocurrent. Two noise sources contribute to error in the measured value of At. S, is the input noise current power spectral density associated primarily with the photodiode, and is the input noise voltage power spectral density associated primarily with the preamplifier. The diode noise is given by the equation:

A

2 Si 2q(2I Ip) (Eq. 6) where I, is the photodiode reverse saturation current and Ip is the photocurrent. As the photocurrent approaches zero. the noise power spectral density approaches a finite value of 4ql, A 2 /Hz. The noise voltage of the preamplifier is determined by its design and has units of V 2 /Hz.

The transfer function from the point where the diode noise is introduced to the output of the integrator is given approximately by the equation: WO 00/33065 PCT/US99/28733 Hi 1 S+O (Eq. 7) where co is the corer frequency of the integrating amplifier and s jco. Ignoring for the moment the effect of the switches, the transfer function from the point where the amplifier noise is introduced to the output of the integrator is given approximately by the equation: H(C) Cd (Eq. 8) The switches perform a correlated double sampling function that attenuates the noise that appears below the switching frequency of the output pulse string. The transfer function of a correlated double sampling circuit is approximated to first order by the equation: H(w) j- (Eq. 9) where At is the average period of the output pulse string. Thus, taking into account the switches, the transfer function from the point where the amplifier noise is introduced to the output of the integrator is approximately given by the equation: H,(co z f (Eq. C s i s+2 This is an important result because the effective zero introduced in the noise voltage transfer function reduces the effect of the flicker noise of the amplifier. This is particularly useful in CMOS implementations of the micro-luminometer where flicker noise can have a dominant effect.

The mean squared output noise at the output of the integrator is given by the equation: (Eq. 11) and the RMS noise voltage is then given by the equation: (Eq. 12) WO 00/33065 PCT/US99/28733 The RMS error in the measured period is determined by the slope of the integrated signal and the noise at the output of the integrator following the relationship: dV (Eq. 13) /dt or, approximately, by the equation: o, (Eq. 14) At The error in measuring At may be reduced by collecting many output pulses and obtaining an average period. The error in the measured average pulse period improves proportionately to the square root of the number of pulses collected, such that o, (V (Eq. At or o, (Eq. 16) At At where is the total measurement time.

Thus, implementation of the micro-luminometer has the following advantages: The low frequency "flicker" noise of the amplifier is reduced by a correlated double sampling process; and, Ideally, the accuracy of the measured photocurrent may be improved without limit by acquiring data for increasing periods of time.

Of course, practical limitations imposed by the lifetime and stability of the signals produced by the luminescent compound under test will ultimately determine the resolution of this implementation.

4.4 READ-OUT ELECTRONICS Several methods of communicating data from the BBIC to external receivers or in vivo drug delivery systems are envisaged. In a preferred embodiment the communication WO 00/33065 PCTIUS99/28733 method is an on-chip wireless communication system that reports the level of the photocurrent to computing circuitry contained within in vivo drug delivery system or an external receiver. In a closed-loop system, this computing circuitry would determine the amount of drug to be delivered by the in vivo drug delivery system. If an external receiver were used, the data from the BBIC along with the user inputs would be used to determine the amount of drug to be administered. The external receiver may include wireless transmission circuitry for communication with the in vivo drug delivery system or the drugs may be administered manually. Other methods of communicating BBIC data include; Generation of a DC voltage level proportional to the photocurrent with a hardwire connection to an in vivo drug delivery system; Generation of a DC current level proportional to the photocurrent with a hardwire connection to an in vivo drug delivery system; Generation of a logical pulse string whose rate is proportional to the photocurrent with a hardwire connection to an in vivo drug delivery system; On-chip implementation of an analog to digital converter that reports a numerical value proportional to the photocurrent with a hardwire connection to an in vivo drug delivery system; On-chip implementation of a serial or parallel communications port that reports a number proportional to the photocurrent with a hardwire connection to an in vivo drug delivery system; Generation of a logical flag when the photocurrent exceeds a predefined level with a hardwire connection to an in vivo drug delivery system; and, Generation of a radio-frequency signal or beacon when the photocurrent exceeds a predefined level.

Wireless communication in vivo may be limited by signal attenuation by body fluids, tissues, and health-related limits on RF signal levels. This may require the BBIC and in vivo drug delivery system to be closely spaced, which may not be the optimum configuration for all cases. In such cases, the BBIC could communicate to an external receiver located ex vivo but closer to the BBIC. This receiver could be connected WO 00/33065 PCT/US99/28733 (hardwired or wirelessly) to a transmitter located ex vivo but closer to the in vivo drug delivery system.

Numerous algorithms are envisioned for controlling an in vivo drug delivery system with a BBIC. These include, but are not limited to a simple look-up table that administers a prescribed drug level that is determined only by a single BBIC data point; a simple look-up table that administers a prescribed drug level when a predetermined number of data points exceed a preset threshold; an algorithm that determines drug dosage by rate of increase or decrease of BBIC signal an algorithm that determines drug dosage by matching BBIC data points to data point patterns stored in memory learning algorithms that use BBIC data point history and user inputs to predict correct drug dosage to achieve desired results Some of these algorithms may require two-way communication between the BBIC and in vivo drug delivery system. In this case, a receiver would be included on the BBIC.

BIOCOMPATIBLE HOUSING AND SEMI-PERMEABLE MEMBRANE The BBIC is enclosed in a biocompatible housing with a semi-permeable membrane covering the bioreporter region. The preparation of biocompatible coverings for implants and prosthetic devices so as to minimize capsule formation and physiological rejection has been an area of extensive investigation. For example, U. S. Patent 5,370,684 and U. S. Patent 5,387,247 (each specifically incorporated herein by reference in its entirety), describe the application of a thin biocompatible carbon film to prosthetic devices. A biocompatible implant material comprising a three-dimensionally woven or knitted fabric of organic fibers is disclosed in U. S. Patent 5,711,960, specifically inlcuded nerein in its entirety. Other coverings for implants constructed to present a biocompatible surface to the body are described in U. S. Patent 5,653,755. U. S. Patent 5,779,734, and U.

S. Patent 5.814,091 (each specifically incorporated herein by reference in its entirety). In addition, collagen coating and albumin coating have been shown to improve the biocomapatibilty of implants and prosthetic devices (Marios et al., 1996; Ksander, 1988).

WO 00/33065 PCT/US99/28733 The present invention contemplates the use of any suitable biocompatible material to either coat or form the housing.

A semi-permeable membrane comprises that part of the BBIC housing that covers the bioreporter and entraps them on the integrated circuit. This membrane allows the selected substance, such as glucose, to pass to the bioreporter, yet prevents the passage of larger molecules. Membranes designed for use with glucose-oxidase based biosensors may also used in the preferred embodiments of the present invention. Membranes investigated and designed for use with glucose-oxidase based biosensors include, but are not limited to: polytetrafluoroethylene membranes (Vaidaya and Wilkins, 1993); perfluorinated ionomer membranes (Moussy et al., 1994); charged and uncharged polycarbonate membranes (Vadiya and Wilkins 1994); and cellulose acetate membranes (Wang and Yuan, 1995; Sternberg et al., 1988). In addition, other membranes have been developed for the use transplantation of islets or other cells bioengineered to produce insulin. The membranes must be permeable to glucose and other metabolites while exclude elements of the host immune system. Such membranes may be adapted for use with the present invention and include, but are not limited to: asymmetric poly(vinyl alcohol) membranes (Young et al., 1996); poly(L-lysine) membranes (Tziampazis and Sambanis, 1995); ployurethane (Zondervan et al., 1992); nucleopore membranes (Ohgawara et al., 1998) and agarose gel (Taniguchi et al., 1997). Biocompatible semipermeable membranes for encapsulation of cells to form an artificial organ are described in U. S. Patent 5,795,790 and U. S. Patent 5,620,883 (each specifically incorporated herein by reference in its entirety). A biocompatible semi-permeable segmented block polyurethane copolymer membrane and its use for permeating molecules of predetermined molecular weight range are disclosed in U.S. Patent No. 5,428,123, (specifcally incorporated herein by reference in its entirety). The present invention contemplates the use of any suitable semi-permeable membrane that allows the selected substance access to the bioreporter yet prevents the passage of larger molecules.

4.6 DRUG DELIVERY DEVICES Numerous drug delivery devices, implantable and external, have been previously described which can be controlled by radio telemetry. For example, U. S. Patent 4,944,659 (specifically incorporated herein by reference in its entirety), describes an WO 00/33065 PCT/IUS99/28733 implantable piezoelectric pump for drug delivery in ambulatory patients. U. S. Patent 5,474,552, specifically included herein in its entirety, describes an implantable pump for use in conjunction with a glucose sensor that can deliver multiple active agents, such as glucose, glucagon, or insulin as required. Separate pumps may be used for delivering each of the agents or a single pump that is switchable between them may be used. U. S.

Patent 5,569,186, specifcally inlcluded herein in its entirety, describes a closed loop infusion pump system controlled by a glucose sensor. U. S. Patent 4,637,391, specifically included herein in its entirety, describes a remote controlled implantable micropump for delivery of pharmaceutical agents. The use of external drug delivery systems is contemplated in other embodiments of the present invention. For example, U. S. Patent 5,800,420, specifically inlcuded herein in its entierty, discloses a pump position topically against the skin surface that delivers a liquid drug, such as insulin, via a hollow delivery needle extending into the dermis. In other embodiments of the present invention, the drug delivery system may be interfaced with the biosensor device and controlled directly, as opposed to remote telemetry control, from the BBIC.

The pump delivery systems described above are examples to facilitate the use of the present invention. Drug delivery devices other than pump systems are also contemplated by the present invention. For example, U. S. Patent 5,421,816, specifcially included herein in its entirety, describes an ultrasonic transdermal drug delivery system.

Ultrasonic energy is used to release a stored drug and forcibly move the drug through the skin of an organism into the blood stream. Thus the invention contemplates the use of any suitable drug delivery system that can be controlled by the BBIC glucose monitor. The factors dictating the choice of such a drug delivery system and its use with the BBIC glucose monitor use will be known to those of skill in the art in light of the present disclosure.

4.7 BIOLUMINESCENT BIOREPORTERS In a preferred embodiment of the invention, the bioreporter for glucose monitoring will be a mammalian bioluminescent reporter cell line that has been genetically engineered to express luminescence in response to glucose concentrations on a continuos basis. An implantable bioluminescent sensor requires a bioluminescent reporter that can function without the exogenous addition of substrate for the luciferase reaction. Current WO 00/33065 PCT/US99/28733 eukaryotic luciferase systems used in molecular biology require the addition of exogenous substrate because of the complex nature for the production of eukaryotic luciferins. Cells must be either permeabilized or lysed and then treated with an assay solution containing luciferin. Thus current eukarvotic luciferases systems are not preferred candidates for online monitoring.

The requirement for the addition of exogenous substrate can be obviated by the use of bacterial lux genes. In a preferred embodiment of the present invention the lux genes of X luminescens, luxAB and luxCDE, are used as the bioluminescent reporter system. The X luminescens luxAB gene encodes the a- and P-subunits of a luciferase enzyme that exhibits greatest thermostability at 37 0 C, while other bacterial luciferases lose significant activity above 30 0 C. The luxCDE genes are required to eliminate the need for the addition of exogenous substrate. The aldehyde substrate of the luciferase encoded by the luxAB genes is generated by a fatty acid reductase complex coded for by the luxCDE genes. The preferred fatty acid for this reaction is myristic acid, which is present in eukaryotic organisms (Rudnick et al., 1993), and thus eukaryotic cells are suitable host cells for this reporter. The enzyme complex reduces the fatty acid to the corresponding aldehyde. The luciferase then oxidizes the aldehyde to back to the fatty acid.

Other bioluminescence nucleic-acid segments may include the lux genes of Vibrio fischerii, luxCDABE, or luciferases from other organisms capable of bioluminescence that can be adapted so not as to require the addition of exogenous substrate. In other embodiments of the invention, nucleic acid segment encodes green fluorescent protein of Aqueorea victoria or Renilla reniformis.

4.8 RECOMBINANT VECTORS EXPRESSING BIOLUMINESCENCE GENES One important embodiment of the invention is a recombinant vector that comprises one or more nucleic-acid segments encoding one or more bioluminescence polypeptides. Such a vector may be transferred to and replicated in a eukaryotic or prokaryotic host.

It is contemplated that the coding DNA segment will be under the control of a recombinant, or heterologous promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a DNA segment encoding a crystal protein or peptide in its natural environment. Naturally, it will WO 00/33065 PCT/US99/28733 be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type, organism, or even animal, chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology (see Sambrook et al., 1989). In a preferred embodiments of this. such promoters are directed by cis-acting glucose response elements.

In one preferred embodiment, the glucose response element is the L4 box which directs the L-pyruvate kinase promoter in liver and islet p-cells. The L4 box consists of a tandem repeat of non-canonical E-boxes (Kennedy et al.. 1997). Glucose enhances the hepatic and pancreatic p-cell by modifying the transactivating capacity of upstream stimulatory factors bound to the L4 box (Kennedy et al., 1997; Doiron et al., 1996).

The exact mechanism by which glucose controls the transactivational capacity of USF proteins is unclear. One possibility is the reversible phosphorylation of USF proteins. Glucose may alter the phosphorylation status through the pentose phosphate shunt via xyulose 5-phosphate (Dorion et al., 1996). An alternative mechanism is via the intracellular concentration of glucose 6-phosphate (Foufelle et al., 1992). Other glucose metabolites may also be implicated. Phosphorylated glucose metabolites include, but are not limited to, fructose 6-phosphate, 6-phosphogluconic acid, 6-phosphoglucono-6lactone, ribulose 5-phosphate. ribose 5-phosphate, erythrose 4-phosphate, sedoheptulose 7-phosphate, glyceraldehyde 3-phosphate and dihdyroxyacetone phosphate. Nonphosphorylated glucose metabolites include, but are not limited to, citric acid, cis-aconitic acid, threo-isocitric acid, succinic acid, fumaric acid, malic acid, oxaloacetic acid, pyruvic acid and lactic acid.

Another glucose response element, similar in arrangement to the L-PK gene L4 box, is the regulatory sequence involved in the transcriptional induction of the rat S14 gene (Shih et al., 1995). Other glucose response elements that have been described include, but are not limited to, the hepatic 6-phosphofructo-2-kinase gene (Dupriez and Rousseau, 1997), the p-islets insulin gene (German and Wang, 1994), the mesangial transforming growth factor-beta gene (Hoffman et al., 1998), and the gene for acetylcoenzyme-A carboxylase (Girard et al., 1997). The present invention contemplates the use any glucose response element that can effectively direct a promoter or otherwise control the expression of the reporter protein in response to glucose.

WO 00/33065 PCT/US99/28733 In a preferred embodiment, the recombinant vector comprises a nucleic-acid segment encoding one or more bioluminescence polypeptides. Highly preferred nucleicacid segments are the ha genes of X luminescens luxAB and luxCDE. Bacterial luciferases may have to be modified to optimize expression in eukaryotic cells.

Almashanu et al. (1990) fused the luxAB genes from V harveyi by removal of the TAA stop codon from luxA, the intervening region between the two genes, and the initial methionine from luxB without disrupting the reading frame. The fusion was successfully expressed in Saccharomyces cerevisiae and Drosophila melanogaster. The same strategy was used with luxAB from X luminescens. The resultant construct has been sequenced to verify the genetic changes to generate the fusion and they were confirmed. The sequence of the fusion region is as follows: 5'-tacctagggagaaagagaatg-3' (SEQ ID NO:7) (end of luxA underlined) (start of luxB underlined) The fusion successfully expresses fused protein in E. coli and has been successfully cloned into the mammalian vector as described in Section 5.1.2.

In a further embodiment, the inventors contemplate a recombinant vector comprising a nucleic-acid segment encoding one or more enzymes that are capable of producing a reaction that yields a luminescent product or a product that can be directly converted to a luminescent signal. For example, substrates of the commonly used pgalactosidase and alkaline phosphates enzymes are commercially available that are luminescent (chemiluminescence) when converted by the respective enzyme.

In another important embodiment, the biosensor comprises at least a forst transformed host cell that expresses one or more of recombinant expression vectors. The host cell may be either prokaryotic or eukaryotic. In a preferred embodiment, the host cell is a mammalian cell. Host cells may include stem cells, P-islets cells or hepatocyte cells.

In a preferred embodiment the host cells are homologous cells, i.e. cells taken from the patient that are cultured, genetically engineered and then incorporated in the BBIC.

Particularly preferred host cells are those which express the nucleic-acid segment or segments comprising the recombinant vector which encode the lux genes of X luminescens, luxAB and luxCDE. These sequences are particularly preferred because the transcribed proteins of the X luminescens lux system have the ability to function at 37°C (ambient human body temperature).

WO 00/33065 PCT/US99/28733 A wide variety of ways are available for introducing a nucleic-acid segment expressing a polypeptide able to provide bioluminescence or chemiluminescence into the microorganism host under conditions that allow for stable maintenance and expression of the gene. One can provide for DNA constructs which include the transcriptional and translational regulatory signals for expression of the nucleic-acid segment, the nucleicacid segment under their regulatory control and a DNA sequence homologous with a sequence in the host organism, whereby integration will occur or a replication system which is functional in the host, whereby integration or stable maintenance will occur or both.

The transcriptional initiation signals will include a promoter and a transcriptional initiation start site. In preferred instances, it may be desirable to provide for regulative expression of the nucleic-acid segment able to provide bioluminescence or chemiluminescence, where expression of the nucleic-acid segment will only occur after release into the proper environment. This can be achieved with operators or a region binding to an activator or enhancers, which are capable of induction upon a change in the physical or chemical environment of the microorganisms. For translational initiation, a ribosomal binding site and an initiation codon will be present.

Various manipulations may be employed for enhancing the expression of the messenger RNA, particularly by using an active promoter, as well as by employing sequences, which enhance the stability of the messenger RNA. The transcriptional and translational termination region will involve stop codon or codons, a terminator region, and optionally, a polyadenylation signal (when used in an Eukaryotic system).

In the direction of transcription, namely in the 5' to 3' direction of the coding or sense sequence, the construct will involve the transcriptional regulatory region, if any, and the promoter, where the regulatory region may be either 5' or 3' of the promoter, the ribosomal binding site, the initiation codon, the structural gene having an open reading frame in phase with the initiation codon, the stop codon or codons, the polyadenylation signal sequence, if any, and the terminator region. This sequence as a double strand may be used by itself for transformation of a microorganism host, but will usually be included with a DNA sequence involving a marker, where the second DNA sequence may be joined to the expression construct during introduction of the DNA into the host.

WO 00/33065 PCT/US99/28733 By "marker" the inventors refer to a structural gene that provides for selection of those hosts that have been modified or transformed. The marker will normally provide for selective advantage, for example, providing for biocide resistance resistance to antibiotics or heavy metals); complementation, so as to provide prototrophy to an auxotrophic host and the like. One or more markers may be employed in the development of the constructs, as well as for modifying the host.

Where no functional replication system is present, the construct will also include a sequence of at least 50 basepairs preferably at least about 100 bp, more preferably at least about 1000 bp, and usually not more than about 2000 bp of a sequence homologous with a sequence in the host. In this way, the probability of legitimate recombination is enhanced, so that the gene will be integrated into the host and stably maintained by the host. Desirably, the nucleic-acid segment able to provide bioluminescence or chemiluminescence will be in close proximity to the gene providing for complementation as well as the gene providing for the competitive advantage. Therefore, in the event that the nucleic-acid segment able to provide bioluminescence or chemiluminescence is lost, the resulting organism will be likely to also have lost the complementing gene, and the gene providing for the competitive advantage, or both.

A large number of transcriptional regulatory regions are available from a wide variety of microorganism hosts, such as bacteria, bacteriophage, cyanobacteria, algae, fungi, and the like. Various transcriptional regulatory regions include the regions associated with the trp gene, lac gene, gal gene, the ?L and R promoters, the tac promoter. See for example, U. S. Patent 4,332,898; U. S. Patent 4,342,832; and U. S.

Patent 4,356,270 (each specifically incorporated herein by reference in its entirety). The termination region may be the termination region normally associated with the transcriptional initiation region or a different transcriptional initiation region, so long as the two regions are compatible and functional in the host. In a preferred embodiment of the present invention, a fragment of the L-pyruvate kinase gene is used that contains the L-PK promoter and the L4 box glucose responsive elements as described by Kennedy et al. (1997). In a highly preferred embodiment, the p.LPK.Lucn plasmid is used (Kennedy et al., 1997), with the exception that the luc gene coding for the firefly luciferase is removed and replaced with the fused X luminescens luxAB genes.

WO 00/33065 PCT/US99/2873-3 Where stable episomal maintenance or integration is desired, a plasmid will be employed which has a replication system that is functional in the host. The replication system may be derived from the chromosome, an episomal element normally present in the host or a different host. or a replication system from a virus that is stable in the host.

A large number of plasmids are available, such as pBR322, pACYC184, RSF1010, pR01614, and the like. See for example, Olsen et al., 1982; Bagdasarian et al., 1981, and U. S. Patent 4,356,270, U. S. Patent 4,362,817, U. S. Patent 4,371,625, and U. S. Patent 5,441,884, each of which is incorporated specifically herein by reference.

The desired gene can be introduced between the transcriptional and translational initiation region and the transcriptional and translational termination region, so as to be under the regulatory control of the initiation region. This construct will be included in a plasmid, which will include at least one replication system, but may include more than one, where one replication system is employed for cloning during the development of the plasmid and the second replication system is necessary for functioning in the ultimate host. In addition, one or more markers may be present, which have been described previously. Where integration is desired, the plasmid will desirably include a sequence homologous with the host genome.

The transformants can be isolated in accordance with conventional ways, usually employing a selection technique, which allows for selection of the desired organism as against unmodified organisms or transferring organisms, when present. The transformants then can be tested for bioluminescence or chemiluminescence activity. If desired, unwanted or ancillary DNA sequences may be selectively removed from the recombinant bacterium by employing site-specific recombination systems, such as those described in U. S. Patent 5,441,884, specifically incorporated herein by reference in its entirety.

4.9 ASSEMBLY AND STORAGE OF THE IN VIVO BIOSENSOR When the biosensor consists of bioengineered cells entrapped in suspension behind a semi-permeable membrane, as opposed to encapsulated in a matrix, the cells may be added to the BBIC any time from immediately to several h before implantation of the biosensor. The biosensor may alternatively consist of cells encapsulated in a polymeric matrix. Matrices will include materials previously shown to be successful in the encapsulation of living cells. including polyvinyl alcohol, sol-gel, and alginate (Cassidy et WO 00/33065 PCT/US99/28733 al., 1996). Prior to encapsulation, prokaryotic cell lines may be lyophilized in a freeze dry system Savant) following the manufacturer's protocol. Lyophilization allows cells to undergo periods of long-term storage (several years) with a simple rehydration protocol being required for cell resuscitation prior to BBIC use (Malik et al.. 1993). S. cerevisiae eukaryotic cells may be similarly lyophilized. Eukaryotic cell lines, preferably consisting of islet P-cells, stem cells, or hepatic cells, may be encapsulated on the IC within polyvinyl alcohol mesh-reinforced or microporous filter supported hydrogels, which have previously been successfully implemented in these types of cell encapsulations (Baker et al., 1997; Burczak et al., 1996; Gu et al., 1994; Inoue et al., 1991).

In the case of the mammalian cell lines, lyophilization, however, is not an alternative. In such cases, mammalian cells may be encapsulated in a sol gel or another immobilization matrix as previously described and attached to the BBIC. The completed BBIC in its enclosure would then be stored in serum or another appropriate maintenance medium and maintained until use. The advantage of using an immortal stem cell line is apparent for both long-term use and storage. Implantation may be performed according to the specific application. In the case of glucose detection, an area where interstitial fluid is accessible would be most appropriate. However an implantable device with the specific application of detecting hormones or other blood bome molecules would have to be accessible to the bloodstream. A synthetic vein or catheter system may need to be employed to allow continuous monitoring of the blood levels of the target molecule. A specific example other than glucose would be the use of the in vivo biosensor device to detect molecules associated with colon cancer. In this case the biosensor would be implanted in the colon.

Integrated circuits may be individually packaged in sterile, static-proof bags.

Prokaryotic-based and yeast eukaryotic biosensors consisting of lyophilized cells may be individually stored in sterile, static-proof, vacuum sealed bags for time periods approaching several years. Cells typically undergo rehydration in a minimal nutrient medium prior to use. Mammalian cell systems will remain frozen for long-term storage (up to 7 years at -150 0 C) or refrigerated for short-term storage (several days), either separately or, if entrapped, frozen or refrigerated in situ on the BBIC. In all cases, cell viability may be checked be exposing the BBIC to a known concentration of the analyte of interest, thus producing a quantitative bioluminescent signal of known magnitude. One WO 00/33065 PCT/US99/2873 or more control vials of analyte(s) or reference "standards" may be included as part of a diagnostic kit, or may be supplied for proper calibration of the implantable device.

4.10 IMPLANTATION AND USE OF THE BIOSENSOR DEVICES In a preferred embodiment of the present invention, the BBIC analyte biosensor is implanted such that it is contact with the interstitial fluid of the animal. For example, in the case of glucose biosensors, it has been shown that glucose kinetics in interstitial fluid can be predicted by compartmental modeling (Gastaldelli et al., 1997). In particular the subcutaneous placement of glucose sensors has been demonstrated (Schmidt et al., 1993; Poitout et al., 1993; Ward et al., 1994; Stenberg et al., 1995; Bantle and Thomas, 1997).

Other potential analyte biosensor tissue implant sites include the peritoneum, pleura and pericardium (Wolfson et al., 1982). In fact, the inventors contemplate that depending upon the particular analyte or metabolite that is being detected, the implantable biosensor may be placed in any convenient location throughout the body using conventional surgical and implant methodologies. For example, the device may be implanted in such as way as to be in contact with interstitial fluid, lymph fluid, blood, serum, synovial or cerebrospinal fluid depending upon the particular analyte to be detected.

In certain embodiments the implantable device msy be placed in contact with particular tissues, organs, or particular organ systems. Likewise, it may be desirable to implant the biosensor such that it contacts particular intracellular fluids, intercellular fluids, or any other body fluid in which the target substance can be monitored.

The present invention also contemplates the use of multiple biosensors for the detection of a plurality of different analytes. For example, in the case of glucose monitoring, one or more devices may be used to monitor various glucose or glucose metabolites, glucagons, insulin, and the like. Likewise, one or more biosensor devices may be employed in controlled drug delivery systems. As such, the device may be operably connected to a drug delivery pump or device that is capable of being controlled by the biosensor and that is able to introduce into the body of the animal an amount of a particular drug, hormone, protein, peptide, or other pharmaceutical composition determined by the concentration of one or more analytes detected by the BBIC device.

Thus, controlled drug delivery systems are contemplated by the inventors to be particularly desirable in providing long-term administration of drugs to an animal such as WO 00/33065 PCT/US99/28733 in the case of chronic or life-long medical conditions or where symptoms persist for a long period of time. The long term controlled delivery of drugs such as pain medications, heart or other cardiac regulators, diuretics, or homones or peptides such as insulin, or metabolites such as glucagon or glucose can be facilitated by such biosensor/pump systems. In cases where it is necessary to deliver more than one drug or metabolite to the animal, multiple drug delivery systems or a single switchable drug delivery system is contemplated to be particularly useful.

Host-rejection effects can be minimized through immunoisolation techniques.

Previous studies have shown that living non-host cells enclosed in hydrogel membranes are protected from immune rejection after transplantation (Baker et al., 1997; Burczak et al., 1996; Inoue et al., 1991). The hydrogels block access by the humoral and cellular components of the host's immune system but will remain permeable to the target substance glucose. A mesh-reinforced polyvinyl alcohol hydrogel bag developed by Gu et al. (1994) may be used to fully encapsulate the BBIC. allowing for transplantation void of immunosuppressive responses.

Host rejection of the implanted biosensor is not an issue if cells from the host are used for the biosensor construction. However if other cell lines are used it may be necessary to provide a barrier between the cells and the appropriate body fluid that permits passage of the signature molecules or analytes but not bioreporter cells or body cells (white blood cells, etc.). Immunosuppressed patients are not affected, as the implant does not contain any kind of pathogenic agent that would affect the patient. In all cases, the surgical methods involved in implantation of the disclosed BBIC devices are well known to one of skill in the surgical arts.

In an illustrative embodiment, the BBIC glucose sensor may be used for monitoring glucose in diabetic patients. However, such a sensor can also be used in other conditions where glucose concentrations are of concern, such as in endurance athletes or other condition involving either hypo- or hyperglycemia. Such measurements may be the end point for investigative or diagnostic purposes or the sensors may be linked via telemetry or directly to a drug delivery system.

The use of implantable BBICs for substances other than glucose can be used in a range of therapeutic situations. With the incorporation of an appropriate cis-activating response element. BBICs could monitor a number of substances and could find use in WO 00/33065 PCT/US99/28733 chronic pain treatment, cancer therapy, chronic immunosuppression, hormonal therapy, cholesterol management, and lactate thresholds in heart attack patients. For example, Section 5.7 describes the use of the BBICs in the detection and diagnosis of cancer.

Individual biosensors can be calibrated to check for viability of the cells as well as performance. The calibration is performed by exposing the sensor to solutions containing varying concentrations of the analyte(s) of interest. The bioreporter may be calibrated by a series of standard analyte concentrations for the specific application after its initial construction. The overall on-line performance can be monitored using microfluidics with a reservoir of the analyte, which would systematically provide a known concentration to the cells this would allow both calibration and test for viability.

The luminescence response is then correlated to concentration and the parameters set. Viability can also be continuously monitored by bioengineered cells in which the reporter exhibits continuous luminescence. Loss of viability results in decreased luminescence. This technique has been used to detect the viability of prokaryotic cells.

Thus the BBIC would contain two bioreporters, the bioreporter detecting the selected substance and the second bioreporter exhibiting a luminescence proportional to cell viability. Measurement of the ratio of the signals from the two bioreporters would give a detection method that would automatically correct for any loss in viability.

Once prepared the bioreporters can be stored in the appropriate maintenance medium standard tissue culture media, sera, or other suitable growth or nutrient formulations), and then calibrated prior to implantation. The viability of the devices may be checked by bioluminescence using microfluidics, or by the quantiation of known standards or other reference solutions to ensure viability and integrity of the system prior to, or after implantation..

In certain embodiments of the invention, the monolithic biosensor devices may be used external to the body of the monitored individual. In some clinical settings the monitor may be used to monitor glucose in body fluids in an extracorporeal fashion. The device may even be used in the pathological or forensic arts to detect the quantity of particular analytes in body tissues or fluids and the like. Likewise, the present invention also contemplates use of the biosensor devices in the veterinary arts. Implantation of such devices in animals for the monitoring of hormone levels in the blood for optimizing milk production), monitoring the onset of estrous (heat) in numerous animals to maximize WO 00/33065 PCT/US99/287-33 artificial insemination efficiency, and monitoring hormone levels in the milk produced online (in the udder) etc. is contemplated to provide particular benefits to commercial farming operations, livestock industries and for use by artisans skilled in veterinary medicine.

4.11 DIAGNOSTICS KITS COMPRISING IN VIVO BIOSENSORS While the individual components of the invention described herein may be obtained and assembled individually, the inventors contemplate that, for convenience, the components of the biosensor may be packaged in kit form. Kits may comprise, in suitable container means, one or more bioreporters and an integrated circuit including a phototransducer. The kit will also preferably contain instructions for the use of the biosensor apparatus, and may further, optionally comprise a drug delivery device or a second biosensor apparatus. The kit may comprise a single container means that contains one or more bioreporters and the integrated circuit including a phototransducer and drug delivery device. Alternatively, the kits of the invention may comprise distinct container means for each component. In such cases, one container would contain one or more bioreporters, either in an appropriate medium or pre-encapsulated in a polymer matrix, another container would include the integrated circuit, and another conatiner would include the drug delivery device. When the bioreporter is pre-encapsulated, the kit may contain one or more encapsulation media. The use of distinct container means for each component would allow for the modulation of various components of the kits. For example, several bioreporters may be available to choose from, depending on the substance one wishes to detect. By replacing the bioreporter, one may be able to utilize the remaining components of the kit for an entirely different purpose, thus allowing reuse of components.

The container means may be a container such as a vial, test tube, packet, sleeve, shrink-wrap, or other container means, into which the components of the kit may be placed. The bioreporter or any reagents may also be partitioned into smaller containers or delivery vehicles, should this be desired.

The kits of the present invention also may include a means for containing the individual containers in close confinement for commercial sale, such as, injection or blow-molded plastic containers into which the desired components of the kit are retained.

WO 00/33065 PCT/US99/28733 Irrespective of the number of containers, the kits of the invention also may comprise, or be packaged with, an instrument for assisting with the placement of the bioreporter upon the integrated circuit. Such an instrument may be a syringe, pipette, forceps, or any other similar surgical or implantation device. The kit may also comprise one or more stents, catheters, or other surgical instrument to facilitate implantation within the body of the target animal. Such kits may also comprise devices for remote telemetry or devices for data storage or long term recordation of the data obtained from the monitoring device. Likewise, in the case of controlled drug delivery systems, the kits may comprise one or more drug delivery pumps as described above, and may also comprise one or more pharmaceutical agents themselves for administration. As an example, in the case of a glucose monitoring system, the system would typically comprise a glucosesensitive BBIC device, a drug delivery pump, instructions for the implantation and/or use of the system, and optionally, reference standards or pharmaceutical formulations of insulin, glucagon or other pharmaceutical composition. The system may also optionally comprise growth and/or storage medium to support the nutritive needs of the bioreporter cells comprised within the BBIC device.

EXAMPLES

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

5.1 EXAMPLE 1 CONSTRUCTION OF A BIOLUMINESCENCE REPORTER FOR MAMMALIAN CELL LINES To facilitate the construction of an implantable bioluminescent glucose sensor it will be necessary to create a bioluminescent reporter system that can function without the exogenous addition of substrate for the luciferase reaction. This exogenous addition is WO 00/33065 PCT/US99/28733 due to the complex nature of the production of luciferins for the various eukaryotic luciferases. Cells must be either permeablized or lysed and then treated with an assay solution containing luciferin. Therefore, the present state of bioluminescence reporters used in eukaryotic molecular biology makes them unsuitable for "on-line" monitoring.

The firefly luciferase has been used in examining the regulation of L-pyruvate kinase promoter activity in single living rat islet p-cells (Kennedy et al., 1997). However, these cells had to be perfused with Beetle luciferin in order to generate a luminescence response.

To alleviate this limitation, a preferred bioluminescent reporter system for the present invention is one that does not require the addition of exogenous substrate. In the case of bacterial luciferase-based detection systems, this may be accomplished using the bioluminescent genes from X luminescens. In this organism, luxA and luxB genes (or a single fused luxAluxB gene encode the a- and p-subunits. respectively, of the luciferase enzyme (Meighen et al., 1991). This luciferase exhibits greatest thermostability at 37 0

C

while other bacterial luciferases lose significant activity above 30 0 C. Therefore, these bacterial luciferases can be expressed in eukaryotic cells with slight modification.

Almashanu et al. (1990) fused the luxAB genes from V. harveyi by removal of the TAA stop codon from luxA, the intervening region between the two genes, and the initial methionine from luxB without disrupting the reading frame. The fusion was successfully expressed in S. cerevisiae and D. melanogaster. Using the same strategy a fused luxAB gene sequence was developed using the genes from X luminescens.

To eliminate the need for the addition of exogenous substrate, cells must themselves supply the appropriate substrate for the luciferase. In the bacterial system the substrate is generated by a fatty acid reductase complex encoded by the luxCDE genes.

This enzyme complex reduces short chain fatty acids to the corresponding aldehyde. The luciferase then oxidizes the aldehyde to the corresponding fatty acid. The preferred fatty acid for this reaction is myristic acid, which is present in eukaryotic organisms (Rudnick et al., 1993). Myristic acid is usually involved in the myristoylation of the amino terminus that is associated with membrane attachment (Borgese et al., 1996, Brand et al., 1996). Thus, to obviate the need for an exogenous supply of the luciferase substrate, the biosensor also preferably comprises a nucleic acid sequence that encodes the three luxC, luxD, and luxE-encoded subunits. As in the case of the luxAluxB gene fusion, the luxC, WO 00/33065 PCTIUS99/28733 luxD, and luxE genes have been fused to produce a single IuxCDE gene fusion that encodes the three subunits of the enzyme complex. The methods of preparing such gene fusions are described below: 5.1.1 FUSION OF THE LUXAB AND LUXCDE GENES The luxAB genes may be fused using conventional molecular biology techniques.

For example, the polymerase chain reaction may be routinely employed for this purpose.

By synthesizing a 5'-primer whose sequence begins with ATG for the start codon for the luxA gene juxtaposed by a 3'-primer ending with the codon immediately preceding the ATT stop codon. These primers may then be used in amplification reactions and the product gel purified. The luxB gene may also be amplified as above using primers that eliminate the ATG initial methionine codon but preserve the reading frame. The PCRTM reactions employ a thermostable polymerase such as the PfuTM polymerase of Stratagene (La Jolla, CA), which does not have terminal deoxytransferase activity and therefore generates a blunt end. The resultant PCRTM products are blunt-end ligated, and the ligation is then subjected to PCRTM using the 5'-primer from luxA and the 3'-primer from luxB using Taq polymerase to facilitate TA cloning (Invitrogen, San Diego, CA). Only ligations with the correct orientation of fragments are amplified. The luxAB amplicon is then gel purified and TA cloned into a suitable vector (such as the PCRIITM vector) and transformed into E. coli using standard manufacturer's protocols.

Transformants are screened for light production by the addition of n-decanal which, when oxidized by the luciferase, generates bioluminescence. Only colonies emitting light are selected since they are in the proper orientation for further genetic manipulation. The IuxCDE fusion is generated using the same strategy as above except transformants are screened by minipreps followed by restriction digestion analysis to determine orientation. Plasmids are amplified in E. coli, recovered and purified twice on CsCI gradients.

5.1.2 EXPRESSION OF LUYAB AND LUXCDE IN HELA CELLS To determine the relative activity of the fused bacterial luciferase components, cloned fragments containing luxAB are cloned into a suitable mammalian expression vector (such as pcDNA 3.1 and luxCDE-containing fragments are cloned into a suitable WO 00/33065 PCT/US99/28733 mammalian expression vector (such as pcDNA/Zeo 3.1) (Invitrogen, Faraday, CA). Both vectors constitutively express inserted genes. HeLa cells are then transfected with luxAB or both luxAB and luxCDE and selected using appropriate antibiotics following the manufacturer's protocol (Promega, Madison, WI). Cells receiving the luxAB fusion are exposed to n-decanal and checked for bioluminescence. These cells cotransfected with luxCDE are then examined for bioluminescence to ascertain the relative expression of the luxCDE fusion. This permits the comparison of bioluminescence via the addition of exogenous aldehyde versus aldehyde that is produced endogenously.

An alternate strategy to enhance bioluminescent expression involves engineering a vector that would contain three copies of the eukaryotic expression machinery contained in pcDNA3.1 (Stratagene, La Jolla, CA). This allows for the expression of the individual components of luxCDE since it has already been shown that the fused luciferase is expressed in eukaryotic cell lines (Almashanu et al., 1990).

5.2 EXAMPLE 2 CONSTRUCTION OF A GLUCOSE BIOLUMINESCENT BIOSENSOR The firefly luciferase has been used in examining the regulation of L-pyruvate kinase promoter activity in single living islet p-cells (Kennedy et al., 1997). A glucose response element designated the L4 box has been determined to be in the proximal promoter. A 200-bp fragment containing this region was cloned in front of the firefly luciferase (luc) in plasmid pGL3Basic resulting in a glucose reporter plasmid designated p.LPK.Luc.F. Results resulted in the detection of single cells that were exposed to 16 mM glucose but not 3 mM glucose. However, these cells had to be perfused with Beetle luciferin making it unacceptable for an on-line biosensor. Therefore, a bioluminescent sensor for glucose was constructed by replacing the firefly luciferase in p.LPK.Luc with the fused luxAB gene as described below.

5.3 EXAMPLE 3 BIOLUMINESCENT REPORTER CONSTRUCTION AND TRANSFECTION OF RAT ISLET P-CELLS The bioluminescent reporter plasmid was constructed by removing the luc gene coding for the firefly luciferase from p.LPK.LucF and replacing it with the fused luxAB gene. This was accomplished by cleaving the luc gene from p.LPK.LucF and cloning in WO 00/33065 PCT/US99/2873 the luxAB gene. The resultant plasmid was amplified in E. coli and the plasmid DNA extracted and double purified on CsCI gradients.

Islet cells were prepared as previously described (German et al., 1990) and transfected by electroporation with the bioluminescent reporter construct and the plasmid containing the constitutively expressed luxCDE construct. This configuration causes the cells to maintain a pool of the aldehyde substrate that is available to the reporter genes (luxAB). Cells were screened for light production in a range of glucose concentrations from 3 mM to 30 mM. Transfected cells were washed, concentrated, and placed in a microwell in a light-tight cell that is then affixed to the integrated circuit. Different concentrations of glucose and assay media (Kennedy et al., 1997) were added to the cells to examine sensitivity and response time of the glucose BBIC.

5.4 EXAMPLE 4 PREPARATION OF BIOLUMINESCENT REPORTER CONSTRUCTS The use of reporter gene technology is widespread in studying gene regulation in both eukaryotic and prokaryotic systems. Various genes are used depending on the cell lines being investigated. However with the BBIC technology the use of reporter genes that result in the emission of light is required. Therefore, reporter genes coding for bioluminescence are utilized. All previously developed reporters utilizing other reporter genes for example the gene coding for P-galactosidase (lacZ) may be converted to the bioluminescent version using standard molecular techniques and the reporter genes utilized in this specific application (modified lux system). Therefore, any currently existing reporter cell line for testing gene expression in mammalian cell lines may be adapted for use as a bioreporter when converted to the lux reporter. The implantable system simply contains the appropriate reporter cell line. Table 1 shows a list of examples of eukaryotic reporter cell lines that may be exploited in an implantable biosensor.

WO 00/33065 PCT/US99/2873 TABLE 1 Reporter Gene Application Reference Fusion ADH4-LUC Monitors expression of alcohol dehydrogenase Edenberg et al., 1999 to increasing concentrations of alcohol TH-lacZ Shows increased gene expression in mice Boundyetal., 1998 subjected to chronic cocaine or morphine exposure Estrogen regulated-LUC Detects estrogens and xenoestrogens by there Balaguer et al., 1999 effect on the estrogen response element Detects the presence of progesterone by the Boonyaratanakorkit et al., 1999 upregulation of the reporter construct CYPIA-/acZ Detects compounds that cause an upregulation Campbell et al., 1996 of cytochrome P450 (potential carcinogens) EXAMPLE 5 CONSTRUCTION AND IMPLANTATION OF A GLUCOSE BIOSENSOR AND INSULIN DELIVERY PUMP In one embodiment, a pair of bioluminescent reporters may be utilized that are in tandem and that specifically respond to deviations in glucose concentrations. One bioreporter utilizes the luxAB and luxCDE genes from X luminescens incorporated into a plasmid-based system designated p.LPK.LucF, which contains a eukaryotic luc gene able to respond to glucose concentrations (increasing bioluminescence corresponds to increasing glucose concentrations). The second bioreporter utilizes a plasmid construct containing the promoter for the phosphoenolpyruvate carboxylase gene (PEPCK) that also responds to glucose concentrations, except increased bioluminescence corresponds to decreased levels of glucose. The incorporation of the luxAB and luxCDE genes into each construct allow for bioluminescence measurements to occur in real-time with deviations in glucose concentrations, negating the requirement for cell destruction and substrate addition.

In this embodiment, the integrated circuit comprises separate photodetector units for each bioreporter (FIG. 9A, FIG. 9B, and FIG. 9C). Bioluminescent responses from each construct can be independently monitored, allowing for the signal processing circuitry to differentiate between one bioreporter's response to increased glucose concentrations and the second bioreporter's response to decreased glucose concentrations.

WO 00/33065 PCT/US99/28733 The signal processing circuitry processes the signals from the photodetectors, converts it to a digital format and relays the information to the implanted insulin pump (FIG. The tandem set of bioreporters allows a more accurate signal as well as redundancy in the detector. Due to the often-fatal outcome of hypoglycemia, this tandem system also allows for more careful monitoring and warning of the onset of hypoglycemia.

The cells used in the tandem bioreporter system may be affixed to each of the photodetectors either directly by attachment or encapsulated in hydrogel (Prevost et al., 1997). It may be necessary to isolate the bioreporters using a semi-permeable membrane to allow the transport of small molecules such as glucose and insulin across the membrane and prohibit the influx of immune effector cells and antibodies (Monaco et al., 1993, Suzuki et al., 1998). However small molecules such as cytokines can still enter the selective membranes and interfere with the bioluminescent reporter cell lines. This approach has been used extensively by those of skill in the art.

When applicable, bioluminescent reporter cell lines may be constructed from cells taken directly from the patient to receive the implant. This approach is particularly desirable in cases of long-term implants such as implantable insulin delivery. Cells may be obtained from the patient, genetically engineered for the appropriate monitoring function, grown in cell culture, evaluated and then preserved for long-term storage. The use of cell lines developed from the patient's own cells, is particularly desirable as it reduces the chance of host rejection and creation of an immune response to the implanted device. Preferably, stem cells (immortal stem cells, if attainable) are used when appropriate, and may be maintained and nourished in suitable culture medium. Such pluripotent, totipotent, or otherwise immortal cell lines provide particular advantage in the creation of suitable long-term implantable devices.

Before implantation the biosensor may be calibrated injecting the chamber containing the cells with various concentrations of glucose delivered from an auxiliary pump and reservoir on the insulin delivery pump (FIG. 10). This permits determination of the appropriate parameters to allow the proper dosage of insulin to be delivered. Once the parameters are set, the pump may be evaluated for insulin delivery. Systematically the glucose biosensor is recalibrated in the patient utilizing the glucose standard contained in the delivery pump.

WO 00/33065 PCT/US99/28733 In the case of drug delivery systems, the glucose biosensor may be operably connected to the delivery pump via a hardwire or wireless connection. The biochip provides digital data that may be input directly to the signal processing circuitry of the pump to proportionally dispense the insulin. Alternatively, the digital data may be converted into analog data and used to control the pump. When a wireless capability is added to the bioreporter device, remote monitoring of the sensor is possible. For example, in this configuration, the patient may place a radio transmitter/receiver outside the body near the implanted device to communicate the data from the implanted device toa remote station. In some applications, the radio transmitter/receiver may be linked to a computer programmed to forward the data to a remote station over a network such as a local area network, a wide area netword, or even the Internet. Such wireless applications allow remote monitoring and maintenance of the patients. There are several pumps currently on the market, which are candidates for interfacing with the biosensor. In one embodiment, the Medtronic Synchronized infusion system may be used as it has extensively used in drug delivery and utilizes a portable computer to allow programming of the pump from outside the body (www.asri.edu/neuro/brochure/pain6.htm). The pump can also be refilled through the skin via a self-sealing septum. The pump is one inch thick and three inches in diameter and weighs approximately six ounces. The biosensor can be integrated into the preexisting electronic circuitry to take advantage of the out-of-body programming by a portable computer. The chip can be powered utilizing the battery that powers the delivery pump.

The biosensor/insulin pump apparatus may be surgically implanted using local anesthesia in the abdominal cavity. Both the sensor and the pump may be implanted in the peritoneal space of the abdomen both for simplicity and to avoid the complications of direct catheter placement in the blood stream. Glucose concentrations are monitored and the insulin delivered peritoneally as required by the patient (FIG. 5.6 EXAMPLE 6 BIOLUMINESCENT REPORTER CONSTRUCTION AND TRANSFECTION OF RAT ISLET P-CELLS AND H4IIE HEPATOMA CELLS The regulation of the PEPCK gene will be exploited in the construction of the bioluminescent reporter for detecting decreased glucose concentrations. This system is highly regulated as the phosphoenolpyruvate carboxylase is the rate-limiting enzyme in WO 00/33065 PCT/US99/28733 gluconeogenesis. PEPCK gene expression is increased in the presence of glucocorticoids and cAMP and decreased in the presence of insulin (Sasaki et al., 1984; Short et al., 1986). In both rat liver and H4IIE hepatoma cells the insulin effect is dominant and the glucocorticoids and cAMP is additive. The promoter region of the PEPCK will be cloned in front of the fused luxAB. The resultant construct will then produce increased bioluminescence in the presence of low glucose concentrations.

The bioluminescent reporter plasmid for detecting increased glucose concentration may be constructed by removing the luc gene coding for the firefly luciferase from p.LPK.LucnF and replacing it with the fused luxAB. This is accomplished by cleaving the luc gene from p.LPK.Lucn and cloning in the luxAB gene. The bioluminescent reporter plasmid for the detection of low glucose concentrations is constructed by replacing the chloramphenicol transferase (CAT) gene in the previously constructed PEPCK promoter CAT fusion (Petersen et al., 1988; Quinn et al., 1988) with the luxAB gene. The resultant plasmid is amplified in E. coli and the plasmid DNA extracted and double purified on CsCI gradients.

Islet and hepatoma cells may be prepared as previously described (German et al., 1990; Petersen et al., 1988) and co-transfected with the bioluminescent reporter construct and the plasmid containing the constitutively expressed luxCDE gene constructed in objective one. This configuration causes the cells to maintain a pool of aldehyde substrate that will be available to the reporter genes (luxAB). Cells are screened for light production in a range of glucose concentrations from 3 mM to 30 mM. Transfected cells are washed, concentrated, and placed in a microwell in a light-tight chamber that is then affixed to the integrated circuit. Different concentrations of glucose and assay media (Kennedy et al., 1997) are added to the cells to examine sensitivity and response time of the glucose BBIC.

After initial characterization, the bioluminescent glucose reporters may also be tested in a flow cell. Cells are placed in an encapsulation medium on the integrated circuit and media containing different concentrations of glucose (3-to-30 mM) is then perfused across the cells to examine dynamic responses.

5.7 EXAMPLE 7 BBICs IN THE DIAGNOSIS AND DETECTION OF CANCER Colon cancer is the second leading cause of cancer death after lung cancer in the United States, and the incidence increases with age in that 97% of colon cancer occurs in WO 00/33065 PCT/US99/28733 persons greater than 40 (Coppola and Karl, 1998). Although most cases of colon cancer are sporadic, in 15% of the patients there is a strong familial history of similar tumors in first-degree relative relatives (Coppola and Karl, 1998). These familial cancers such as hereditary nonpolyposis colon cancer (HNPCC) and familial adenomatous polyposis (FAP) result from autosomal dominant inheritable genetic mutations in putative tumor suppressor genes, and a spectrum of lesions occurs from hyperplasia-dysplasia-adenomacarcinoma (Coppola and Karl, 1998). Because much of the early molecular lesions are known about inherited colonic cancer, they represent a useful model for development of a novel biosensor strategy for early clinical detection. Biosensors are hybrid devices combining a biological component with a computerized measuring transducer.

This example describes the adaptation of the implantable biosensor device to permit early detection of cancers, and to permit means for monitoring remission and recurrence of cancer. Because the miniaturized biosensors of the present invention are small enough to be implantable, and can be combined with a reporter system engineered to produce light without the need for cellular lysis or additional substrate, a powerful tool for early diagnosis of colon cancer in the form of an implantable device is now possible for the first time.

As described above for glucose and other metabolite biosensors, the inducible reporter system utilized is based on the luxAB and luxCDE genes from X luminescens placed in a eukaryotic reporter cell so that expression of certain genes or their products can be detected by expression of bioluminescence by the BBICdevice. The eukaryotic reporter cell is treated with mitomycin C so it is unable to divide, but is still able to respond metabolically and produce a quantitative bioluminescent signal.

Colon cancer is the second leading cause of cancer death in the United Sates, with at least 50% of the population developing a colorectal tumor by the age of 70 (Kinzler and Vogelstein, 1996). Although most cases of colorectal cancer are sporadic, 15% are the result of heritable cancer syndromes, familial adenomatous polyposis (FAP) and hereditary nonpolyposis coiorectal cancer (HNPCC) (Kinzler and Vogelstein, 1996).

Familial adenomatous polyposis is a syndrome characterized by the development of hundreds to thousands of adenomas or polyps in the colon and rectum, only a small number of which develop into invasive cancer (Kinzler and Vogelstein, 1996). Loss of function of both alleles of the adenomatous polyposis coli (APC) tumor suppressor gene WO 00/33065 PCT/US99/28733 predisposes persons to develop malignant cancer (Coppola and Marks, 1998). In addition, most sporadic colon cancers are also found to contain mutations in the APC gene (Kinzler and Vogelstein, 1996). In hereditary nonpolyposis colorectal cancer, there is marked microsatellite instability secondary to mutations in DNA mismatch repair genes such as hMSH2 and hMSH1; single, high grade tumors develop at a young age and are usually confined to the right colon (Coppola and Karl, 1996; Smyrk, 1994). Whereas cells with mutations in APC are generally aneuploid from loss of whole sections of chromosomes, cells with mutations in hMSH2 or hMSH are euploid (Lengauer et al., 1998).

The molecular events leading to the development of colonic neoplasia are fairly well understood (Kinzler and Vogelstein, 1996). Persons with complete loss of APC develop lesions in the colon called dysplastic aberrant crypt foci that progress to early adenomas (Kinzler and Vogelstein, 1996). Other mutations begin to accumulate, such as those in K-Ras or p53, and the tumors progress to late adenomas, carcinomas, and metastatic carcinomas (Kinzler and Vogelstein, 1996). A similar progression is seen in HNPCC as well. Because the sequence of genetic events is fairly well understood for both these types of cancer, they represent excellent models for development of sensitive and specific diagnostic tests that can be used to detect one or more altered cells in vitro.

The APC gene encodes a cytoplasmic protein that localizes to the ends of microtubules at focal adhesion complexes (Kinzler and Vogelstein, 1996). As cells migrate up through the crypts, expression of APC increases until the terminally differentiated and located colonic epithelial cells undergo apoptosis (Kinzler and Vogelstein. 1996). Cadherins are transmembrane proteins that are localized to focal adhesion plaques in most epithelial cells (Aplin et al., 1998). The carboxy terminus of each cadherin interacts with cytoplasmic structural proteins known as catenins (Aplin et al., 1998). There are three types of catenins: P-catenin binds to the cytoplasmic domain of cadherin; -catenin binds to p-catenin and the actin cytoskeleton via -actinin; -catenin functions in place of p-catenin in some cell types (Aplin et al., 1998). p-Catenin also is part of a signal transduction pathway involving the secreted glycoprotein Wnt and glycogen synthase kinase 3 (GSK3) (Aplin et al., 1998). APC interacts with several components of the Wnt-p-catenin-GSK3 pathway, including p- and y-catenins, GSK3, and tubulin (Aplin et al.. 1998). Most of the mutations in colorectal cancer are in the carboxy terminal region of APC so that it can no longer bind p-catenin (Aplin et al., 1998). In WO 00/33065 PCT/U S99/28733 fact, -catenin lies downstream of APC and is critical for its function as a tumor suppressor gene (Aplin et al., 1998). When the Wnt pathway is inactivated, GSK3 phosphorylates the N-terminus of p-catenin, targeting it for degradation by the ubiquitin pathway (Munemitsu et al., 1996). When p-catenin accumulates, it activates gene transcription via the transcription factor Lef-1/TCF (Morin et al.. 1997). APC works in concert with GSK3 to inhibit -cateAin-mediated transcriptional activity (Kinzler and Vogelstein, 1996).

In hereditary nonpolyposis colon cancer, microsatellite instability is the result of mutations in one or more DNA-mismatch repair genes (Jiricny 1998; Nicholaides et al., 1994). At least 90% of HNPCC tumors have microsatellite instability (Karran 1996; Smyrk 1994). One potential marker for microsatellite instability in colorectal tumors is inactivation of the type II receptor for TGF-p (Markowitz et al., 1995). Loss of function of PRII is associated with loss of growth regulation and tumor progression in colorectal adenomas in HNPCC (Wang et al., 1995). Other signaling components of the TGF-p pathway that are involved in colorectal tumorigenesis include mutations in Smad 3 and Smad 4, both of which result in the development of colorectal adenocarcinomas in mice (Zhu et al., 1998; Takaku et al., 1998). Loss of function of PRII is a useful marker for early lesions in HNPCC (Markowitz et al., 1995).

Because mutations in APC are the most common mutations in colorectal cancer, a reporter construct for T cell transcription factor (Tcf) was devised to screen multiple colon cancer cell lines for activation of transcriptional activity. Mutations in either APC or Pcatenin result in activation of Tcf-responsive transcription through the accumulation of unphosphorylated cytoplasmic p-catenin (Morin et al., 1997) and detecting activation of a reporter construct is useful as a marker for mutations in either of these genes. The vector pDISPLAY (Invitrogen) permits expression of the promoter for Tcf on the surface of the bioreporter cell; this construct consists of a tandem set of Tcf promoters: one upstream of the genes for luxAB. the other upstream of the luxCDE. In the presence of excess Pcatenin the promoter constructs will stimulate activity of the reporter and bioluminescence will result.

Once the HepG2 and HeLa cells have been transfected with pcDNA3 encoding the luxAB genes. the cells are attached to the biosensor chip. It is necessary to insure that these cells are incapable of dividing, so after transfection and selection, the cells are irradiated with 6,000 rads y-radiation from a 60 Co source (UT College of Veterinary WO 00/33065 PCT/U S99/28733 Medicine). In some embodiment it may be necessary to attach the cells to the biochip prior to irradiation so that efficient attachment can occur. An alternative is to treat the cells with mitomycin C to prevent further mitosis. Biochips may be coated with Matrigel, a basement membrane material that promotes attachment of epithelial cells. An alternative approach suspends the cells in Matrigel and allows it to form a gel on the surface of the 'biochip. The cells are then immobilized in the basement membrane material and are not subject to dislodgement by friction. Optionally, the surface of the chip may be altered by adding a net charge poly-L-lysine), coating the surface with surgical tissue glue, or by adding some other surface modification that allows the biopolymers to adhere tightly to the surface. Because mutations in APC are the most common mutations in colorectal cancer, a reporter construct for T cell transcription factor (Tcf) may be devised to screen multiple colon cancer cell lines for activation of transcriptional activity.

The present invention also provides a biosensor that may be used for endoscopic screening of the colonic mucosa to detect the presence of mutated cells prior to the onset of gross morphological alterations. It may be necessary to attempt detection of more than one abnormality at a time for the degree of sensitivity needed to detect small foci of malignant transformation. For example, many colonic tumors, especially those with mutations in APC, overexpress cyclooxygenase-2 (COX-2) and secrete large amounts of prostaglandins (Kutchera et al., 1996; Sheng et al., 1997; Coffey et al., 1997; Kinzler and Vogelstein, 1996). Cyclooxygenase-2 is an early response gene that not constitutively expressed, but is turned on in colonic epithelial cells by growth factors and tumor promoters (Kutchera et al., 1996; Sheng et al., 1997; Coffey et al., 1997). It may be possible to bioengineer reporter cells to bioluminesce in the presence of increased levels of prostaglandins in the intestinal lumen. Prostaglandins freely pass the cell membrane and would be able to enter the cytoplasm of the reporter cell to activate a reporter construct. Engineering a reporter cell to detect increased levels of prostaglandins through the use of the cyclooxygenase-2 promoter fused to the luxAB genes could also be of benefit in early detection of colon cancer. Because the levels of prostaglandins may be elevated in inflammation as well as neoplasia, this approach lacks appropriate specificity for diagnosing cancer. It would, however, be useful in determining which patients would benefit from treatment with specific cyclooxygenase inhibitors.

WO 00/33065 PCT/US99/28733

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All of the compositions, methods, devices, apparatus and systems disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the methods, devices, apparatus and systems of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods, devices, apparatus and systems and in the steps or in the sequence of steps of the methods described herein without departing form the 20 concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such 25 similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. Accordingly, the exclusive rights sought to be patented area as described in the claims below.

30 For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.

H:\janel\Keep\Speci\20408-00.doc 11/04/02 EDITORIAL NOTE APPLICATION NUMBER 20408/00 The following Sequence Listing pages are part of the description. The claims follow on pages 63 to WO 00/33065 WO 03065PCT/US99/28733 SEQUENCE LISTING <110> SAYLER, GARY S.

SIMPSON, MICHAEL L.

APPLEGATE, BRUCE M.

RIPP, STEVEN A.

<120> IN VIVO BIOSENSOR APPARATUS AN~D METHOD OF USE <130> 4300.004300 <140> UNKNOWN <141> 1999-12-02 <150> 60/110,684 <151> 1998-12-02 <160> 7 <170> Patentln Ver. <210> 1 <211> 7669 <212> DNA <213> Xenorhabdus luminescens <220> <221> CDS <222> (1215) .(2657) <223> LUXC <220> <221> CDS <222> (2671) (3594) <223> LUXD <220> <221> CDS <222> (3776) (4858) <223> LUXA <22 0> <221> CDS <222> (4873) .(5847) <223> LUXB <220> <221> CDS <222> (6160) .(7272) <223> LUXE <400> 1 gaattctcag actcaaatag aacaggattc taaagactta agagcagctg tagatcgtga ttttagtacg atagagccaa cattgagaaa ttatggggca acggaagcac aacttgaaga 120 WO 00/33065 WO 0/3065PCT/US99/2873-3..__ cgccagagcc acagaggcac attccttata gtgaaagagg ttaagattgc ttgaagcatt ataccaccga gatgaaatca ttgcttacat tgctttgttg gttcacttgc gaaaattgcc ttatcacgta tttttattaa taaacggaga aattgcagag tctttgtata aaaatacaca taataaacag ggaataagcc ggatttttt t ttagagacga ctatggatag gacaaatttt gtatgcagat cagacgtgag gctgctttct cgccgcgctg tgatttatat taaaaaaatt caaatcttgt aggcagcatg tatgatcgca ttttgttttg agcttaacca cctaggcaag aagtggttct gtcgtttgaa taaagctttg gggatgggt t aaaaaggcag ttcttatgat ttggtaattt ctcaccccag caacttgaaa ataacttaac gcgattcttt tgatgtgaaa ttgatgattc ttctgataaa ttatttacgt agaacagagg tcctaccaag cctggggtgC cggaatagta tttatatttc agagaaaatt tttggttgga ttacgtgaac aatatatata tcacatagtt tctattgggt ttgtaaacca taatttgaaa attttcgttt actcagccag ggttacaggt tgcagcaaga ttatacaaat aaatttttga ctaatatttg aaatattaaa caaatgaatt taggtgatct aagatctata aggttaattc cccttcatcc atctatgctc atatgctatt gataataatt tagttcaatt gctattttaa actgacagtt cactcgcaac caaaaataga gacagttaat tgaagggcta tattgacatg cgaaattact gccatcaccg gattaaatca tcgttttacq agtgactttc ttcaagttgc ctggggattc ggtaattatg tacatgaata taattgaaac cagatattgt ttaagcggaa cagaatttca agaaacaaat 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 atttatacaa cccgtttgca agagggttaa acagcaattt aagttgaaat tgccctatta 1200 aatggatggc aaat atg aac aaa aaa att tca ttc att att aac ggt cga Met Asn Lys Lys Ile Ser Phe Ile Ile Asn Gly Arg att aat ttt Ile Asn Phe gtt gaa ata Val Giu Ile ggt gat aat Gly Asp Asn ttt cct gaa agt Phe Pro Glu Ser gat tta gtg caa Asp Leu Val Gin tcC Ser agt gtt cat Ser Val His ttg Leu 35 gaa Glu cca gta ttg aat Pro Val Leu Asn gat Asp tct caa gta aaa Ser Gin Vai Lys 1250 1298 1346 1394 1442 1490 aac Asn atc Ile att att gat tat Ile Ile Asp Tyr aat aat gaa Asn Asn Giu t tg Leu tgg Trp, caa ttg cat aac Gin Leu His Asn aac ttt ctc Asn Phe Leu gta ggg caa cga Val Gly Gin Arg aaa aat gaa Lys Asn Giu gaa tat Glu Tyr tca aga cgc agg tca aa cg agg tat att cgt gat cta aaa aga tat atg gga tat WO 00/33065 Ser Arg Arg tca gaa gaa Ser Glu Glu PCT/US99/28733 Thr Tyr Ile Arg Asp Leu Lys Arg Tyr Met Gly Tyr atg att ttg Met Ile Leu atg got aag cta Met Ala Lys Leu gag Glu 100 gcc aac tgg ata Ala Asn Trp Ile tct Ser 105 1538 tgc tct Cys Ser 110 aaa ggt ggc ctt tat gat ctt gta aaa Lys Gly Gly Leu Tyr Asp Leu Val Lys 115 aat Asn 120 gaa ctt ggt tct Glu Leu Gly Ser 1586 cat att atg gat His Ile Met Asp tgg cta cct cag Trp Leu Pro Gin gat Asp 135 gaa agt tat att Glu Ser Tyr Ile aga Arg 140 1634 1682 get ttt ccg aaa Ala Phe Pro Lys gga Gly 145 aaa tcc gta cat Lys Ser Val His ttg acg ggt aat Leu Thr Gly Asn gtg cca Val Pro 155 tta tct ggt Leu Ser Gly tgc att ata Cys Ile Ile 175 gtg Val 160 ctg tct ata ttg Leu Ser Ile Leu cgt Arg 165 gca att tta aca Ala Ile Leu Thr aag aat caa Lys Asn Gin 170 aat gca tta Asn Ala Leu 1730 1778 aaa acc tca tca Lys Thr Ser Ser act Thr 180 gat cct ttt acc Asp Pro Phe Thr get Ala 185 gcg cta Ala Leu 190 agt ttt atc gat Ser Phe Ile Asp gtg Val 195 gac cct cat cat Asp Pro His His ccg Pro 200 gta acg cgt tct Val Thr Arg Ser tca gtc gta tat Ser Val Val Tyr caa cat caa ggc Gin His Gin Gly gat Asp 215 ata tca ctc gca Ile Ser Leu Ala 1826 1874 1922 gag att atg caa Glu Ile Met Gin cat His 225 gcg gat gtc gtt Ala Asp Val Val gct tgg gga ggg Ala Trp Gly Gly gaa gat Glu Asp 235 gcg att aat Ala Ile Asn aag ttt ggt Lys Phe Gly 255 tgg Trp 240 get gta aag cat Ala Val Lys His gca Ala 245 cca ccc gat att Pro Pro Asp Ile gac gtg atg Asp Val Met 250 cct gtt gat Pro Val Asp 1970 2018 cct aaa aag agt Pro Lys Lys Ser ttt Phe 260 tgt att att gat Cys Ile Ile Asp aac Asn 265 tta gta Leu Val 270 tec gca get aca Ser Ala Ala Thr ggg Gly 275 gcg gct cat gat Ala Ala His Asp gtt Val 280 tgt ttt tac gat Cys Phe Tyr Asp 2066 2114 caa gct tgt ttt Gin Ala Cys Phe acc caa aat ata Thr Gin Asn Ile tat Tyr 295 tac atg gga agt Tyr Met Gly Ser tat gaa gag ttt aag cta gcg ttg ata gaa aaa ttg aac tta tat gcg Tyr Glu Glu Phe Lys Leu Ala Leu Ile Glu Lys Leu Asn Leu Tyr Ala 2162 WO 00/33065 WO 0/3065PCT/US99/2873.

315 cat ata tta His Ile Leu tcc tta gtt Ser Leu Val 335 cca Pro 320 aac acc aaa aaa Asn Thr Lys Lys ttt gat gaa aag Phe Asp Giu Lys gcg gcc tat Ala Ala Tyr 330 gta gag gtt Val Giu Val 2210 2258 caa aaa gaa tgt Gin Lys Glu Cys tta Leu 340 ttt gct gga tta Phe Ala Gly Leu gat gtt Asp Val 350 cat cag cgc tgg His Gin Arg Trp gtt att gag tca Val Ile Giu Ser aat Asn 360 gcg ggt gta gaa Ala Gly Val Giu aat caa cca ctt Asn Gin Pro Leu ggc Gly 370 aga tgt gtg tat Arg Cys Vai Tyr ctt Leu 375 cat cac gtc gat His His Vai Asp 2306 2354 2402 att gag caa ata Ile Giu Gin Ile ttg Leu 385 cct tat gtg cga Pro Tyr Val Arg aaa Lys 390 aat aaa acg caa Asn Lys Thr Gin acc ata Thr Ile 395 tct gtt ttt Ser Vai Phe tta aaa ggt Leu Lys Gly 415 tgg gag gcc gcg Trp Giu Ala Ala ctt Leu 405 aag tat cga gac Lys Tyr Arg Asp tta tta gca Leu Leu Ala 410 aat ata ttt Asn Ile Phe 2450 2498 gca gaa agg att Ala Giu Arg Ile gta Val 420 gaa gca gga atg Giu Ala Gly Met aat Asn 425 cgg gtt Arg Val 430 ggt ggt gct cat Gly Gly Ala His gga atg aga cct Gly Met Arg Pro tta Leu 440 caa cga ttg gtg Gin Arg Leu Val tat act tcc cat Tyr Ile Ser His gaa Giu 450 aga cca tcc cac Arg Pro Ser His tat Tyr 455 act gct aaa gat Thr Ala Lys Asp gcg gtc gaa ata Ala Val Glu Ile gaa Giu 465 cag act cga ttc Gin Thr Arg Phe c tg Leu 470 gaa gaa gat aag Giu Giu Asp Lys ttc ctg Phe Leu 475 gta ttt gtc Val Phe Val cca Pro 480 taa taggtaaaag aat atg gaa aat aaa Met Giu Asn Lys 485 aaa acc Lys Thr 490 atc gac cat gtt Ile Asp His Val att Ile 495 tgt gtt gaa gaa Cys Val Giu Giu aat Asn 500 ag Ar tcc aga tat Ser Arg Tyr a aaa att cat g Lys Ile His 2546 2594 2642 2691 2739 2787 2835 gtc Val 505 tgg gag acg ctg Trp Glu Thr Leu cca Pro 510 aaa gaa aat Lys Giu Asn agt cca aag aga aaa aat acc Ser Pro Lys Arg Lys Asn Thr 515 520 agg atg gat cat ttt gcc ggt Arg Met Asp His Phe Ala Giy 530 535 ctt att att gcg Leu Ile Ile Ala tog Ser 525 ggt ttt gcc cgc Gly Phe Ala Arg WO 00/33065 WO 0033065PCT/US99/2873-3 ctg gca gag Leu Ala Glu tct ctt cac Ser Leu His 555 ttg tcg cag aat Leu Ser Gin Asn gga Gly 545 ttt cat gtg atc Phe His Val Ile cgc tat gat Arg Tyr Asp 550 gaa ttt aca Glu Phe Thr 2883 2931 cac gtt gga ttg His Val Gly Leu tca ggg aca att Ser Gly Thr Ile atg tcc Met Ser 570 ata gga aaa cag Ile Giy Lys Gin agt Ser 575 tta tta gca gtg Leu Leu Ala Val gtt gat tgg tta aat Val Asp Trp Leu Asn 580 tca agc tta tct gcg Ser Ser Leu Ser Ala aca Thr 585 cga aaa ata aat Arg Lys Ile Asn ctc ggt atg ctg Leu Gly Met Leu gct Ala 595 2979 3027 3075 cgg ata gct tat Arg Ile Aia Tyr agt cta tct gaa Ser Leu Ser Giu att Ile 610 aat gtc tcg ttt Asn Val Ser Phe tta. att Leu Ile 615 acc gca gtc Thr Ala Val gga ttt gat Gly Phe Asp 635 ggt Gly 620 gtg gtt aac tta Val Val Asn Leu aga Arg 625 tat act ctc gaa Tyr Thr Leu Giu aga gct tta Arg Ala Leu 630 gat aat tta Asp Asn Leu 3123 3171 tat ctc agc tta Tyr Leu Ser Leu cct Pro 640 att gat gaa ttg Ile Asp Giu Leu gat ttt Asp Phe 650 gaa ggt cat aaa Giu Giy His Lys ttg Leu 655 ggt gct gag gtt Giy Ala Giu Val ttt Phe 660 gcg aga gat tgc Ala Arg Asp Cys ttt Phe 665 gat tct ggc tgg Asp Ser Gly Trp gat tta act tct Asp Leu Thr Ser aca Thr 675 att aat agt atg Ile Asn Ser Met atg Met 680 3219 3267 3315 cat ctt gat ata.

His Leu Asp Ile ccg Pro 685 ttt att gct ttt Phe Ile Ala Phe gca aat aat gac Ala Asn Asn Asp gat tgg Asp Trp 695 gta aag caa.

Val Lys Gin caa tgt aag Gin Cys Lys 715 gat Asp 700 gaa gtt att aca Giu Val Ile Thr cta tca agc atc Leu Ser Ser Ile cgt agt cat Arg Ser His 710 ttg ggt gag Leu Gly Giu 3363 3411 ata tat tct tta Ile Tyr Ser Leu gga agc tca cat Gly Ser Ser His aac tta Asn Leu 730 gtg gtc ctg cgc Val Vai Leu Arg aat Asn 735 ttt tat caa tcg Phe Tyr Gin Ser acg aaa gcc gct Thr Lys Aia Ala 3459 3507 atc Ilie 745 gcg atg gat aat Ala Met Asp Asn 750 tgt ctg gat att Cys Leu Asp Ilie gat Asp 755 gtc gat att att Val Asp Ile Ile gag Glu 760 WO 00/33065 WO 0/3065PCT/US99/287373 ccg toa ttc gaa cat tta acc att gcg gca gtc Pro Ser Phe Glu His Leu Thr Ile Ala Ala Val 765 770 aaa att gag att gaa aat caa gtg att tog ctg Lys Ile Glu Ile Giu Asn Gin Val Ilie Ser Leu 780 785 aat gaa cgc cga atg Asn Giu Arg Arg Met 775 tct taa aacctatacc Ser 3555 3604 aatagatttc gagttgcagc gcggcggcaa gtgaacgcat tcooaggagc atagataact. 3664 ctqtgactgg ggtgcgtgaa agcagccaac aaagcagcaa cttgaaggat gaagggtata 3724 ttgggataga tagttaactc tatcactcaa atagaaatat aaggactctc t atg aaa Met Lys 790 3781 ttt gga aac Phe Gly Asn gag gta atg Glu Val Met 810 ttg ott aca tac Leu Leu Thr Tyr coo coo caa. ttt Pro Pro Gin Phe tot caa aca Ser Gin Thr 805 gag gaa. tgo Giu Glu Cys 3829 3877 aaa cgg ttg gtt Lys Arg Leu Val tta ggt cgc atc Leu Giy Arg Ile ggt ttt Gly Phe 825 gat acc gta tgg Asp Thr Val Trp tta Leu 830 ctt gag cat cat Leu Glu His His ttc Phe 835 acg gag ttt ggt Thr Giu Phe Giy 3925 ttg Leu 840 ctt ggt aac cct tat gtg gct got gct.

Leu Gly Asn Pro Tyr Val Ala Ala Ala 845 tat Tyr 850 tta ctt ggc gca Leu Leu Gly Ala acc Thr 855 3973 aag aaa ttg aat Lys Lys Leu Asn gta Val 860 ggg act gcg gct Gly Thr Ala Ala gtt ctc coo acc Val Leu Pro Thr got cat Ala His 870 4021 cca gtt cgc Pro Val Arg gga cga ttt Gly Arg Phe 890 cag Gin 875 ott gaa gag gtg Leu Giu Glu Val ttg ttg gat caa Leu Leu Asp Gin atg tca aaa Met Ser Lys 885 aaa gat ttt Lys Asp Phe 4069 4117 cga ttt ggt att Arg Phe Gly Ile cgg ggg ctt tac Arg Gly Leu Tyr cgc gta Arg Val 905 ttt ggc aca gat Phe Gly Thr Asp atg Met 910 aat aac agt cgt Asn Asn Ser Arg gcc Ala 915 tta atg gag tgt Leu Met Giu Cys tgg Trp 920 tat aag ttg ata Tyr Lys Leu Ilie aat gga atg act Asn Gly Met Thr gag Giu 930 gga tat atg gaa Gly Tyr Met Glu got Ala 935 4165 4213 4261 gao aac gaa oat Asp Asn Glu His aag tto cat aag Lys Phe His Lys aaa gtg ctg ccg Lys Val Leu Pro acg gca Thr Ala 950 tat agt oaa ggt ggt gca cot att tat gto gtt got gaa too got. too 4309 WO 00/33065 Tyr Ser Gin acg act gaa Thr Thr Giu 970 PCT/US99/28733 Gly 955 Gly Ala Pro Ile Tyr 960 Val Val Ala Glu Ser Ala Ser 965 tgg gcc gct caa Trp Ala Ala Gin ggt tta ccg atg Gly Leu Pro Met tta agt tgg Leu Ser Trp 4357 att ata Ile Ile 985 gtc gct Val Ala 1000 aat act aac gaa Asn Thr Asn Giu caa gaa tat gga Gin Giu Tyr Gly 1005 aag Lys 990 aaa gca caa att Lys Ala Gin Ile gag Giu 995 ctt tat aac gag Leu Tyr Asn Giu 4405 4453 cac gat att His Asp Ile cat aat His Asn 1010 atc gac cat Ile Asp His tgc tta Cys Leu 1015 tca tat ata aca Ser Tyr Ile Thr tgc cgg aat ttt Cys Arg Asn Phe 1035 tcg Ser 1020 gta gac cat gac tca Val Asp His Asp Ser 1025 atg aaa gcg Met Lys Ala aaa gaa att Lys Giu Ile 1030 ctg ggg cat Leu Giy His tgg tat Trp, Tyr 1040 gat tcc tat Asp Ser Tyr gtt aat gcc aca Val Asn Ala Thr 1045 4501 4549 4597 acc att ttt Thr Ile Phe 1050 gat gat tca Asp Asp Ser gac aaa Asp Lys 1055 aca aag ggc Thr Lys Gly tat gat Tyr Asp 1060 ttc aat aaa Phe Asn Lys gga caa Gly Gin 1065 cgc gtt Arg Val 1080 tgg cgc gac Trp, Arg Asp gat tac agt Asp Tyr Ser ttt gtc Phe Vai 1070 tta aaa gga cat aaa Leu Lys Gly His Lys 1075 aat act aat cgt Asn Thr Asn Arg 4645 4693 tac Tyr 1085 gaa atc aat Glu Ile Asn ccg gtg Pro Val 1090 gga acc ccg cag gaa Gly Thr Pro Gin Giu 1095 aca gga ata tca aat Thr Gly Ile Ser Asn 1110 tgt att gat Cys Ile Asp ata att Ile Ile 1100 caa aca gac Gin Thr Asp att gac gcc Ile Asp Ala 1105 4741 att tgt tgt ggg ttt gaa gct aat gga aca gta gat gaa att atc tct Ile Cys Cys Gly Phe Giu Ala Asn Gly Thr Val Asp Gu Ile Ile Ser 4789 1120 1125 tcc atg aag ctc ttc Ser Met Lys Leu Phe 1130 caa cgt tcg cta tta Gin Arg Ser Leu Leu 1145 ttc ttc ctt aac ttt Phe Phe Leu Asn Phe 1160 cag tct gat gta atq ccg Gin Ser Asp Vai Met Pro 1135 tat tag ctaaggaaaa tgaa Tyr 1150 atc aat tca aca act att Ile Asn Ser Thr Thr Ile 1165 ttt ctt aaa gaa aaa Phe Leu Lys Giu Lys 1140 atg aaa ttt ggc ttg Met Lys Phe Gly Leu 1155 caa gag caa agt ata Gin Glu Gin Ser Ile 1170 4837 4887 4935 gct cgc atg cag gaa ata aca gaa tat gtc gac aaa ttg aat ttt gag Ala Arg Met Gin Giu Ile Thr Giu Tyr Val Asp Lys Leu Asn Phe Giu 4983 WO 00/33065 PCT/US99/28733 1175 1180 1185 cag att ttg Gin Ile Leu 1190 gtg tgt gaa aat cat Val Cys Glu Asn His 1195 ttt tca gat aat ggt Phe Ser Asp Asn Gly 1200 gtt gtc ggc Val Val Gly 5031 get cct Ala Pro 1205 aaa att Lys Ile 1220 ttg act gtt tct ggt ttt Leu Thr Val Ser Gly Phe 1210 ggt tca ttg aat cat gtc Gly Ser Leu Asn His Val 1225 tta ctt ggc cta Leu Leu Gly Leu 1215 aca gaa aaa att Thr Glu Lys Ile 5079 5127 att Ile aca act Thr Thr 1230 cat cat cct His His Pro gtc cgc Val Arg 1235 ata gcg gaa Ile Ala Glu att tta gga Ile Leu Gly 1 aat cgc cct Asn Arg Pro 1270 gaa gcg Glu Ala 1240 tgc tta ttg Cys Leu Leu gat cag Asp Gin 1245 tta agc gaa Leu Ser Glu gga aga ttt Gly Arg Phe 1250 ttt Phe .255 agt gat tgc gag aga Ser Asp Cys Glu Arg 1260 aag gat gaa Lys Asp Glu atg cat ttt ttc Met His Phe Phe 1265 5175 5223 5271 gaa caa tac Glu Gin Tyr cag cag Gin Gin 1275 caa tta ttt Gmn Leu Phe gaa gaa Glu Glu 1280 tgc tat gac Cys Tyr Asp att att Ile Ile 1285 ttt tat Phe Tyr 1300 aac gat gct Asn Asp Ala tta aca Leu Thr 1290 aca ggc tat Thr Gly Tyr tgt aat Cys Asn 1295 cca aat ggc gat Pro Asn Gly Asp 5319 5367 aat ttc ccc aaa Asn Phe Pro Lys 1305 ata tcc gtg Ile Ser Val aat ccc Asn Pro 1310 cat gct tat acg caa His Ala Tyr Thr Gin 1315 aac ggg cct cgg aaa tat gta aca gca aca agt tgt cat gtt gtt gag Asn Gly Pro Arg Lys Tyr Val Thr Ala Thr Ser Cys His Val Val Glu 5415 1320 1325 1330 tgg gct gct aaa aaa ggc att cct cta Trp Ala Ala Lys Lys Gly Ile Pro Leu 1335 1340 ate ttt aag Ile Phe Lys tgg gat gat tcc Trp Asp Asp Ser 1345 5463 aat gaa gtt Asn Glu Val 1350 aaa cat gaa tat gcg Lys His Glu Tyr Ala 1355 aaa aga tat caa gcc Lys Arg Tyr Gin Ala 1360 ata gca ggt Ile Ala Gly 5511 gaa tat Glu Tyr 1365 gtt aac Val Asn 1380 ggt gtt gac ctg gca Gly Val Asp Leu Ala 1370 gag ata gat cat cag Glu Ile Asp His Gin 1375 tta atg ata ttg Leu Met Ile Leu 5559 5607 tat agt gaa gac Tyr Ser Glu Asp 1385 agt gag aaa Ser Glu Lys gct aaa Ala Lys 1390 gag gaa acg cgt gca Glu Glu Thr Arg Ala 1395 ttt ata agt gat tat att ctt gca atg cac cct aat gaa aat ttc gaa Phe Ile Ser Asp Tyr Ile Leu Ala Met His Pro Asn Glu Asn Phe Glu 5655 1400 1405 1410 WO 00/33065 WO 0033065PCT/US99/2873 aag aaa ctt gaa Lys Lys Leu Giu 1415 gaa tgt aca act Giu Cys Thr Thr 1430 ggt ata tta ttg Giy Ile Leu Leu 1445 aac gca att gat Asn Ala Ile Asp 1460 gaa ata atc aca gaa aac Glu Ile Ile Thr Glu Asn 1420 tcc gtc gga gat tat atg Ser Val Gly Asp Tyr Met 1425 5703 gcg gct aaa ttg gca atg gag aaa tgt ggt gca aaa Ala Ala Lys Leu Ala Met Glu Lys Cys Gly Ala Lys 5751 1435 1440 tcc ttt gaa tca atg agt Ser Phe Glu Ser Met Ser 1450 gat ttt aca cat caa ata Asp Phe Thr His Gin Ile 1455 5799 att gtc aat gat aat att aaa aag tat cac atg taa Ile Vai Asn Asp Asn Ile Lys Lys Tyr His Met 5847 1465 1470 1475 tataccctat. ggatttcaag gtgcatcgcg acggcaaggg agcgaatccc cgggagcata 5907 tacccaatag atttcaagtt gcagtgcggc ggcaagtgaa cgcatcccca ggagcataga 5967 taactatgtg actggggtaa gtgaacgcag ccaacaaagc agcagcttga aagatgaagg 6027 gtatagataa cgatgtgacc ggggtgcgtg aacgcagcca acaaagaggc aacttgaaag 6087 ataacgggta taaaagggta tagcagtcac tctgccatat cctttaatat. tagctgccga 6147 ggtaaaacag gt. atg act tca tat gtt gat aaa caa gaa atc aca gca agt 6198 Met Thr Ser Tyr Val Asp Lys Gin Giu Ile Thr Ala Ser 1480 1485 tca gaa Ser Giu 1490 tac gac Tyr Asp 1505 att gat gat ttg att Ile Asp Asp Leu Ile 1495 ttt tcg agt gat cca Phe Ser Ser Asp Pro 1500 tta gtc tgg tct Leu Val Trp Ser gaa cag gaa aag Giu Gin Giu Lys 1510 att aga aaa aaa Ile Arg Lys Lys ctt gtg ctt gat Leu Val Leu Asp .515 cgt cac tac tgt Arg His Tyr Cys gcg ttt Ala Phe 1520 cag gca Gin Ala -535 6246 6294 6342 cgt cat cac tat aaa Arg His His Tyr Lys 1525 cat tgt caa gaa tac His Cys Gin Giu Tyr 1530 cat aaa gta gat His Lys Val Asp 1540 gac aat att.

Asp Asn Ile acg gaa Thr Glu 1545 att gat gat ata Ile Asp Asp Ile *cct gta ttc Pro Val Phe 1550 6390 cca aca tca Pro Thr Ser 1555 gtg ttt aag ttt act cgc tta tta act tct aat gag aac Val Phe Lys Phe Thr Arg Leu Leu Thr Ser Asn Glu Asn 6438 1560 1565 gaa att Giu Ile 1570 gaa agt tgg t:tt acc Giu Ser Trp Phe Thr 1575 agt agt ggc Ser Ser Giy aca aat Thr Asn 1580 ggc tta aaa agt Gly Leu Lys Ser 6486 cag gta cca cgt gac aga cta agt att gag agg ctc tta ggc tct gta Gin Val Pro Arg Asp :krg Leu Ser Ile Giu Arg Leu Leu Gly Ser Val 6534 WO 00/33065 PCTUS99/28733 1585 1590 1595 1600 agt tat ggt Ser Tyr Gly atg aaa Met Lys 1605 tat att ggt agt Tyr Ile Gly Ser 1 tgg ttc gat cat Trp Phe Asp His L610 aat gct cat aat Asn Ala His Asr caa atg gaa Gin Met Glu 1615 att tgg ttt 1 Ile Trp Phe 1630 6582 6630 ttg gtc aac ctg Leu Val Asn Leu 1620 gga cca gat Gly Pro Asp aga ttt Arg Phe 1625 aaa tat gtt Lys Tyr Val 1635 atg agc ttg gta gag Met Ser Leu Val Glu 1640 tta tta tat cct acg tca ttc acc Leu Leu Tyr Pro Thr Ser Phe Thr 1645 6678 gta aca Val Thr 1650 cga ata Arg Ile 1665 gaa gaa cac Glu Glu His ata gat Ile Asp 1655 ttc gtt cag Phe Val Gin aca tta Thr Leu 1660 aat agt ctt gag Asn Ser Leu Glu aaa cat caa ggg Lys His Gin Gly 1670 aaa gat att Lys Asp Ile tgt ctt Cys Leu 1675 att ggt tcg Ile Gly Sex cca tac Pro Tyr 1680 tca ttt Ser Phe 1695 6726 6774 6822 ttt att tat ttg ctc Phe Ile Tyr Leu Leu 1685 tgc cgt tat Cys Arg Tyr atg aaa Met Lys 1690 gat aaa aat atc Asp Lys Asn Ile tct gga gat aaa Ser Gly Asp Lys 1700 agt ctt tat Ser Leu Tyr att ata Ile Ile 1705 acg ggg gga ggc Thr Gly Gly Gly tgg aaa agt Trp Lys Ser 1710 ctt tta ttc Leu Leu Phe 6870 6918 tac gaa aaa Tyr Glu Lys 1715 gaa tct ttg Glu Ser Leu aag cgt Lys Arg 1720 aat gat ttc aat Asn Asp Phe Asn cat His 1725 gac act Asp Thr 1730 caa gtt Gin Val 1745 ttc aac ctc agt aat Phe Asn Leu Ser Asn 1735 att aac cag Ile Asn Gin atc cgt Ile Arg 1740 gat ata ttt aat Asp Ile Phe Asn gaa ctc aac act Glu Leu Asn Thr 1750 tgt ttc ttt Cys Phe Phe gag gat Glu Asp 1755 gaa atg caa cgt aaa Glu Met Gin Arg Lys 1760 6966 7014 7062 cat gtt ccg ccg tgg His Val Pro Pro Trp 1765 gta tat gcg Val Tyr Ala cga gca Arg Ala 1770 ctt gat cct gaa aca ttg Leu Asp Pro Glu Thr Leu 1775 aaa ccg gta cct gat ggg atg cct ggt Lys Pro Val Pro Asp Gly Met Pro Gly 1780 1785 ttg atg agt tat Leu Met Ser Tyr atg gat gca Met Asp Ala 1790 7110 tca tca acg agt tat ccg Ser Ser Thr Ser Tyr Pro 1795 gca ttt Ala Phe 1800 att gtt acc gat gat atc gga ata Ile Vai Thr Asp Asp Ile Gly Ile 1805 7158 att agc aga gaa tat ggt caa tat cct ggt gta ttg gtt gaa att tta Ile Ser Arg Glu Tyr Gly Gin Tyr Pro Gly Val Leu Val Glu Ile Leu 7206 1810 1815 1820 WO 00/33065 WO 0033065PCT/US99/2873-3 cgt cgc gtt aat acg agg aaa caa aaa ggt tgt gct tta agc tta act Arg Arg Val Asn Thr Arg Lys Gin Lys Gly Cys Ala Leu Ser Leu Thr 1825 1830 1835 1840 gaa gca ttt ggt agt tga tagtttcttt ggaaagagga gcagtcaaag Giu Ala Phe Gly Ser 1845 7254 7302 gctcatttgt t t tccc cgca atgttgattt tatcaacggt gcggagagct tacacgatac cgaattc tcaatgcttt tcaggggtat t taagtatga cttttctgct gccaagtact taaacgttga tgcgaaacgt atacaagtaa gatacatggg ttatcgaggc tgtgacagtt accgtagagg tttgtcgaac aaaagctcag cggatttaaa tataagtttc ttattgccat gagcaacatt tctaggcgaa ggggtaaacc taacggagtc ttgcagtttt ctctggcgtg caatgcccgc ggttctcgac tgagcttggg agtttggaaa aaccacaacc actgctgctt gctaagttca 7362 7422 7482 7542 7602 7662 7669 <210> 2 <211> 480 <212> PRT <213> Xenorhabdus luminescens <400> 2 Met 1 Pro Asn Lys Lys Ilie Ser Phe Ile Ile Gly Arg Val Glu Ile Phe Glu Ser Asp Asp Leu Val Gin Ser 25 Ser Asn Phe Gly Val His Leu Tyr Asn Glu Pro Val Leu Asn Gin Val Lys Asp Asn Ser Ile Ile Asp Asn Phe Leu Asn Asn Glu Leu His Asn Tyr Thr Ile Tyr Val Gly Gin Lys Asn Giu Thr Giu Gly Ser Arg Arg Tyr Ile Arg Asp Al a Leu Lys Arg Tyr Met 90 Met Tyr Ser Glu Giu Met Ala Lys Leu Gly Leu Tyr 115 Asn Trp Ile Ile Leu Cys Ser Lys Gly 110 His Ile Met Leu Val Lys Asn 120 Leu Gly Ser Asp Giu Trp Leu Pro Gin Asp Giu Ser Tyr Ile Arg Ala Phe Pro Lys 130i 135 140 WO 00/33065 PCT/US99/28733.-- Val His Leu Leu Thr Gly Asn Vai Pro Leu Ser Giy Vai Gly 145 Leu Thr Ile Tyr His 225 Ala Lys Ala Phe Lys 305 Asn Lys Arg Leu Leu 385 Trp Giu Lys Ser Ser Asp Trp 210 Ala Val Lys Thr Ser 290 Leu Thr Giu Trp Gly 370 Pro Giu Arg Ser Ile Ser Val 195 Gin Asp Lys Ser Gly 275 Thr Ala Lys Cys Met 355 Arg Tyr Ala Ile 150 155 160 Leu Thr 180 Asp His Vai His Phe 260 Ala Gin Leu Lys Leu 340 Val Cys Val Ala Val 420 Arg Ala 165 Asp Pro Pro His Gin Gly Val Val 230 Aia Pro 245 Cys Ile Ala His Asn Ile Ile Glu 310 Asp Phe 325 Phe Ala Ile Giu Val Tyr Arg Lys 390 Leu Lys 405 Glu Ala Ile Phe His Asp 215 Ala Pro Ile Asp Tyr 295 Lys Asp Gly Ser Leu 375 Asn Tyr Gly Leu Thr Pro 200 Ile Trp Asp Asp Val 280 Tyr Leu Glu Leu Asn 360 His Lys Arg Met Thr Ala 185 Vai Ser Gly Ile Asn 265 Cys Met Asn Lys Lys 345 Al a His Thr Asp Asn 425 Lys 170 Asn Thr Leu Giy Asp 250 Pro Phe Gly Leu Ala 330 Val Gly Val Gin Leu 410 Asn Asn Ala Arg Al a Giu 235 Vai Val Tyr Ser Tyr 315 Ala Glu Val Asp Thr 395 Leu Ile Gin Leu Ser Lys 220 Asp Met Asp Asp His 300 Ala Tyr Val Giu Asn 380 Ile Ala Phe Cys Al a Leu 205 Glu Ala Lys Leu Gin 285 Tyr His Ser Asp Leu 365 Ile Ser Leu Arg Ile Leu 190 Ser Ile Ile Phe Val 270 Gin Glu Ile Leu Val 350 Asn Glu Val Lys Val 430 Ile 175 Ser Val Met Asn Gly 255 Ser Al a Glu Leu Val 335 His Gln Gln Phe Gly 415 Gly Lys Phe Val Gin Trp 240 Pro Ala Cys Phe Pro 320 Gin Gin Pro Ile Pro 400 Al a Gly Ala His Asp Gly Met Arg Pro Leu Gln Arg Leu Val Thr Tyr Ile Ser 435 440 44S WO 00/33065 PCTJUS99/28733.-- His Glu Arg Pro Ser His Tyr Thr Ala Lys Asp Val Ala Val Glu Ile 450 455 460 Glu Gin Thr Arg Phe Leu Glu Glu Asp Lys Phe Leu Val Phe Val Pro 465 470 475 480 <210> 3 <211> 307 <212> PRT <213> Xenorhabdus luminescens <400> 3 Met Giu Asn Lys Ser Arg Tyr Lys Thr Ile Asp His Val Ile Cys 1 5 10 Val Glu Glu Asn Arg Lys Ile His Val Trp Giu Thr Leu Pro Lys Glu 25 Asn Ser Pro Lys Arg Lys Asn Thr Leu Ile Ile Ala Ser Gly Phe Ala 40 Arg Arg Met Asp His Phe Ala Gly Leu Ala Giu Tyr Leu Ser Gin Asn 55 Gly Phe His Val Ile Arg Tyr Asp Ser Leu His His Val Gly Leu Ser 70 Ser Gly Thr Ile Asp Giu Phe Thr Met Ser Ile Gly Lys Gin Ser Leu 85 90 Leu Ala Val Val Asp Trp Leu Asn Thr Arg Lys Ile Asn Asn Leu Gly 100 105 110 Met Leu Ala Ser Ser Leu Ser Ala Arg Ile Ala Tyr Ala Ser Leu Ser 115 120 125 Giu Ile Asn Val Ser Phe Leu Ile Thr Ala Val Giy Val Val Asn Leu 130 135 140 Arg Tyr Thr Leu Glu Arg Ala Leu Giy Phe Asp Tyr Leu Ser Leu Pro 145 150 155 Ile Asp Glu Leu Pro Asp Asn Leu Asp Phe Glu Gly His Lys Leu Gly 165 170 175 Ala Glu Val Phe Ala Arg Asp Cys Ph-e Asp Ser Gly Trp Glu Asp Leu 180 185 190 Thr Ser Thr ile Asri Ser Met Met His Leu Asp Ile Pro Phe Ile Ala 195 200 205 Phe Thr Ala Asn Asn Asp Asp Trp Val Lys Gin Asp Glu Val Ile Thr 210 215 220 Leu Leu Ser Ser Ile Arg Ser His Gin Cys Lys Ile Tyr Ser Leu Leu 225 230 235 WO 00/33065 WO 0033065PCT/US99/287-33 Gly Ser Ser His Asp Leu Gly Giu Asn Leu Val Val Leu Arg Asn Phe 245 250 255 Tyr Gin Ser Val Thr Lys Ala Ala Ile Ala Met Asp Asn Gly Cys Leu 260 265 270 Asp Ilie Asp Val Asp Ilie Ilie Giu Pro Ser Phe Giu His Leu Thr Ile 275 280 285 Ala Ala Val Asn Glu Arg Arg Met Lys Ile Glu Ile Giu Asn Gin Vai 290 295 300 Ile Ser Leu Ser 305 <210> 4 <211> 360 <212> PRT <213> Xenorhabdus luminescens <400> 4 Met Lys Phe Gly Asn 1 Gin Glu Phe Ala Ala Ser Asp Giu Giu 145 Thr Ala Thr Cys Giy so Thr His Lys Phe Cys 130 Al a Ala Ser Giu Gly Leu Lys Pro Gly Arg 115 Trp Asp Tyr Thr Vai Phe Leu Lys Val Arg 100 Val Tyr Asn Ser Thr 5 Met Asp Gly Leu Arg Phe Phe Lys Giu Gin 165 Glu Phe Lys Thr Asn Asn 70 Gin Arg Gly Leu His 150 Gly Trp Leu Arg Val Pro 55 Val Leu Phe Thr Ile 135 Ile Gly Ala Leu Thr Tyr Leu Val Lys 25 Trp Leu Leu 40 Tyr Val Ala Gly Thr Ala Giu Glu Val 90 Gly Ile Cys 105 Asp Met Asn 120 Arg Asn Gly Lys Phe His Ala Pro Ile 170 Ala Gin His Gin Leu Glu Ala Ala 75 Asn Arg Asn Met Lys 155 Tyr Pro Gly His Ala Ile Leu Gly Ser Thr 140 Val Val Pro Arg His Tyr Val Leu Leu Arg 125 Giu Lys Val Gin Ile Phe Leu Leu Asp Tyr 110 Ala Gly Val Ala Phe Ser Thr Leu Pro Gin Asn Leu Tyr Leu Glu Ser Glu Giu Gly Thr Met Lys Met Met Pro 160 Ser 175 Gly Leu Pro Met Ile Leu WO 00133065 WO 0033065PCT/US99/28733 Ser Asn Cys 225 Giu Ala Asn Asn Gin 305 Ser Ile Giu Trp Glu 210 Leu Ile Thr Lys Arg 290 Giu Asn S er Lys Ile Ile 195 Val Ala Ser Tyr Cys Arg Thr Ile 260 Giy Gin 275 Arg Val Cys Ile Ile Cys Ser Met 340 Gin Arg 355 Asn Thr Gin Giu Ile Thr 230 Asn Phe 245 Phe Asp Trp Arg Asp Tyr Asp Ile 310 Cys Gly 325 Lys Leu Ser Leu Asn Tyr 215 Ser Leu Asp Asp Ser 295 Ile Phe Phe Leu 185 Giu Lys 200 Giy His Val Asp Gly His Ser Asp 265 Phe Vai 280 Tyr Giu Gin Thr Giu Ala Gin Ser 345 Tyr 360 Lys Asp His Trp 250 Lys Leu Ile Asp Asn 330 Ala Gin Ile Glu Leu Tyr 205 Ile Asp 235 Tyr Thr Lys Asn Ile 31i5 Gly His 220 Ser Asp Lys Gly Pro 300 Asp Thr Asn Met Ser Gly His 285 Val Al a Val Ile Lys Tyr Tyr 270 Lys Gly Thr Asp Asp Ala Val 255 Asp Asn Thr Gly Glu His Lys 240 Asn Phe Thr Pro Ile 320 Ile 335 Asp Vai Met Pro Phe Leu Lys <210> <211> 324 <212> PRT <213> Xenorhabdus luminescens <400> Met Lys Phe Gly Leu Phe Phe 1 5 Ile Gin Giu Gin Ser Ile Ala Arg Asp Lys Leu Asn Phe Giu Gin Ile Asp Asn Gly Val Val Gly Ala Pro 55 Gly Leu Thr Giu Lys Ilie Lys Ile 70 Leu Met Leu 40 Leu Giy Asn Gin Val Thr Ser Phe Giu Cys Val Leu Ile Ile Giu Ser Asn Asn Thr Asn Gly His 350 Ser Glu His Phe Val WO 00/33065 PCT/US99/28733 Thr His His Pro Val Arg Ile Ala Glu Glu Ala Cys Leu Leu Asp Gin 85 90 Leu Ser Glu Gly Arg Phe Ile Leu Gly Phe Ser Asp Cys Glu Arg Lys 100 105 110 Asp Glu Met His Phe Phe Asn Arg Pro Glu Gin Tyr Gin Gin Gin Leu 115 120 125 Phe Glu Glu Cys Tyr Asp Ile Ile Asn Asp Ala Leu Thr Thr Gly Tyr 130 135 140 Cys Asn Pro Asn Gly Asp Phe Tyr Asn Phe Pro Lys Ile Ser Val Asn 145 150 155 Pro His Ala Tyr Thr Gin Asn Gly Pro Arg Lys Tyr Val Thr Ala Thr 160 165 170 175 Ser Cys His Val Val Glu Trp Ala Ala Lys Lys Gly Ile Pro Leu Ile 180 185 190 Phe Lys Trp Asp Asp Ser Asn Glu Val Lys His Glu Tyr Ala Lys Arg 195 200 205 Tyr Gin Ala Ile Ala Gly Glu Tyr Gly Val Asp Leu Ala Glu Ile Asp 210 215 220 His Gin Leu Met Ile Leu Val Asn Tyr Ser Glu Asp Ser Glu Lys Ala 225 230 235 Lys Glu Glu Thr Arg Ala Phe Ile Ser Asp Tyr Ile Leu Ala Met His 240 245 250 255 Pro Asn Glu Asn Phe Glu Lys Lys Leu Glu Glu Ile Ile Thr Glu Asn 260 265 270 Ser Val Gly Asp Tyr Met Glu Cys Thr Thr Ala Ala Lys Leu Ala Met 275 280 285 Glu Lys Cys Gly Ala Lys Gly Ile Leu Leu Ser Phe Glu Ser Met Ser 290 295 300 Asp Phe Thr His Gin Ile Asn Ala Ile Asp Ile Val Asn Asp Asn Ile 305 310 315 Lys Lys Tyr His Met 320 <210> 6 <211> 370 <212> PRT <213> Xenorhabdus luminescens <400> 6 Met Thr Ser Tyr Val Asp Lys Gin Glu Ile Thr Ala Ser Ser Glu Ile 1 5 10 WO 00/33065 PCT/US99/28733 Asp Gin Tyr Asp Val Ser Arg Met Leu 145 Met Glu His Leu Lys 225 Glu Asn Leu Pro Pro 305 Asp Glu Lys Asp Phe Trp Asp Lys 130 Gly Ser His Gin Leu 210 Ser Ser Leu Asn Trp 290 Leu Lys His Asn Lys Phe Arg 115 Tyr Pro Leu Ile Gly 195 Cys Leu Leu Ser Thr 275 Val Ile Ile Cys Ile Phe Thr 100 Leu Ile Asp Val Asp 180 Lys Arg Tyr Lys Asn 260 Cys Tyr Phe Arg Gin Thr Thr Ser Ser Gly Arg Glu 165 Phe Asp Tyr Ile Arg 245 Ile Phe Ala Ser Lys Glu Glu 70 Arg Ser Ile Ser Phe 150 Leu Val Ile Met Ile 230 Asn Asn Phe Arg Ser Lys Tyr 55 Ile Leu Gly Glu Trp 135 Asn Leu Gin Cys Lys 215 Thr Asp Gin Glu Ala 295 Asp Leu 40 Arg Asp Leu Thr Arg 120 Phe Ala Tyr Thr Leu 200 Asp Gly Phe Ile Asp 280 Leu Pro 25 Val His Asp Thr Asn 105 Leu Asp His Pro Leu 185 Ile Lys Gly Asn Arg 265 Glu Asp Ser Leu Leu Tyr Ile Ser 90 Gly Leu His Asn Thr 170 Asn Gly Asn Gly His 250 Asp Met Pro Tyr Val Asp Cys Pro 75 Asn Leu Gly Gin Ile 155 Ser Ser Ser Ile Trp 235 Leu Ile Gin Glu Met 315 Trp Ala Gln Val Glu Lys Ser Met 140 Trp Phe Leu Pro Ser 220 Lys Leu Phe Arg Thr 300 Asp Ser Phe Ala Phe Asn Ser Val 125 Glu Phe Thr Glu Tyr 205 Phe Ser Phe Asn Lys 285 Leu Ala Tyr Arg His Pro Glu Gin 110 Ser Leu Lys Val Arg 190 Phe Ser Tyr Asp Gin 270 His Lys Ser Asp His Lys Thr Ile Val Tyr Val Tyr Thr 175 Ile Ile Gly Glu Thr 255 Val Val Pro Ser Glu His Val Ser Glu Pro Gly Asn Val 160 Glu Lys Tyr Asp Lys 240 Phe Glu Pro Val Thr 320 Asp Gly Met Pro Gly Leu Met 310 WO 00/33065 WO 0033065PCT/US99/28733.- Ser Tyr Pro Ala Phe Ile Val Thr Asp Asp Ile Gly Ile Ile Ser Arg 325 330 335 Giu Tyr Gly Gin Tyr Pro Gly Val Leu Val Giu Ile Leu Arg Arg Val 340 345 350 Asn Thr Arg Lys Gin Lys Gly Cys Ala Leu Ser Leu Thr Glu Ala Phe 355 360 365 Gly Ser 370 <210> 7 <211> 21 <212> DNA <2i3> Synthetic <400> tacctaggga gaaagagaat g 21

Claims (47)

1. An implantable monolithic bioelectronic device for detecting analyte within the body of an animal, said device comprising: an integrated circuit including at least one transducer for generating an electrical signal in response to light incident thereon; and a bioreporter capable of metabolizing said analyte and emitting light when exposed to said analyte, said bioreporter positioned so that at least a portion of said emitted light reaches said transducer.

2. An implantable monolithic bioelectronic device as claimed in claim 1, further comprising a biocompatible container in which said integrated circuit and said bioreporter are located, whereby said implantable monolithic bioelectronic device can be implanted in the body of said animal by implanting said biocompatible container.

3. The implantable monolithic bioelectronic device of claim 2, wherein said biocompatible container comprises a polymeric matrix.

4. The implantable monolithic bioelectronic device of claim 3, wherein said polymeric matrix comprises polyvinyl alcohol, poly-L-lysine, or alginate. 0 30

5. The implantable monolithic bioelectronic device of claim 3, wherein said polymeric matrix further comprises a microporous, mesh-reinforced or filter- supported hydrogel.

6. The implantable monolithic bioelectronic device of claim 1, wherein said integrated circuit comprises a phototransducer. H:\janel\Keep\Speci\20408-OO.doc 11/04/02 65

7. The implantable monolithic bioelectronic device of claim 6, further comprising a transparent, biocompatible, bioresistant separator operably positioned between the phototransducer and the bioreporter.

8. The implantable monolithic bioelectronic device of claim 1, wherein said bioreporter comprises a plurality of eukaryotic or prokaryotic cells that produce a bioluminescent reporter polypeptide in response to the presence of said analyte.

9. The implantable monolithic bioelectronic device of claim 8, wherein said plurality of prokaryotic cells comprise bacteria.

The implantable monolithic bioelectronic device of claim 8, wherein said plurality of eukaryotic cells comprise mammalian cells.

11. The implantable monolithic bioelectronic device of claim 10, wherein said plurality of eukaryotic cells comprise islet p-cells, immortal stem cells, or hepatic cells.

12. The implantable monolithic bioelectronic device of claim 11, wherein said plurality of eukaryotic cells comprise recombinant human immortal stem cells.

13. The implantable monolithic bioelectronic device of claim 8, wherein said plurality of cells comprise a nucleic acid segment that encodes a luciferase polypeptide or a green fluorescent protein that is produced by said cells in response to the presence of said analyte.

14. The implantable monolithic bioelectronic device of claim 13, wherein said nucleic acid segment encodes an H:\janel\Keep\Speci\20408-00.doc 11/04/02 *i 66 Aqueorea victoria or a Renilla reniformis green fluorescent protein.

The implantable monolithic bioelectronic device of claim 13, wherein said nucleic acid segment encodes a humanized green fluorescent protein.

16. The implantable monolithic bioelectronic device of claim 13, wherein said nucleic acid segment encodes a bacterial Lux polypeptide.

17. The implantable monolithic bioelectronic device of claim 16, wherein said nucleic acid segment encodes a bacterial LuxA, LuxB, LuxC, LuxD, or LuxE polypeptide, or a LuxAB, or LuxCDE fused polypeptide.

18. The implantable monolithic bioelectronic device of claim 17, wherein said nucleic acid segment encodes a Vibrio fischerii or a Xenorhabdus luminescens LuxA, LuxB, 20 LuxC, LuxD, or LuxE polypeptide, or a LuxAB, or Lux CDE fused polypeptide.

19. The implantable monolithic bioelectronic device of claim 18, wherein said nucleic acid encodes a S" 25 Senorhabdus luminescens LuxA, LuxB, LuxC, LuxD, or LuxE polypeptide, or a LuxAB, or LuxCDE fused polypeptide.

The implantable monolithic bioelectronic device Seof claim 19, wherein said polypeptide is encoded by a sequence comprising at least 25 contiguous nucleotides from SEQ ID NO:1.

21. The implantable monolithic bioelectronic device of claim 20, wherein said polypeptide is encoded by a sequence comprising at least 30 contiguous nucleotides from SEQ ID NO:1. H\janel\Keep\Speci\20408-00.doc 11/04/02 67

22. The implantable monolithic bioelectronic device of claim 21, wherein said polypeptide is encoded by a sequence comprising at least 35 contguous nucleotides from SEQ ID NO:1.

23. The implantable monolithic bioelectronic device of claim 17, wherein the expression of said nucleic acid segment is regulated by a nucleic acid sequence comprising a cis-acting element that is responsive to the presence of said analyte.

24. The implantable monolithic bioelectronic device of claim 23, wherein said cis-acting response element is a nucleotide sequence selected from the group consisting of an S14 gene sequence, a hepatic L-pyruvate kinase gene sequence, a hepatic 6-phosphofructo-2-kinase gene sequence, a 3-islets insulin gene sequence, a mesangial transforming growth factor-3 gene sequence, and an acetyl- coenzyme-A carboxylase gene sequence.

25. The implantable monolithic bioelectronic device of claim 24, wherein said cis-acting response element comprises a contiguous nucleotide sequence from a p-islets insulin gene sequence or a hepatic L-pyruvate kinase gene 25 sequence.

26. The implantable monolithic bioelectronic device of claim 13, wherein expression of said nucleic acid sequence is regulated by a promoter sequence derived from an L-pyruvate kinase-encoding gene.

27. The implantable monolithic bioelectronic device of claim 1, wherein said analyte is glucose, glucagon or insulin.

28. The implantable monolithic bioelectronic device of claim 8, further comprising a source of nutrients H:\janel\Keep\Speci\20408-0 0 .doc 11/04/02 68 capable of sustaining said cells.

29. The implantable monolithic bioelectronic device of claim 1, further comprising a wireless transmitter.

The implantable monolithic bioelectronic device of claim 1, further comprising an antenna.

31. The implantable monolithic bioelectronic device of claim 1, further comprising an implantable drug delivery pump capable of being controlled by said device, and capable of delivering said drug to the body of said animal.

32. The implantable monolithic bioelectronic device of claim 1, wherein said biocompatible container further comprises a membrane that is permeable to said analyte but not to said bioreporter. 20

33. The implantable monolithic bioelectronic device of claim 1, wherein said bioreporter expresses said light- emitting polypeptide following the metabolism of said analyte by said bioreporter. 25

34. The implantable monolithic bioelectronic device of claim 3, wherein said biocompatible container comprises silicon nitride or silicon oxide.

The implantable monolithic bioelectronic device of claim 1, wherein said integrated circuit is a complementary metal oxide semiconductor (CMOS) integrated circuit.

36. The implantable monolithic bioelectronic device of claim 6, wherein said phototransducer comprises a photodiode. H:\janel\Keep\Speci\20408-00.doc 11/04/02 69

37. The implantable monolithic bioelectronic device of claim 1, wherein said integrated circuit further comprises a photodiode and a current to frequency converter.

38. The implantable monolithic bioelectronic device of claim 1, wherein said integrated circuit further comprises a current to frequency converter and a digital counter.

39. The implantable monolithic bioelectronic device of claim 1, further comprising a transmitter.

The implantable monolithic bioelectronic device of claim 39, wherein said transmitter is capable of transmitting digital data.

41. An implantable controlled drug delivery system, comprising the device of claim 1, and an implantable drug delivery pump capable of being operably controlled by said :device.

42. A method of providing a controlled supply of a drug to a patient in need thereof, comprising implanting 25 within the body of said patient the controlled drug delivery system of claim 41.

43. A method of determining the amount of a drug required by a patient in need thereof, comprising implanting within the body of said patient the device of claim 1, and determining the amount of drug required by said patient based upon the output from said device.

44. A kit for the detection of an analyte comprising the device of claim 1 and instructions for using said device.

H\janel\Keep\Speci\20408-00.doc 11/04/02 70 The kit of claim 44, further comprising a standardized reference solution.

46. A method of regulating the blood glucose level of an animal in need thereof, comprising monitoring the level of glucose in the bloodstream or inerstitial fluid of said patient using the device of claim 1 or the kit of claim 44, and administering to said patient an effective amount of an insulin composition sufficient to regulate said blood glucose level.

47. An implantable monolithic bioelectronic device as claimed in claim 1 and substantially as herein described with reference to the accompanying drawings. Dated this 11th day of April 2002 UT-BATTELLE, LLC and THE UNIVERSITY OF TENNESSEE RESEARCH CORPORATION 20 By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia eee H,\janel\Keep\Speci\20408-00.doc 11/04/02