KR20010102951A - In vivo biosensor apparatus and method of use - Google Patents

Abstract

The present invention describes a bioluminescent bioreporter integrated circuit device for detecting selected analytes in a fluid when implanted in the body of an animal. The device includes a bioreporter genetically generated to contain a nucleic acid fragment comprising a cis-activating response element that is reactive to a selected material suitably bound to a gene encoding a bioluminescent reporter polypeptide. In a preferred embodiment, the target analyte is glucose, glucagon or insulin. Exposure of the bioreporter to the target material results in upregulation of the nucleic acid sequence encoding the reporter polypeptide that produces a luminescent reaction that is detected and quantified. In an exemplary embodiment, the bioreporter device processes the signal obtained by encapsulating on an integrated circuit capable of detecting emitted light, and then remotely reporting the result. In addition, controlled drug delivery systems are described that can be controlled directly or indirectly by a detection device that provides a drug (eg, insulin) to an animal in response to the amount of the target analyte present in the body fluid.

Description

In vivo biosensor device and method of use {IN VIVO BIOSENSOR APPARATUS AND METHOD OF USE}

1. Related Technology

A. Biosensor

Biosensors are hybrid devices that combine biological components with analytical measurement elements. Biological components react and / or interact with important analyte (s) to produce a response measurable by electronic, optical or mechanical transducers. The most common structures currently available are the use of immobilized macromolecules (eg enzymes or antibodies) to produce biological components. Examples of analytes and immobilized macromolecules include: glucose and immobilized glucose oxidase (eg Wilkins et al., 1995); Nitrates and immobilized nitrate reductases (Wu et al., 1997); Hydrogen peroxide and 2,3-dichlorophenoxyacetic acid and immobilized horseradish peroxidase (Rubtsova et al., 1998); And aspartate and immobilized L-aspartase (Campanella et al., 1995).

B. Whole-cell Biosensors

Further refinement has recently been developed for biosensors using intact living cells (eg microorganisms) or eukaryotic cells or cell cultures as replacements for immobilized enzymes. Microbial cells are particularly well suited for biosensor technology; They are physically robust and can exist under extremely harsh and widely varying environmental conditions, and they have various repertoires of responses to their environment, and they can also be generated by genetic engineering to create reporter systems that are very sensitive to these environmental reactions. Can be. Polynucleotide sequences comprising specific promoter sequences are suitably bound to a gene or a plurality of genes encoding the desired reporter enzyme (s), which are then introduced into and maintained in living cells. If a target analyte is present, the reporter gene is expressed to produce the enzyme (s) that are responsible for generating the measured signal. Commonly used reporter systems used β-galactosidase ( lac Z) or catechol-2,3-dioxygenase ( xyl E) enzymes (Kricka, 1993).

Unfortunately, these systems have a limitation that following exposure to the target substance (s), the cells are disruptively lysed and the enzyme (s) are isolated. This lysis, in turn, is followed by the addition of one or more secondary metabolites to produce a colorimetric signal that is proportional to the concentration of enzyme (s) in solution, providing a means of quantifying the concentration of the original target substance.

A more recent improvement in these sensors is the use of green fluorescent proteins as reporter systems, with an important advantage that cells do not require disruptive analytical techniques to generate colorimetric signals. However, since the substrate is added to the green fluorescent protein construct to initiate the photoreaction first, these systems are very complex and provide little benefit for detecting analytes in situ (Prasher, 1995).

C. In Vivo Sensor

The development of an integrated in vivo implantable glucose monitor was first reported by Wilkins and Atanasov (1995). This system uses glucose oxidase immobilized in the microbioreactor. The enzyme and catalyst to the oxidation of β-D- glucose by molecular oxygen to produce gluconolactone and hydrogen peroxide, wherein the concentration of glucose is proportional to the consumption and generation of H ₂ O ₂ in the O _2. Unfortunately, the presence of glucose oxidase inhibitor molecules in human blood flow tends to offset proportional constants, making the device unsatisfactory with accurate glucose monitoring and control (Gough et al., 1997). In addition, the limitation is that the relatively large size of the device ( 5 x 7 cm), which negates its usefulness as an implantable device.

Many small needle and microdialysis glucose sensors have since been developed with enveloping size limitations (Gough et al., 1997, Selam, 1997), but their dependence on glucose oxidase enzyme-based systems has been shown to improve its overall utility and reliability. Restrict.

Many nonspecific electrochemical sensors have also been studied as possible glucose sensors (eg Yao et al., 1994; Larger et al., 1994), but problems involving limited sensitivity, instability and limited long-term reliability have prevented their widespread use. Interference (Patzer et al., 1995). According to Atanasov et al. (1997), a continuous variable implantable glucose biosensor with long term stability has not yet been completed.

2. Problems in the prior art

Despite the significant miniaturization of biosensors over the past decade, they are still relatively large and protruding for use as ideal implantable devices. Current methods using mammalian bioluminescent reporter cells require cell lysis and the addition of exogenous substrates to produce measurable responses. As a result, these cells cannot be used as a continuous direct monitoring device.

Thus, a small, implantable monolithic (i.e., biologically constructed on a single substrate layer) that is durable, inexpensive, wireless, capable of communicating remotely to a drug delivery system to provide controlled delivery of a therapeutic agent (e.g. insulin). There is a continuing need for the development of bioelectronic monitors that contain both electrical components.

This application is a continuous application claiming priority over US Provisional Application No. 60 / 110,684, filed December 2, 1998, in particular the entire contents of which are incorporated herein by reference.

The US government has certain rights in the present invention in accordance with number R21RR14169-01, licensed from the National Institutes of Health (NIH).

The present invention generally relates to the field of implantable diagnostic devices (ie, devices placed in vivo in an animal) for monitoring one or more target substances, analytes or metabolites in an animal. In particular, the present invention provides implantable biosensor devices for monitoring and regulating levels of analytes in human tissues and the circulatory system. In an exemplary embodiment, such devices include biosensors that monitor blood sugar levels in diabetic or hypoglycemic patients. In addition, the sensors described herein can be used to control or regulate the delivery of drugs or other pharmaceutical agents from external or implantable drug delivery systems. For example, the device herein may form part of an artificial pancreas to adjust insulin dose in response to the level of glucose detected in situ .

The following drawings form part of this specification and are included to further illustrate certain aspects of the invention. The invention may be better understood with reference to one or more of these figures in conjunction with the detailed description of the specific aspects presented herein. Exemplary aspects of the invention are shown in the drawings, wherein like numerals are used to refer to like and corresponding parts of the several views.

1 illustrates a perspective view of one exemplary embodiment of the present invention.

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

3 shows a block diagram of an exemplary aspect of an integrated circuit.

4A shows a high quality photodetector that can be fabricated using standard N-well CMOS methods.

4B shows two photodetector structures fabricated by the insulator silicon CMOS method: a device similar to the left side (right side) except the side PIN detector (left) and the junction formed with a Schottky junction.

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

5B shows a circuit for supplying a large-scale photodiode for efficient light collection and a forward bias current that dissipates the photocurrent using a small diode in the feedback loop.

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

6 shows the current-to-frequency converter structure of the device.

7 shows a circular BBIC biosensor.

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

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

10A shows a schematic representation of an implantable biosensor containing two separate photodetectors with a bioreporter that responds to an increase or decrease in glucose concentration.

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

10C shows a schematic representation of the use of selective permeable membranes to protect bioreporters from immune responses.

Summary of the Invention

The present invention overcomes these and other inherent limitations in the prior art by providing implantable devices and methods for detecting and quantifying specific analytes in the body of an animal. In particular, the present invention provides an apparatus for detecting and quantifying in vivo metabolites, drugs, hormones, toxins or microorganisms (such as viruses) in humans or animals. In an exemplary embodiment, the present invention provides a BBIC device useful for the detection of glucose in humans. Such a device first provides an accurate direct detector for glucose monitoring and confers the ability to control the administration of the pharmaceutical agent via an external or implantable drug delivery system. Also described are BBIC devices for detecting concentrations of signal molecules (ie, proteins released from cancer cells, etc.), blood clotting factors, enzymes, and the like and other analytes present in the bloodstream or interstitial fluid. In the field of oncology, biosensor devices find utility in both initiation and re-release surveillance, in direct measurements of the effects of chemotherapy, and in stimulation / activity of the immune system. Similarly, biosensor devices can be directly controlled for enzymes associated with the development of blood clots (seizures, heart attacks, etc.), detection and quantification of clotting factors (maintenance levels), hormone replacement, continuous drug monitoring (prisoners, military forces, etc.). Tests on substances), surveillance of military or sublingual exposure to neurological and other debilitating agents, monitoring of levels of compounds affecting mental illness, and the like.

In one aspect of the invention, an implantable monolithic bioelectronic device for detecting an analyte in the body of an animal is provided. In a general sense, the device includes a bioreporter that is suitably disposed on a substrate over an integrated circuit. The bioreporter can metabolize the target analyte and, when in contact with the analyte, emit light by this metabolism. The apparatus further includes a sensor disposed in proximity to the integrated circuit that detects the emitted light and generates an electrical signal in proportion to the amount of light generated by the bioreporter. Preferably the entire implantable device is contained in a biocompatible container implanted into the body of an animal in need of analyte detection.

Biocompatible containers may consist of silicon nitride, silicon oxide or a suitable polymer matrix, examples of which are polyvinyl alcohol, poly-L-lysine, with alginates being particularly preferred. In addition, the polymer matrix may further comprise a microporous, mesh-reinforced or filter-supported hydrogel.

In certain embodiments, it may also be desirable to provide a transparent, biocompatible, bioresistant separator that is suitably disposed between the photoconverter and the bioreporter.

Bioreporters preferably include a number of eukaryotic or prokaryotic cells that produce a bioluminescent reporter polypeptide in response to the presence of a target analyte. Prokaryotic cells (eg, one or more bacterial strains) and eukaryotic cells (eg, : Mammalian cells) are particularly preferred. Examples of mammalian cells are human cells (e.g., β-cell islands, stem cells or hepatic cells), and hepatocytes that do not die are particularly preferred.

Preferably, these cells comprise one or more nucleic acid fragments encoding luciferase polypeptides or green fluorescent proteins produced in response to the presence of analytes by the cells. Preferably, the nucleic acid fragment is Aqueorea victoria, Renilla reniformis or humanized green fluorescent protein, or more preferably bacterial Lux polypeptide (eg, LuxA, LuxB, LuxC, LuxD or LuxE polypeptide) or as described herein. Encode LuxAB or LuxCDE fused polypeptide.

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

Examples of lux gene sequences that can be used to prepare the genetic constructs described herein include the gene sequences described in SEQ ID NO: 1. Examples of Lux polypeptide sequences are described in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

Preferably, the Lux polypeptide comprises 10 or more contiguous amino acid sequences from one or more polypeptide sequences described in SEQ ID NO: 2 to SEQ ID NO: 6. More preferably, the Lux polypeptide comprises 15 or more contiguous amino acid sequences from one or more polypeptide sequences described in SEQ ID NO: 2 to SEQ ID NO: 6, more preferably still SEQ ID NO: 2 To 20 or more contiguous amino acid sequences from one or more polypeptide sequences described in SEQ ID NO: 6.

Such polypeptides are preferably encoded by nucleic acid sequences comprising 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. do.

The expression of Lux-encoding nucleic acid fragments is preferably controlled by nucleic acid regulatory sequences that are suitably bound to the Lux-encoding segment. Preferably, this regulatory sequence comprises a cis-acting element that is reactive to the presence of the target analyte. Exemplary cis-acting response elements include the S14 gene sequence, hepatic L-pyruvate kinase gene sequence, hepatic 6-phosphofructo-2-kinase gene sequence, β-islet insulin gene sequence, glomerular interstitial transforming growth factor-β The gene sequence and the acetyl-coenzyme-A-carboxylase gene sequence.

In an exemplary embodiment, the cis-functional response element comprises a contiguous nucleotide sequence from a β-islet insulin gene sequence or a liver L-pyruvate kinase gene sequence. Expression of nucleic acid sequences is preferably controlled by promoter sequences such as those derived from the L-pyruvate kinase-coding genes described herein.

The device of the present invention may further comprise a source of nutrients capable of holding the radio transmitter, antenna and bioreporter cells. Likewise, the biocompatible container surrounding the bioreporter may further comprise a membrane that is permeable to the analyte but impermeable to the bioreporter cell itself. Such semipermeable membranes allow the analyte to move freely from body fluid to the detector device, but restrict the movement of bioreporter cells from the device to the surrounding tissue or circulation of the body into which the device is implanted.

In one aspect of the invention, the integrated circuit is a complementary metal oxide semiconductor (CMOS) integrated circuit. An integrated circuit may include one or more photoconverters, which may themselves consist of one or more photodiodes. Likewise, integrated circuits may further include photodiodes, current-to-frequency converters, digital counters, and / or transmitters capable of transmitting digital or analog data.

The invention also provides an implantable controlled drug delivery system comprising a BBIC device and an implantable drug delivery pump that can be suitably controlled by the BBIC and delivers the drug to the body of the animal in response to control by the device. to provide. The present invention also relates to a method of providing a controlled supply of drugs to a patient in need thereof. The methods of the invention generally involve the implantation of a controlled drug delivery system into the body of a patient.

The present invention also provides a method of measuring the amount of drug required by a patient in need of the drug, such as providing a suitable amount of insulin to a diabetic patient. The methods of the present invention generally implant one or more BBIC devices responsive to glucose, glucagon, insulin or other glucose metabolites in the body of a diabetic patient and based on the levels of analytes detected by the device in body fluids, Measuring the amount of insulin required. When the device of the present invention indicates that a high level of insulin is needed, appropriate control signals are sent to the drug delivery system and large amounts of insulin can be administered. When the device of the present invention indicates that a low level of insulin is needed, a suitable control signal is sent to the drug delivery system and a small amount of insulin can be administered. This “real time” monitoring of glucose in the body of the animal allows for controlled release of insulin over the day, eliminating the need for daily or more frequent injections of insulin, which may be too much or too little for a particular time of administration. This provides more cost-effective administration of the drug and gives the patient a more suitable dose of insulin on a "if needed" basis.

The present invention also provides a kit for analyte detection, which kit generally includes one or more of the described BBIC devices with appropriate instructions for using the detection device. In addition, such kits may generally contain one or more standardized reference solutions for calibrating the device, and may contain suitable reservoirs or nutrient media for maintaining the bioreporter cells during storage or once implanted in the body of the animal. It may include. For therapeutic kits, these kits also generally include one or more controlled delivery systems for administering the drug to the body of the animal.

The present invention also provides a method of controlling blood glucose levels in an animal as required. This method is generally characterized by monitoring the level of glucose in a patient's blood or interstitial fluid using a BBIC device and administering to the patient an effective amount of an insulin composition sufficient to control blood glucose levels.

This new class of bioluminescent-based bioreporters can monitor target substances without the disadvantageous requirement that cells break down to produce measurable signals. This allows continuous on-line and real-time monitoring of occurrence (Simpson et al., 1998a, 1998b). These cells rely on the luciferase gene (directed by lux in prokaryotes and luc in eukaryotes) for the reporter enzyme system. U.S. Patent Application No. 08 / 978,439 and International Patent Application No. PCT / US98 / 25925, each of which are hereby incorporated by reference in their entirety, disclose specific molecular targets ex situ or ex vivo (ex vivo). A self-contained small bioluminescent bioreporter integrated circuit (“BBIC”) designed to detect is described.

The present invention relates to an implantable, in situ or in vivo BBIC device capable of being implanted into the body of an animal and detecting the concentration of one or more analytes present within the animal. Implantable monolithic bioelectronic devices of the present invention generally include a substrate, a bioreporter capable of reacting to a specific substance by luminescence, a container attached to a substrate capable of supporting the bioreporter, and a concentration of the substance in response to light. An integrated circuit on a substrate comprising a photoconverter operable to generate an electrical signal representative of the light source; And a biocompatible housing that can be implanted into the body of the animal, wherein the housing portion covers the bioreporter container including a semipermeable membrane that allows the analyte to pass from the body of the animal and contact the biosensor, It restricts the diffusion of molecules into the body of the animal containing the implanted device. The bioreporter may be present in a solution, ie a cell suspension, may be trapped by a semipermeable membrane in a container, or the bioreporter may be enclosed in an optional permeable polymer matrix that may allow selected materials in the solution to reach the bioreporter. have. Preferably, the matrix is optically transparent.

The device of the present invention may further comprise a layer of bioresistant / biocompatible material (eg, silicon nitride layer) between the substrate and the container. The integrated circuit is preferably a CMOS integrated circuit and the photoconverter is preferably a photodiode.

In addition, integrated circuits may include current-to-frequency converters and / or digital counters. In addition, the integrated circuit may include one or more transmitters. Such a transmitter may be wireless or typically wired. In a preferred aspect, the device of the present invention also includes a drug delivery device capable of receiving a transmission from a transmitter.

A further aspect of the invention is an implantable device for detecting selected materials in a solution, which includes an integrated circuit suitable for inputting an electrical signal into the circuit in response to light, reacting to the selected material in solution by luminescence Bioreporter, reporter suitable for contact with a substance; And a transparent, biocompatible and bioresistant separator disposed between the photoconverter and the bioreporter such that light emitted from the bioreporter can reach the photoconverter. In a preferred embodiment of the invention, the substance selected is glucose. The bioreporter may be a mammalian cell containing a nucleotide sequence encoding one or more luminescent reporter molecules. Such nucleotide sequences may comprise one or more lux genes. In a preferred embodiment, the lux gene comprises both the luxCDE gene and the fused luxAB gene. In one embodiment, these lux genes are derived from Xenorhabdus luminescens. The lux gene can be regulated by a nucleic acid sequence comprising one or more cis-acting glucose response elements. In one exemplary embodiment, the glucose response element can be derived from β-islet or hepatic L-pyruvate kinase gene. In a very preferred embodiment, the p.LPK.Luc _FF plasmid is used to direct the expression of one or more lux genes by providing one or more glucose response elements and an L-pyruvate kinase promoter. The cells that make up the bioreporter are in suspension and can be trapped by a semipermeable membrane in place on the IC. Alternatively, the cells making up the bioreporter may be enclosed in a polymer matrix attached to the IC. Such matrices may be permeable to selected materials in solution.

A further aspect of the invention relates to an implantable monolithic bioelectronic device for detecting selected substances in body fluids. The device generally includes a biocompatible housing; A bioreporter capable of reacting with a material selected by luminescence; And a sensor capable of generating an electrical signal in response to receiving light emission. Such a device may also include a transparent, bioresistant and biocompatible separator disposed between the bioreporter and the sensor, and a semipermeable membrane disposed in the biocompatible housing, allowing the selected material to access the bioreporter.

Standard integrated circuits (ICs) are covered with a layer of insulating material such as silicon dioxide or silicon nitride. This process is called passivation and is used to protect the chip's surface from moisture, contamination and mechanical damage. The BBIC must be biocompatible and bioresistant, must protect the OASIC from chemical stress, optically tuned to efficiently transmit light from the test material, adhere to an oxide coating, and pin-hole It needs to be absent and any structure needed to hold the second coating and bioreporter or to collect the sample that must be patterned to form a hole over the bond pad.

The present invention contemplates that the components of the biosensor can be packaged in kit form. The kit may comprise an integrated circuit comprising one or more bioreporters and a photoconverter in suitable container means. The kit may further comprise a drug delivery device.

Description of Example Aspects

Luciferase systems are suitable for use in biosensors in vivo. In prokaryotes, the lux system consists of luciferase consisting of two subunits encoded by the genes luxA and luxB, which oxidize long-chain fatty acid aldehydes to the corresponding fatty acids to emit blue-green light at a wavelength close to 490 nm (Tu and Mager, 1995). The system also contains three proteins, a reductase encoded by luxC, a transferase encoded by luxD, and a synthetase encoded by luxE, which converts and recycles fatty acids to an aldehyde substrate, and a polyenzyme fatty acid reductase encoded by luxE. do. The gene is contained in a single operon, allowing cloning of the full lux gene cassette downstream from the user-specific promoter for the use of bioluminescence to monitor gene expression. Most bioluminescent bioreporters consist of Gram-negative organisms created to detect and monitor critically important chemical and environmental stressors (Ramanathan et al., 1997, Steinberg et al., 1995). Luciferase fusion has also been successfully performed in yeast cell lines as well as in Gram-positive bacteria (Andrew and Roberts, 1993, Srikantha et al., 1996).

Eukaryotic luciferase genes cloned with bacterial reporters include firefly luciferase-generating light near 560 nm and firefly luciferase-emitting light near 595 nm (Cebolla et al., 1995, Hastings, 1996). Eukaryotic bioreporters are characterized by glucose concentrations in rat β-cell islets (Kennedy et al., 1997), steroid activity in HeLa cells (Gagne et al., 1994), and ultraviolet effects in mouse fibroblasts (Filatov et al., 1996). Toxic effects in human liver cancer cells (Anderson et al., 1995), estrogen and antiestrogens compounds in breast cancer cell lines (Demirpence et al., 1995) and erythropoietin gene induction in human liver cancer cells (Gupta and Goldwasser, 1996). To date, most eukaryotic bioluminescent reporters require the addition of cell disruption and exogenous substrates, generally luciferin, which produce measurable luminescent responses.

In addition, green fluorescent protein ("GFP") is commonly used as a reporter system, where an important advantage is that cells do not require harmful assay techniques to generate colorimetric signals (Hanakam et al., 1996; Grygorezyk et al. , 1996; Siegel and Isacoff, 1997; Biondi et al., 1998). However, the substrate must be added to the GFP construct to initiate the photoreaction first (Prasher, 1995). Humanized GFP cDNAs have been generated that are particularly suitable for high levels of expression in mammalian cells, especially those derived from humans (Zolotukhin 1996). Humanized GFP can be efficiently inserted into mammalian cells using viral vectors (Levy et al., 1996; Gram et al., 1998).

Detection of bioluminescent signals from reporter organisms is performed through the use of optical transducers, including photomultipliers, photodiodes, microchannel plates, photographic films, and charge-coupled devices. Light is collected and traveled to the transducer through a lens, fiber optic cable or liquid light guide. However, applications requiring small amounts, remote detection or multiple parallel sensing require a new class of devices that are compact and portable while maintaining high sensitivity.

A. Overview of the System of the Invention

The present invention describes implantable BBICs that detect selected materials. Bioreporters are genetically engineered cell lines in which nucleic acid sequences contain cis-activating response elements that are reactive to selected materials. In a preferred embodiment, the substance selected is glucose. The bioreporter is exposed to the selected material, causing the reaction component to upregulate the nucleic acid sequence encoding one or more polypeptides that produce a luminescent reaction. In a preferred embodiment, the luminescence reaction is produced by a prokaryotic lux system.

The function of the IC portion of the BBIC is to detect, filter, amplify, digitize and report bioluminescent signals. In practice, the IC is used as a complete laboratory instrument (microphotometer) on a chip.

Silicon-based ICs can detect optical signals using PN junctions commonly used to fabricate transistors in the near, visible and near infrared regions (Simpson et al., 1999a). Using an n-well / p-substrate photodiode in a 0.5 μm bulk CMOS IC method, about 66% quantum efficiency was measured at 490 nm bioluminescence wavelength (Simpson et al., 1999b). Multiple signal processing schemes may be used. However, by counting pulses from the current-to-frequency converter circuit, a long time constant integrator is formed, which is the causal part of the matched filter for low bioluminescence signals in white noise. Using the photodiode described above by this signal processing method, an rms noise level of 175 electrons / second is measured for an integration time of 13 minutes, which corresponds to a detection limit of about 500 photons / second (Simpson et al., 1999b).

Circular BBIC is prepared by placing a toluene sensitive bioreporter, P. putida TVA8, on a conventional integrated microphotometer. FIG. 6 shows circular BBIC (including bioreporter inclusions) used for characterization studies (Simpson et al., 1998b; Simpson et al., 1998c; Simpson et al., 1998d).

In the absence of a luminescent signal from the cells, measurements are made several times at an integration time set to 1 minute. The leakage current generates a signal of about 6 counts / minute, with a standard deviation σ of 0.22 counts / minute. As expected, σ decreases with the square root of the integration time. Long integration times are generated by totaling 1 minute measurements off-line.

Bioluminescence is induced by exposure to toluene vapor in BBIC cells and control samples of cells. From control sample measurements, we estimate that the toluene concentration is 1 ppm or less. A signal of 12 counts / minute (6 counts / minute on the background) is measured. From the previous measurements, it is known that P. putida TVA8 exhibits a linear response to toluene concentration until saturated when the concentration reaches a level of about 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 photodiode area.

Naphthalene-sensitive BBICs are prepared using the microphotometer and bioreporter P. fluorescens 5RL. Using the same experimental method as above, this BBIC is exposed to a concentration of about 10 ppm of nattalene vapor. A signal of 240 counts / minute is recorded.

To eliminate the need for the addition of exogenous substrates, the cells themselves must supply a substrate suitable for luciferase. In bacterial systems, the substrate is produced by fatty acid reductase complexes encoded by the luxCDE gene. This enzymatic complex reduces the short chain of fatty acids to the corresponding aldehyde. Luciferase then oxidizes the aldehyde to the corresponding fatty acid. Preferred fatty acids for this reaction are myristic acid, which is present in eukaryotic organisms (Rudnick et al., 1993). Myristic acid is generally included in myristoylation of the amino terminus associated with membrane adhesion (Borgese et al., 1996, Brand et al., 1996).

In a preferred embodiment, the glucose report bioreporter is a mammalian bioluminescent reporter cell line genetically generated to express luminescence continuously in response to glucose concentration, without the need for cell disruption and exogenous substrate addition. Current methods using mammalian bioluminescent reporter cells require cell lysis and the addition of exogenous substrates to produce measurable responses. As a result, these cells cannot be used as a continuous direct monitoring device. In a preferred embodiment, this new cell line uses the lux AB and lux CDE genes from X. luminescens incorporated into a plasmid based system designated p.LPK.Luc _FF containing eukaryotic luc genes capable of responding to glucose concentrations. It is constructed using a bioluminescent reporter. Replacing the luc gene with the lux AB gene allows bioluminescence to be measured in real time with glucose concentration, thus eliminating the need for cell disruption and substrate addition.

To produce implantable glucose monitoring BBICs, the bioreporter can trap them in a container behind a semipermeable membrane that holds them in place over the IC photodetector. Alternatively, the bioreporter can be placed in a polymer matrix. The BBIC is enclosed in a biocompatible housing having a semipermeable membrane covering the bioreporter region. This membrane allows glucose to pass through the bioreporter, but blocks the passage of large molecules that can interfere with glucose measurements. When glucose reaches the bioreporter, it is metabolized and the cells emit visible light. The IC detects these rays, amplifies and filters these signals and then reports these measurements. Such measurements may be reported to the patient (eg, with a wristwatch receiver) or with an insulin pump in a closed loop system that works very similarly to the pancreas.

1 shows a perspective view of the present invention. The detected glucose 10 enters the BBIC 11 through the semipermeable membrane 12 covering the bioreporter.

2 shows a side view of the present invention. The BBIC is enclosed in a biocompatible housing 20 having a semipermeable membrane 21 covering the bioreporter held in the vessel 22. The cells that make up the bioreporter may be in suspension or enclosed in a polymer matrix. The bioreporter is separated from the photodetector 23 by a protective coating 24. Single material 25 contains photodetectors as well as additional circuits 26 for processing and transmitting signals.

3 shows a block diagram of one aspect of an integrated circuit (IC). The photodetector is a photodiode 33 connected to the current to frequency converter 30. Photodiodes respond to light by sinking current. The current is converted into a series of pulses accumulated in the digital counter 31. The number of counts at a fixed amount of time in the counter is directly proportional to the amount of light collected by the photodiode, which is directly proportional to the concentration of glucose. The digital processing circuit in the digital counter determines the next step suitable for the insulin pump based on the measured glucose level. The next indication for the measured concentration or insulin pump may be reported via the wireless transmitter 32. All these circuits (photodiodes, signal processing and wireless transmission) can be fabricated on one IC.

4 shows a bioreporter fed with water and nutrients. Fluid and nutrient reservoir 141 is connected to the microfluidic pump 142 to allow nutrients and fluid 144 to flow through the polymer matrix 143 surrounding the bioreporter. These devices may each be configured on a single substrate 140.

5A shows a high quality photodetector fabricated using a standard N-well CMOS method. The photodetector consists of two reverse bias diodes in parallel. The upper diode is formed between the P + active layer 45 and the N-well 46 and the lower 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 has good long wavelength sensitivity (500-1100 nm). Thus, a complete diode is sensitive in the 400-1100 nm or more range. The light emitting compound 41 to be tested is separated from the photodetector by a layer of Si ₃ N _{4 and} a layer of SiO ₂ .

10A, 10B and 10C show schematic representations of implantable biosensors containing two separate photodetectors with bioreporters in response to an increase or decrease in glucose concentration.

10B shows a side view of a biosensor showing a plastic sheath.

10C shows a schematic representation of the use of selective permeable membranes to protect bioreporters from immune responses.

B. Photodetector

The first element in the microphotometer signal processing chain is a photodetector. The main requirements for photodetectors are:

Sensitivity to the wavelength of light emitted by the bioluminescent or chemiluminescent compounds tested;

Low background signal (ie, leakage current) due to the eddy current reverse bias diode;

Coatings suitable for preventing the materials in the semiconductor device from interfering with the bioluminescent or chemiluminescent methods studied and for preventing the method under study from degrading the performance of the microphotometer; And

Compatibility with fabrication methods used to form microphotometer circuits.

Two photodetector structures that meet these requirements are described below. However, it should be understood that alternative methods of making such photodetectors can be used by those skilled in the art without departing from the spirit and scope of the invention as defined in the claims.

In a first aspect, the photodetector is manufactured by standard N-well CMOS methods. This detector shown in FIG. 5A is fabricated 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 detector obtained is sensitive to light between about 400 nm and about 1100 nm, and this range includes the 450 to 600 nm emission range of the most commonly used bioluminescent and chemiluminescent compounds or organisms. In order to meet the requirement that the device has a low background signal, the device is operated with zero bias and sets the operating voltage of the diode equal to the substrate voltage. The photodiode coating may be formed of a deposited silicon nitride layer or other material suitable for semiconductor processing techniques.

In a second photodetector embodiment, the detector is manufactured by an insulator silicon (SOI) CMOS method. The internal leakage current in the SOI method is two to three times lower than in standard CMOS due to the presence of an oxide insulating layer buried between the active layer and the substrate. Two photodetector structures are designed with the SOI method. The first structure shown on the left of FIG. 5B consists of a side PIN detector, where the P layer is formed by a P + contact layer, the I (native) region is formed by a slightly doped active layer, and the N region is Formed by the N + contact layer of the SOI CMOS method. The spectral sensitivity of this side detector is determined by the thickness of the active layer, which can be tuned for specific bioluminescent and chemiluminescent compounds.

The second structure shown on the right side of FIG. 3B is similar to the first structure, but is formed as a Schottky junction between a layer of cobalt silicide (CoSi ₂ ) or other suitable material to which the junction is attached and a slightly doped active layer.

We contemplate that other photodetector structures may be designed with silicon or other semiconductor methods that meet the above standards.

C. Low noise electronics

Low-noise electronics are the second element in microphotometer signal processing. The requirements for low-noise electronics are as follows:

Sensitivity to very low signal levels provided by photodetectors;

Immunity or compensation for electronic noise in the signal processing chain;

Minimal sensitivity to temperature changes;

Minimum sensitivity to changes in power supply voltage (for battery supply applications);

For certain applications, the electronics must have sufficient linearity and dynamic range to accurately record the detected signal levels; And

In other applications, electronic products must simply detect the presence of signals even in the presence of electronic and environmental noise.

Three aspects of meeting these requirements are described below. However, it should be understood that alternative methods of detecting small signals while meeting these requirements may be used without departing from the spirit and scope of the invention as defined in the claims.

6A schematically illustrates a first approach to detecting very small signals. The device uses a P-diffusion / N-well photodiode, a structure suitable for standard CMOS IC methods, in an open circuit manner with a read amplifier (made on the same IC as the photodiode). The luminescent signal generates electron-hole pairs in the P-diffusion and N-wells. The light-generating electrons in the P-diffusion section are injected into the N-well, while the light-generating holes in the N-well are injected into the P-diffusion section. The N-well is coupled 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 the CMOS amplifier (near infinity at low frequency), the positive charge collects in this region. Thus, the voltage on the P-diffusion node begins to rise.

As the P-diffusion voltage rises, the P-diffusion / N-well photodiode becomes a forward bias, thereby generating a current in the opposite direction to the photo-generated current. The system reaches a steady state when the voltage on the P-diffusion node produces a forward bias current (but opposite polarity) that is exactly the same size as the photocurrent. If this PN junction does not deviate from the ideal diode equation, the output voltage is given by:

[Equation 1]

V _out = V _t ln (I _p / (AI _S ) + 1)

In the above formula,

V _t is the thermal voltage (approximately 26 mV at room temperature),

I _p is photocurrent,

A is the cross-sectional area of the PN junction,

I _S is the reverse saturation current for the PN junction with unit cross section.

The value of I _S is highly dependent on the IC method and the material parameters.

Two major error currents-recombination current and generated current-are present in the PN junction operating at low current densities. Except at very low temperatures, free carriers are optionally produced 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 produce a current component (generated current) in the same direction as the photocurrent. In addition, the carrier crossing the space-charge region has a finite opportunity to recombine. This produces a different current component (recombination current) in the opposite direction to the photocurrent. Therefore, considering these error currents, Equation 1 becomes the following Equation 2:

[Equation 2]

V _out = V _t ln ((I _p + I _g -I _r ) / (AI _S ) + 1)

This output voltage is generally a function of parameters beyond the inventors' control. However, we control over the junction region, A. Unfortunately, in order to make our output signal large, we want a small A, while we want a large A for high quantum efficiency (QE).

6B shows a second microphotometer embodiment that meets both of these needs. The circuit uses large photodiodes for effective light collection, but uses small diodes in a feedback loop that provides forward bias current to dissipate photocurrent. Once again, amplifiers and feedback diodes are manufactured in the same IC as the photodiodes. For this circuit, the following equation 3 is obtained:

[Equation 3]

V _out = 3 V _t ln ((I _p + I _g -I _r / (A _fb I _S ) + 1)

In the above formula,

A _fb is the small cross sectional area of the feedback diode.

One or more diodes are used in the feedback path to produce a large output signal compared to the DC offset of subsequent amplifier stages. This technique allows for the efficient collection of light using large photodiodes, but produces large output voltages due to the small diodes in the feedback path.

The feedback circuit of FIG. 6B maintains the photodiode at zero bias. In the absence of application potential, the recombination and generated current dissipate. When small recombination and generated currents are ignored in the small feedback current, Equation 3 becomes Equation 4:

[Equation 4]

V _out = 3 V _t ln ((I _p / (A _fb I _S ) + 1)

Key benefits of the second microphotometer embodiment shown in FIG. 6B include the following:

SNR is measured entirely by photodiodes, and noise from small diodes and amplifiers is minor;

The diode may enter the feedback path until the signal level at the output of the amplifier is comparable to the offset voltage (and offset voltage drift) of the subsequent stages;

This method is fully suitable for standard CMOS methods without adding masks, materials or fabrication steps;

This detection method can be manufactured in the same IC as similar and digital signal processing circuits and RF communication circuits;

Measurements can be taken even when no power is applied to the circuit. Measurements can be read after applying power, but measurements can be obtained in the absence of power.

The third microphotometer implementation shown in FIG. 6C uses correlated double sampling (CDS) to minimize time- or temperature-dependent changes in amplifier offset voltage as well as low frequency (blink) amplifier noise effects. As shown in FIG. 6C, a photodiode with capacitance C _d and noise power spectral density S _t is provided with a feedback capacitance C _f through a set of switches controlled by a partial level of flip-flop output and It is connected to an integrated preamplifier with input noise power spectral density S _V. When the flip-flop output is low, the switch is positioned so that photocurrent flows out of the preamplifier to increase the output voltage of the integrator. If the low pass-filtered integrator output voltage exceeds the threshold, V _HI , the upper comparator "stimulates" the setting of the flip-flop and causing the rise of its output. The detector opens and closes the change position, causing current to flow into the integral amplifier, which in turn reduces the amplifier output voltage. If the integrator output is below the second threshold, V _LO , the lower comparator "stimulates" resetting the flip-flop and causing the output to decrease again. The method repeats itself as long as there is a photocurrent.

The average duration of the output pulses, Δt, is given by:

[Equation 5]

In the above formula,

V _HI and V _LO are the threshold voltages of the comparator,

I _P is a diode photocurrent.

The two noise sources are sources of error in the measurements of Δt, where S _i is basically the input noise current power spectral density associated with the photodiode and S _V is basically the input noise voltage power spectral density associated with the preamplifier.

Diode noise is given by Equation 6:

[Equation 6]

In the above formula,

I _S is the photodiode reverse saturation current,

I _P is a photocurrent.

As the photocurrent approaches zero, the noise power spectral density approaches a finite value of 4qI _S A ² / Hz. The noise voltage S _V of the preamplifier is determined by its design and has a unit of V ² / Hz.

The transfer function from the point at which diode noise is introduced at the integrator's output is given by Equation 7:

[Equation 7]

In the above formula,

ω ₁ is the nominal frequency of the integral amplifier,

s = jω.

Neglecting the effect of the switch for a while, the transfer function from the point where the amplifier noise is introduced at the integrator's output is given by:

[Equation 8]

The switch performs a correlated double sampling function that attenuates noise that appears below the open and close frequencies of the output pulse string. The transfer function of a correlated double sampling circuit is first approached by:

[Equation 9]

In the above formula,

Δt is the average duration of the output pulse string.

Thus, considering the switchgear, the transfer function from the point where the amplifier noise is introduced at the output of the integrator is given by approximately:

[Equation 10]

This is an important result as the useful zero introduced into the noise voltage transfer function reduces the effect of the flicker noise of the amplifier. This is particularly useful for CMOS performance in microphotometers where flicker noise can have pronounced effects.

The square of the mean output noise at the output of the integrator is given by Equation 11, and the RMS noise voltage is given by Equation 12:

[Equation 11]

[Equation 12]

The RMS error in the measured period is determined according to the relationship of equation (13) or approximately by equation (14) by the slope of the integral signal and the noise at the output of the integrator:

[Equation 13]

[Equation 14]

The error in Δt measurements can be reduced by collecting many output pulses and obtaining the average duration. The error within the measured average pulse duration is improved in proportion to the square root of the number of pulses collected, resulting in equation (15) or (16).

[Equation 15]

[Equation 16]

In the above formula,

t _meas is the total measurement time.

Thus, performance of microphotometers has the following advantages:

Low frequency “blink” noise of the amplifier is reduced by the correlated double sampling method;

Ideally, the accuracy of the measured photocurrent can be improved indefinitely by acquiring data on the increase in duration.

Of course, the practical limitations imposed by the lifetime and stability of the signal produced by the luminescent compounds tested ultimately determine the resolution of this performance.

D. READ-OUT ELECTRONICS

Many methods of communicating data from a BBIC to an external receiver or to an in vivo drug delivery system are envisioned. In a preferred embodiment, the method of communication is a wireless communication system of a semiconductor chip that reports the level of photocurrent to a computational circuit contained in an in vivo drug delivery system or an external receiver. In a closed loop system, this computational circuit measures the amount of drug delivered by the in vivo drug delivery system. If an external receiver is used, the data from the BBIC is used to determine the amount of drug administered, depending on user input. The external receiver may include a wireless transmission circuit for communicating with the in vivo drug delivery system or may administer the drug by hand. Other methods of communicating BBIC data include:

Generation of DC voltage levels proportional to photocurrent using wiring connections with in vivo drug delivery systems;

Generation of DC current levels proportional to photocurrent using wiring connections with in vivo drug delivery systems;

The generation of logical pulse strings whose speed is proportional to photocurrent using wiring connections to the in vivo drug delivery system;

Completion of a semiconductor chip of an analog-to-digital converter that reports numerical values proportional to photocurrent using wire connections as an in vivo drug delivery system;

Semiconductor chip completion of a series or parallel communication ports that report numbers proportional to photocurrent using wiring connections with an in vivo drug delivery system;

The generation of logical markers if the photocurrent exceeds previously limited levels using wiring connections to the in vivo drug delivery system; And

Generation of radio frequency signals or beacons when photocurrent exceeds a predefined level.

In vivo wireless communication may be limited by signal attenuation by body fluids, and health-related restrictions on RF signal levels. This may require closely spaced BBICs and in vivo drug delivery systems, which may not be the optimal structure for all cases. In this case, the BBIC is disjoint but can communicate to an external receiver placed in close proximity to the BBIC. Such a receiver may be connected (by wiring or wirelessly) to a transmitter ex vivo but placed proximate to an in vivo drug delivery system.

Many computational methods are expected to control in vivo drug delivery systems using BBIC. These include, but are not limited to:

A simple look-up table for administering prescribed drug levels measured only by a single BBIC data point;

A simple tour table for administering the prescribed drug level if the predetermined data point number exceeds a preset threshold;

An algorithm for determining drug dose by the rate of increase or decrease of the BBIC signal;

An algorithm that matches the BBIC data points to data point patterns stored in memory to determine drug dose; And

A learning algorithm that uses BBIC data point transitions and user input to estimate the correct drug dose to achieve the desired result.

Some of these algorithms may require two way communication between the BBIC and the in vivo drug delivery system. In this case, the receiver is included above the BBIC.

E. Biocompatible Housings and Semi-Permeable Membranes

The BBIC is enclosed in a biocompatible housing having a semipermeable membrane covering the bioreporter region. The manufacture of biocompatible covers for implants and prosthetic devices to minimize capsule formation and body rejection has been an extensive area of research. For example, US Pat. No. 5,370,684 and US Pat. No. 5,387,247, each of which is specifically incorporated herein by reference, describe the application of thin biocompatible carbon films to prosthetic devices. Biocompatible implants comprising woven or knitted fabrics of three-dimensional organic fibers are described in US Pat. No. 5,711,960, which is specifically incorporated herein in its entirety. Other covers for implants configured to provide a biocompatible surface to the body are described in US Pat. No. 5,653,755, US Pat. No. 5,799,734 and US Pat. No. 5,814,091, each of which is specifically incorporated herein by reference in its entirety. In addition, collagen and albumin coatings have been shown to improve the biocompatibility of implants and prosthetic devices (Marios et al., 1996; Ksander, 1988). The present invention contemplates the use of suitable biocompatible materials to coat or form the housing.

The semipermeable membrane includes a BBIC housing portion that covers the bioreporter and traps them over the integrated circuit. This membrane allows selected materials (eg glucose) to pass through the bioreporter, but prevents the passage of large molecules. In addition, membranes designed for use as glucose-oxidase based biosensors may be used in preferred embodiments of the present invention. Membranes studied and designed for use as 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 discharged 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 use in transplantation of islets or other cells that have been biotechnically produced to produce insulin. Such membranes are permeable to glucose and other metabolites, while excluding elements of the host immune system. Such membranes may be suitable for use in the present invention, including but not limited to: asymmetric poly (vinyl alcohol) membranes (Young et al., 1996); Poly (L-lysine) membranes (Tziampazis and Sambanis, 1995); Polyurethanes (Zondervan et al., 1992); Nuclear pore membranes (Ohgawara et al., 1998); And agarose gels (Taniguchi et al., 1997). Biopermeable membranes that are biocompatible to enclose cells producing artificial organs are described in US Pat. No. 5,795,790 and US Pat. No. 5,620,883, each of which is specifically incorporated herein by reference in its entirety. Biocompatible semipermeable segmented block polyurethane copolymer membranes and their use for permeation of molecules in a range of molecular weights are described in US Pat. No. 5,428,123 (in particular incorporated herein by reference in its entirety). Although the selected material allows access to the bioreporter, it contemplates the use of suitable semipermeable membranes that hinder the passage of macromolecules.

F. Drug Delivery Device

Many drug delivery devices, both implantable and external, that can be controlled by wireless telemetry have been described previously. For example, US Pat. No. 4,944,659, which is specifically incorporated herein by reference in its entirety, describes implantable piezoelectric pumps for drug delivery in outpatients. US Pat. No. 5,474,552, which is specifically incorporated herein in its entirety, describes implantable pumps for use with glucose sensors that can deliver multiple active agents (eg, glucose, glucagon, or insulin), if necessary. Separate pumps can be used to deliver each formulation or a single pump that can be opened and closed between them can be used. US Pat. No. 5,569,186, which is specifically incorporated herein in its entirety, describes a closed loop infusion pump controlled by a glucose sensor. US Pat. No. 4,637,391, which is specifically incorporated herein in its entirety, describes a remotely controlled implantable micropump for delivering pharmaceutical agents. The use of an external drug delivery system is contemplated in another aspect of the present invention. For example, US Pat. No. 5,800,420, which is specifically incorporated herein in its entirety, describes a local pump position relative to the skin surface for delivering a liquid drug (eg insulin) through a hollow delivery needle extending into the dermis. . In another aspect of the invention, the drug delivery system may be directly connected and controlled with the biosensor device, as opposed to telemetry control from the BBIC.

The pump delivery system is an example that makes use of the present invention useful. In addition to the pump system, other drug delivery systems are also contemplated in the present invention. For example, US Pat. No. 5,421,816, which is specifically incorporated herein in its entirety, describes an ultrasound transdermal drug delivery system. Ultrasound energy is used to release stored drugs and to force them into the bloodstream through the skin of the organism. Accordingly, the present invention contemplates the use of a suitable drug delivery system that can be controlled by a BBIC glucose monitor. The selection of such drug delivery systems and the factors that direct their use with the use of BBIC glucose monitors are known in the light of the art to those skilled in the art.

G. Bioluminescent Bio Reporter

In a preferred embodiment of the invention, the glucose report bioreporter is a mammalian bioluminescent reporter cell line genetically generated to continuously express luminescence in response to glucose concentration. Implantable bioluminescent sensors require bioluminescent reporters that can function without the addition of an exogenous substrate for the luciferase reaction. Current eukaryotic luciferase systems used in molecular biology require the addition of exogenous substrates due to the complex nature of producing eukaryotic luciferins. Cells must be permeated or lysed and then treated with assay solution containing luciferin. Thus, current eukaryotic luciferase systems are not preferred candidates for direct monitoring.

The requirement for the addition of exogenous substrates can be removed by the use of the bacterial lux gene. In a preferred embodiment of the invention, the lux genes of X. luminescens , lux AB and lux CDE are used as bioluminescent reporter systems. The X. luminescens lux AB gene encodes the α- and β-subunits of the luciferase of an enzyme that exhibits maximum thermal stability at 37 ° C, while other bacterial luciferases lose significant activity above 30 ° C. The lux CDE gene is needed to eliminate the need for the addition of exogenous substrates. The aldehyde substrate of luciferase encoded by the lux AB gene is produced by the fatty acid reductase complex encoded by the lux CDE gene. Preferred fatty acids for this reaction are myristic acid, which is present in eukaryotic organisms (Rudnick et al., 1993), and eukaryotic cells are therefore suitable host cells for such reporters. Enzyme complexes reduce fatty acids to the corresponding aldehydes. Luciferase then oxidizes the aldehyde back to fatty acids.

Other bioluminescent nucleic acid fragments may include luciferase from Vibrio fischerii , the lux gene of lux CDABE, or other bioluminescent capable organisms that may be suitable to not require the addition of exogenous substrates. In another embodiment of the invention, the nucleic acid fragment encodes a green fluorescent protein of Aqueorea victoria or Renilla reniformis .

H. Recombinant Vectors Expressing Bioluminescent Genes

One important aspect of the present invention is a recombinant vector comprising one or more nucleic acid fragments encoding one or more bioluminescent polypeptides. Such vectors can be transferred to and replicated in eukaryotic or prokaryotic hosts.

The coding DNA segment is considered to 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 generally associated with a DNA segment that encodes a crystalline protein or peptide in its natural environment. Inherently, it is important to use a promoter that effectively directs the expression of DNA segments in the cell species, organism or even animal selected for expression. The use of promoter and cell species combinations for protein expression is generally known to experts in the field of molecular biology (eg Sambrook et al., 1989). In its preferred embodiment, such promoters are indicated by cis-acting glucose response elements. In one preferred embodiment, the glucose response element is an L4 box that directs the L-pyruvate kinase (“L-PK”) promoter in the liver and β-cell islets. L4 boxes consist of series repetitions of non-standard E-boxes (Kennedy et al., 1997). Glucose alters the interactivation performance of the upward stimulating factor ("USF") bound to the L4 box to promote liver and pancreatic β-cells (Kennedy et al., 1997; Doiron et al., 1996).

The exact mechanism by which glucose controls the interactivation performance of USF proteins is unclear. One possibility is reversible phosphorylation of USF proteins. Glucose can alter the phosphorylation state via xylose-5-phosphate via pentose phosphate shorts (Dorion et al., 1996). An alternative mechanism is via intracellular concentrations of glucose 6-phosphate (Foufelle et al., 1992). In addition, other glucose metabolites may be included. Phosphorylated glucose metabolites include, but are not limited to: fructose 6-phosphate, 6-phosphogluconic acid, 6-phosphoglucono-δ-lactone, ribulose 5-phosphate, ribose 5- Phosphate, erythrose 4-phosphate, sedoheptulose 7-phosphate, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Non-phosphorylated glucose metabolites include, but are not limited to, citric acid, cis-aconic 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 described include, but are not limited to: liver 6-phosphopructo-2-kinase gene (Dupriez and Rousseau, 1997), β-islet insulin gene (German and Wang. 1994), Gene for glomerular interstitial transforming growth factor-β gene (Hoffman et al., 1998) and acetyl-coenzyme-A carboxylase (Girard et al., 1997). The present invention contemplates the use of a glucose response element that can effectively direct a promoter or otherwise control the expression of a reporter protein in response to glucose.

In a preferred embodiment, the recombinant vector comprises a nucleic acid fragment encoding one or more bioluminescent polypeptides. Highly preferred nucleic acid fragments are the lux genes of X. luminescens lux AB and lux CDE. Bacterial luciferase can be altered to optimize expression in eukaryotic cells. Almashanu et al. (1990) remove lux A, the TAA stop codon from the intervening region between the two genes, and the original methionine from lux B, and the lux AB gene from V. harveyi without destroying the reading frame. By fusion. Fusion is successfully expressed in Saccharomyces cerevisiae and Drosophila melanogaster . The same method is used for lux AB from X. luminescens . The resulting constructs are sequenced to demonstrate genetic changes, resulting in fusions, which are identified. The sequence of the fusion region is as follows:

5'- tac ctagggagaaagaga atg-3 '(SEQ ID NO: 7)

(End point of underlined lux A) (starting point of underlined lux B)

Fusions successfully expressed fused proteins in E. coli and were cloned successfully into mammalian vectors as described in Example 1.

In a further aspect, we contemplate recombinant vectors comprising nucleic acid fragments encoding one or more enzymes capable of producing a reaction that produces a luminescent product or a product that can be converted directly into a luminescent signal. For example, substrates of commonly used β-galactosidase and alkaline phosphate enzymes are commercially available, which, when converted by each enzyme, are luminescent (chemiluminescent).

In another important aspect, the biosensor comprises one or more first transformed host cells that express one or more recombinant expression vectors. The host cell can be a prokaryotic or eukaryotic cell. In a preferred embodiment, the host cell is a mammalian cell. Host cells can include hepatocytes, β-islet cells, or hepatocytes. In a preferred embodiment, the host cell is a homologous cell, ie a cell obtained from a patient who has been cultured, genetically generated and then incorporated into BBIC. Particularly preferred host cells are those expressing nucleic acid segments comprising recombinant vectors encoding the lux genes of X. luminescens , lux AB and lux CDE. These sequences are particularly preferred because the transcribed proteins of the X.luminescens lux system have the ability to act at 37 ° C. (ambient human temperature).

A wide variety of methods are useful for introducing nucleic acid fragments that express polypeptides that can provide bioluminescence or chemiluminescence to microbial hosts under conditions that allow stable maintenance and expression of the gene. One can provide a DNA construct comprising transcriptional and translational control signals for the expression of a nucleic acid fragment, wherein the nucleic acid fragment is under regulatory control and the DNA sequence is homologous to the sequence in the host organism. Or a replication system acting on the host, whereby assembly or stable maintenance occurs, or both.

The transcription initiation signal includes a promoter and a transcription initiation start site. If desired, it may be necessary to provide regulated expression of nucleic acid fragments that can provide bioluminescence or chemiluminescence, where expression of the nucleic acid fragments occurs only after release into a suitable environment. This can be done using a region that is bound to an actuator or activator or enhancer, which can be induced upon changes in the body or chemical environment of the microorganism. For initiation of translation, ribosomal binding sites and initiation codons are present.

Many manipulations can be used to enhance expression of messenger RNA, in particular by using sequences as well as using active promoters, which enhance the stability of messenger RNA. Transcriptional and translational termination regions include stop codon (s), species stage regions, and optionally, polyadenylation signals (if used in eukaryotic systems).

In the direction of transcription (ie, 5 'to 3' direction of the coding or sense sequence), the construct, if present, comprises a transcriptional regulatory region, and a promoter, wherein the regulatory region is the 5 'or 3' of the promoter, Ribosome binding site, start codon, structural gene with open reading frame in the phase with start codon, stop codon (s), if present, polyadenylation signal sequence and terminator region. As a double strand, this sequence can be used alone in the transformation of a microbial host, but is generally included with a DNA sequence comprising a label, where the second DNA sequence is to be bound to the expression construct during the introduction of the DNA into the host. Can be.

The term "marker" as used herein refers to structural genes that provide for the selection of altered or transformed hosts. The label may be, for example, biocidal resistance (eg resistance to antibiotics or heavy metals); The selective benefit of providing complementarity is generally provided to provide prototrophy to the host of the strain, for example. One or more labels can be used to alter the host as well as the generation of the construct.

In the absence of a functional replication system, the construct also comprises a sequence of at least 50 basepairs (bp), preferably at least about 100 bp, more preferably at least about 1000 bp, and generally at most about 2000 bp Include sequences that are homologous to In this way, the possibility of suitable recombination is enhanced, allowing the gene to be assembled into the host and maintained stable by the host. Preferably, nucleic acid fragments that can provide bioluminescence or chemiluminescence are present in close proximity to genes that provide complementarity as well as genes that provide competitive advantages. Thus, when the nucleic acid fragments that can provide bioluminescence or chemiluminescence are hosts, the organisms obtained are also likely to lose complementary genes, and genes that provide competitive advantages, and both.

Many transcriptional regulatory regions are useful from a wide variety of microbial hosts (eg, bacteria, bacteriophages, cyanobacteria, algae, fungi, etc.). Many transcriptional regulatory regions include regions associated with trp genes, lac genes, gal genes, λ _L and λ _R promoters, and tac promoters. See, for example, US Pat. No. 4,332,898; US Patent No. 4,342,832; And US Pat. No. 4,356,270, each of which is incorporated herein by reference in its entirety. The terminator region may be a transcription initiation region or a terminator region generally associated with two regions, as long as different transcription initiation regions are suitable and functional in the host. In a preferred embodiment of the invention, fragments of the L-pyruvate kinase gene are used, which contain the L-PK promoter and L4 box glucose reactive elements described by Kennedy et al. (1997). In a very preferred embodiment, the p.LPK.Luc _FF plasmid is used (Kennedy et al., 1997) and only the luc gene encoding firefly luciferase is removed and replaced with the fused X. luminescens lux AB gene.

Where stable episomal maintenance or assembly is desired, plasmids with replication systems functional in the host are used. The replication system may be derived from a replication system from a chromosome, host or episomal element present in a different host, or from a virus stable in the host. Many plasmids are useful, for example pBR322, pACYC184, RSF1010, pR01614 and the like. See, eg, Oslen et al., 1982; Bagdasarian et al., 1981, US Pat. No. 4,356,270, US Pat. No. 4,362,817, US Pat. No. 4,371,625 and US Pat. No. 5,441,884, respectively. Specifically cited herein by reference.

Preferred genes can be introduced between the transcriptional and translational initiation region and the transcriptional and translational terminator region to regulate and control the initiation region. This construct is included in the plasmid, which includes one or more replication systems, but may include more than one, where one replication system is used for cloning during the development of the plasmid and the second replication system is in the last host. It is essential to make it work. In addition, more than one label may be present, as previously described. If assembly is preferred, the plasmid preferably comprises sequences homologous to the host genome.

Transformants can be isolated using conventional screening techniques, according to conventional methods, thereby allowing selection of non-altering or transferring organisms, where present and the desired organisms. The transformant can then be tested for bioluminescent or chemiluminescent activity. If desired, unwanted or concomitant DNA sequences can be selectively removed from recombinant bacteria by using site-specific recombination systems, see, for example, US Pat. No. 5,441,884, which is specifically incorporated herein by reference in its entirety. Described.

I. Assembly and Storage of In Vivo Biosensors

In contrast to biosensors encapsulated in a matrix, when the biosensors consist of biotechnology-generated cells trapped in a suspension beyond a semipermeable membrane, the cells can be immediately added to the BBIC at a time point of several hours before implanting the biosensors. Biosensors can also consist of cells enclosed in a polymer matrix. The matrix includes materials previously shown to be successful in the encapsulation of living cells, including polyvinyl alcohol, sol-gels and alginates (Cassidy et al., 1996). Prior to encapsulation, prokaryotic cell lines can be lyophilized with a lyophilization system (eg Savant) according to the manufacturer's protocol. Lyophilization allows cells to undergo long term storage periods (years), where a simple rehydration protocol is required for cell reactivation prior to BBIC use (Malik et al., 1993). S. cerevisiae eukaryotic cells can similarly be lyophilized. Eukaryotic cell lines, preferably consisting of β-cell islets, hepatocytes or hepatocytes, may be enclosed in polyvinyl alcohol mesh-reinforced or microporous filter supported hydrogels on IC, which previously contained cell sorts of this kind (Baker et al., 1997; Burczak et al., 1996; Gu et al., 1994; Inoue et al., 1991).

However, for mammalian cell lines, lyophilization is not an alternative. In such cases, mammalian cells may be enclosed in a sol gel or other fixed matrix and attached to BBIC as previously described. The finished BBIC in the enclosure is then stored in serum or other suitable maintenance medium and maintained until use. The benefits of using intact hepatocyte lines are evident for both long term use and storage. Transplantation can be performed depending on the particular application. For glucose detection, the area where the interstitial fluid is available is most suitable. However, implantable devices with specific applications for detecting hormones or other blood containing molecules are available for the bloodstream. Synthetic venous or catheter systems may need to be used to allow continuous monitoring of the blood levels of the target molecule. Another particular example besides glucose is the use of an in vivo biosensor device to detect molecules associated with colon cancer. In this case, the biosensor is implanted into the colon.

Integrated circuits may be individually packaged in sterile growth prevention bags. Prokaryotic and yeast eukaryotic biosensors, consisting of lyophilized cells, can be stored individually in sterile, growth resistant, vacuum sealed bags for close to several years. Cells are generally rehydrated in minimal nutrient medium before use. Mammalian cell lines, either separately or when trapped, are frozen or frozen on the BBIC at the site of development, remain frozen for long term storage (up to 7 years at -150 ° C.) or frozen for short term storage (several days). In all cases, BBIC is exposed to known concentrations of important analytes, which allows testing of cell viability to produce quantitative bioluminescent signals of known size. One or more control vials of the analyte (s) or reference “standard” may be included as part of a diagnostic kit or supplied in a suitable assay of an implantable device.

J. Implantation and Use of Biosensor Devices

In a preferred embodiment of the present invention, the BBIC analyte biosensor is implanted in contact with the interstitial fluid of the animal. For example, in the case of glucose biosensors, it has been shown that glucose dynamics in interstitial fluid can be predicted by compartmental modeling (Gastaldelli et al., 1997). In particular, subcutaneous placement of glucose sensors has been demonstrated (Schmidt et al., 1993; Poitour et al., 1993; Ward et al., 1994; Stenberg et al., 1995; Bantle and Thomas, 1997). Other possible analyte biosensor tissue transplant sites include the peritoneum, pleura and pericardium (Wolfson et al., 1982). Indeed, we can place implantable biosensors at convenient locations throughout the body, using conventional surgical and implantation methods, depending on the particular analyte or metabolite detected. For example, the device of the present invention may be implanted in accordance with a particular analyte detected in such a way as to be in contact with interstitial fluid, lymph, blood, serum, synovial fluid or cerebrospinal fluid.

In certain embodiments, the implantable device described above may be placed in contact with a particular tissue, organ or particular organ system. Similarly, it may be desirable to implant the biosensors into contact with certain intracellular fluids, interstitial fluids, or other body fluids from which the target material can be detected.

In addition, the present invention contemplates the use of multiple biosensors to detect multiple different analytes. For example, for glucose monitoring, one or more devices may be used to monitor various glucose or glucose metabolites, glucagon, insulin, and the like. Likewise, one or more biosensor devices can be used in a controlled drug delivery system. In this way, such a device can be controlled by a biosensor and determine the amount of a particular drug, hormone, protein, peptide or other pharmaceutical composition measured by the concentration of one or more analytes detected by the BBIC device in the body of the animal. Introducing biosensors can be suitably connected to a controlled drug delivery pump or device. Accordingly, controlled drug delivery systems are considered by the inventors to be particularly desirable for providing long term drug administration to animals, such as in the case of chronic or lifelong medical conditions or when symptoms persist for a long time. Prolonged controlled delivery of pain medications, heart or other cardiac modulators, diuretics, or drugs such as hormones or peptides (such as insulin) or metabolites (such as glucagon or glucose) can be facilitated by such biosensor / pump systems. have. If it is necessary to deliver one or more drugs or metabolites to the animal, it is contemplated that multiple drug delivery systems or single gated drug delivery systems are particularly useful.

Host-rejection effects can be minimized through immunoisolation techniques. Previous studies have found that bio-host cells sealed to hydrogel membranes are protected from immune rejection after transplantation (Baker et al., 1997; Burczak et al., 1996; Inoue et al., 1991). The hydrogel block is accessed by the humoral and cellular components of the host's immune system but remains permeable to the target substance glucose. The mesh-reinforced polyvinyl alcohol hydrogel bag developed by Gu et al. (1994) is used to fully encapsulate BBIC to allow graft porosity of the immunosuppressive response.

If cells from the host are used for biosensor fabrication, host rejection of the implanted biosensor is not a problem. However, where other cells are used, it may be necessary to provide a barrier between suitable body fluids that allow passage of cells and signal molecules or analytes but do not allow passage of bioreporter cells or somatic cells (such as white blood cells). Immunosuppressed patients are not infected because the implant does not contain a pathogen agent of the kind that invades the patient. In all cases, the surgical methods involved in the implantation of the described BBIC device are well known to those skilled in the surgical art.

In an exemplary embodiment, a BBIC glucose sensor can be used to monitor glucose in diabetic patients. However, such sensors can be used in other conditions where glucose concentration is important, such as in sustained exercise or other conditions, including hypoglycemia or hyperglycemia. Such measurements may be endpoints for research or diagnostic purposes, or such sensors may be connected via telemetry or directly to the drug delivery system.

The use of implantable BBICs for other substances besides glucose can be used for a range of therapeutic situations. By incorporating suitable cis-activating response elements, BBIC can monitor a number of substances and find use in the treatment of chronic pain, cancer treatment, chronic immunosuppression, hormone therapy, cholesterol management, and lactate limits in heart attack patients. have. For example, Example 7 below describes the use of BBIC in the detection and diagnosis of cancer.

Individual biosensors can be assayed to examine the performance as well as the viability of the cells. The assay is performed by exposing the sensor to a solution containing a change in concentration of the important analyte (s). The bioreporter can be assayed by a series of standard analyte concentrations for a particular application after its initial construction. Overall on-line performance can be monitored with analyte reservoirs using microfluidics, which systematically provide known concentrations for cells, allowing for assays and viability testing.

At this time, the luminescence reaction is correlated with the concentration and parameter set. Viability can also be monitored continuously by cells produced by biotechnology, where the reporter exhibits continuous luminescence. Loss of viability reduces luminescence. This technique was used to detect the viability of prokaryotic cells. Thus, BBIC contains two bioreporters, a bioreporter that detects selected substances, and a second bioreporter that exhibits luminescence in proportion to cell viability. A detection method can be provided that measures the ratio of signals from two bioreporters and automatically corrects for loss of viability.

Once prepared, the bioreporter can be stored in a suitable maintenance medium (eg, standard tissue culture medium, serum, or other suitable growth or nutrient formulation) and then assayed prior to transplantation. The viability of this device can be examined by bioluminescence using microfluidics or by quantification of known standards or other reference solutions to ensure the viability and integrity of the system before and after implantation.

In certain aspects of the invention, the monolithic biosensor device can be externally used as the body of the monitored individual. In certain clinical settings, monitors may be used to monitor glucose in body fluids in a decongested manner. The device may even be used in the pathology or forensic field to detect the amount of a particular analyte, such as in tissues or body fluids. Likewise, the present invention also contemplates the use of biosensor devices in the field of veterinary medicine. Monitor hormone levels in the blood (i.e. to optimize milk production), monitor the onset (heat) of estrus in many animals to maximize artificial insemination efficiency, and directly monitor hormone levels in the resulting milk Implantation of such devices in animals is considered to provide certain benefits for use by professionals in commercial farming operations, animal husbandry and veterinary medicine.

K. Diagnostic Kits Including Biosensors In Vivo

While the individual components of the invention described herein can be obtained and configured individually, the inventors conveniently contemplate that the components of the biosensor can be packaged in kit form. The kit may comprise an integrated circuit comprising one or more bioreporters and a photoconverter in suitable container means. In addition, the kit preferably includes instructions for the use of the biosensor device, and may optionally further comprise a drug delivery device or a second biosensor device. The kit may comprise a single container means and drug delivery device containing an integrated circuit comprising one or more bioreporters and a photoconverter. Alternatively, the kits of the present invention may comprise separate container means for each component. In this case, one container contains one or more bioreporters in a suitable medium or pre-encapsulated in a polymer matrix, another container comprising an integrated circuit, and another container containing a drug delivery device. If the bioreporter is pre-encapsulation, the kit may contain one or more encapsulation media. The use of separate container means for each component allows for modification of the various components of the kit. For example, many bioreporters may be useful to select, depending on the desired material for detection. By replacing the bioreporter, the components of the kit can be retained for completely different purposes, and the components can be reused.

The container means described above can be a container such as a vial, test tube, bag, sleeve, shrink wrap or any other container means in which the components of the kit can be placed. The bioreporter or any reagent may be dispensed into a small container or delivery vehicle, if necessary.

In addition, the kits of the present invention may include means for containing individual containers in a closed range on a commercial scale, for example injection or blow-molded with the desired components of the kit. It is a plastic container.

Regardless of the number of containers, the kit of the present invention may also include or use a device for assisting the placement of the bioreporter in an integrated circuit. Such device may be a syringe, pipette, tweezers or other similar surgical or implantable device. The kit may also include one or more stents, catheters or other surgical instruments that facilitate implantation in the body of the target animal. Such kits may also include devices for long term recording of data obtained from telemetry devices or data storage or monitoring devices. Likewise, in the case of a controlled drug delivery system, the kit may include one or more drug delivery pumps as described above, and may include one or more pharmaceutical agents to administer themselves. For example, in the case of a glucose monitoring system, the system may include glucose-sensitive BBIC devices, drug delivery pumps, instructions for implantation and / or use of the system, and optionally, reference standards or pharmaceutical formulations of insulin, glucagon or other pharmaceutical compositions. Generally includes. In addition, the system may optionally include growth and / or storage medium to support the nutritional needs of the bioreporter cells included in the BBIC device.

The following examples are included to demonstrate preferred embodiments of the present invention. It is understood by those of ordinary skill in the art that the techniques disclosed in the following examples represent techniques that work well in the practice of the present invention, as revealed by the inventors, and thus may be considered to constitute a preferred manner for performance. However, one of ordinary skill in the art appreciates that many changes can be made in the light of the present technology without departing from the spirit and scope of the invention in certain embodiments that are described and still yield similar results.

Example 1

Construction of Bioluminescent Reporters for Mammalian Cell Lines

To facilitate the construction of an implantable bioluminescent glucose sensor, it is essential to form a bioluminescent reporter system that can function without exogenous addition of a substrate for the luciferase reaction. This exogenous addition is due to the complex nature of luciferin production for many eukaryotic luciferases. Cells must be permeated or lysed and then treated with assay solution containing luciferin. Thus, the current state of bioluminescent reporters used in eukaryotic molecular biology makes them unsuitable for "on-line" monitoring. Firefly luciferase has been used to test the regulation of L-pyruvate kinase promoter activity in single living β-cell islets (Kennedy et al., 1997). However, these cells must be perfused with Beetle luciferin to produce a luminescent response.

In order to alleviate this limitation, preferred bioluminescent reporter systems in the present invention do not require the addition of an exogenous substrate. In the case of bacterial luciferase-based detection systems, this can be done using bioluminescent genes from X. luminescens . In this organism, the lux A and lux B genes (or single fused luxAluxB genes) respectively encode the α- and β-subunits of the luciferase enzyme (Meighen et al., 1991). This luciferase shows maximum stability at 37 ° C., while other bacterial luciferases lose significant activity above 30 ° C. Thus, these bacterial luciferases can be expressed in slightly altered eukaryotic cells. Almashanu et al. (1990) fused the luxAB gene from V. harveyi by removing V. luxA, the TAA stop codon from the intervening region between the two genes, and the original methionine from luxB without disrupting the reading frame. Fusion is successfully expressed in S. cerevisiae and D. melanogaster . The luxAB gene sequence fused using the same method is generated using genes from X. luminescens .

To eliminate the need for the addition of exogenous substrates, the cells themselves must supply a substrate suitable for luciferase. In bacterial systems, the substrate is produced by fatty acid reductase complexes encoded by the luxCDE gene. Such enzyme complexes reduce short chain fatty acids to the corresponding aldehydes. Luciferase then oxidizes the aldehyde to the corresponding fatty acid. Preferred fatty acids for this reaction are myristic acid, which is present in eukaryotes organisms (Rudnick et al., 1993). Myristic acid is generally involved in myristoylation of the amino terminus associated with membrane adhesion (Borgese et al., 1996, Brand et al., 1996). Thus, in order to eliminate the need for exogenous supply of luciferase substrates, the biosensor also preferably comprises a nucleic acid sequence encoding three luxC, luxD and luxE-encoded subunits. As in the case of the luxAluxB gene fusion, the luxC, luxD and luxE genes were fused to produce a single luxCDE gene fusion encoding the three subunits of the enzyme complex. Methods of generating such gene fusions are described below.

A. Fusion of the luxAB and luxCDE Genes

The luxAB gene can be fused using conventional molecular biology techniques. For example, polymerase chain reactions can generally be used for this purpose. By synthesizing a 5'-primer whose sequence is initiated by ATG for the start codon for the luxA gene placed side by side with the 3'-primer end, where the codon immediately precedes the ATT stop codon. These primers can then be used for amplification reactions and gel purified products. The luxB gene can also be amplified as above using primers that remove the ATG initiating methionine codons but preserve the reading frame. The PCR ™ reaction uses a thermostable polymerase, such as Pfu ™ polymerase from Stratagene (La Jolla, Calif.), Which has no terminal deoxytransferase activity and thus produces a blunt end. The PCR ™ product obtained is blunt-end linked, and this linkage is then applied by PCR using 5'-primer from luxA and 3'-primer from luxB and Taq polymerase to PCR ™. Promotes cloning (Invitrogen, San Diego, Calif.). Only linkages in which the fragments are correctly oriented are amplified. Next, the luxAB amplicon is gel purified and the TA is cloned into a suitable vector (eg PCRII ™ vector) and transformed into E. coli using standard manufacturer protocols.

Transformants are screened for light production by the addition of n-decanal, which, when oxidized by luciferase, produces bioluminescence. Only colonies that emit light are selected because they exist in a suitable orientation for further genetic manipulation. luxCDE fusions are generated using the same method as above, only transformants are selected by miniprep and then subjected to restriction lysis analysis to determine orientation. Plasmids are amplified in E. coli, recovered and purified twice with CsCl gradient.

B. Expression of luxAB and luxCDE in HeLa Cells

To determine the relative activity of the fused bacterial luciferase component, the cloned fragment containing luxAB is cloned into a suitable mammalian expression vector (eg pcDNA 3.1) and the luxCDE-containing fragment is converted to a suitable mammalian expression vector (eg pcDNA). / Zeo 3.1) (Invitrogen, Faraday, Calif.). Both of these vectors constitutively express the inserted gene. HeLa cells are then transfected with luxAB or luxAB and luxCDE and selected using a suitable antibiotic according to the manufacturer's protocol (Promega, Madison, Wisconsin). Cells receiving luxAB fusion are exposed to n-decane and bioluminescence is examined. Next, these cells co-transfected with luxCDE are examined for bioluminescence to confirm the relative expression of luxCDE fusion. This makes it possible to compare the addition of exogenous aldehydes versus bioluminescence through aldehydes produced internally.

An alternative method of enhancing bioluminescent expression involves the generation of a vector containing three copies of the eukaryotic expression group contained in pcDNA 3.1 (Stratagene, La Jolla, Calif.). This allows for the expression of individual components of luxCDE, since it has already been shown that fused luciferases are expressed in eukaryotic cell lines (Almashanu et al., 1990).

Example 2

Construction of Glucose Bioluminescent Biosensors

Firefly luciferase was used to test the regulation of L-pyruvate kinase promoter activity in single living β-cell islets (Kennedy et al., 1997). The glucose response element indicated by the L4 box was determined to be present in the proximity promoter. The 200 bp fragment containing this region was cloned in front of firefly luciferase (luc) in plasmid pGL3Basic to generate a glucose reporter plasmid designated p.LPK.Luc _FF . The result detects single cells exposed to 16 mM glucose but not exposed to 3 mM glucose. However, these cells must be perfused with Beetle luciferin to render it unacceptable to direct biosensors. Thus, a bioluminescent sensor for glucose is constructed by replacing firefly luciferase in p.LPK.Luc _FF with the fused luxAB gene described below.

Example 3

Bioluminescent Reporter Construction and Transfection of Rat β-Islet Cells

Bioluminescent reporter plasmids are constructed by removing the luc gene encoding firefly luciferase from p.LPK.Luc _FF and replacing it with the fused luxAB gene. This is done by separating the luc gene from p.LPK.Luc _FF and cloning in the luxAB gene. The resulting plasmid is amplified in E. coli, plasmid DNA is extracted and purified twice with CsCl gradient.

Islet cells are prepared as described above (German et al., 1990) and transfected by electroporation with plasmids containing bioluminescent reporter constructs and essentially expressed luxCDE constructs. . This structure allows cells to maintain a pool of aldehyde substrates available for the reporter gene (luxAB). Cells are selected for light production in a glucose concentration range of 3 mM to 30 mM. The transfected cells are washed, concentrated, placed in slightly dense cells in the microwells and then attached to the integrated circuit. Different concentrations of glucose and assay medium (Kennedy et al., 1997) are added to the cells to examine the sensitivity and response time of glucose BBIC.

Example 4

Preparation of Bioluminescent Reporter Constructs

The use of reporter gene technology is widespread in gene regulation research in both eukaryotes and prokaryotic systems. As cell lines are studied, a number of genes are used. However, there is a need for the use of reporter genes that emit light using BBIC technology. Therefore, reporter genes encoding bioluminescence are used. All reporters previously generated using other reporter genes, eg, genes encoding β-galactosidase (lacZ), can be prepared using standard molecular techniques and reporter genes (modified lux system) used herein. Can be converted to bioluminescent transformations. Thus, existing reporter cell lines for testing gene expression in mammalian cell lines may be suitable for use as bioreporters when converted to lux reporters. Implantable systems simply contain suitable reporter cell lines. Table 1 shows a list of examples of eukaryotic reporter cell lines that can be used in implantable biosensors.

TABLE 1

Reporter Gene Application Reference

fusion

Alcohols that increase the concentration of ADH4-LUC alcohols Edenberg et al., 1999

To monitor the expression of dehydrogenase

Boundy et al., 1998 exposed to TH-lacZ chronic cocaine or morpholine

Shows increased gene expression in mice

Estrogens on the effects on estrogen response factors Balaguer et al., 1999

Detects Estrogens and Heterologous Estrogens by Regulated-LUC

Boonyaratanakomkit by Upregulation of IGFBP-5-LUC Reporter Constructs

Detect the presence of progesterone et al., 1999

Campbell et al., 1996 of CYPIA-lacZ cytochrome P450 (potent carcinogen)

Detect compounds that cause upregulation

Example 5

Construction and Transplantation of Glucose Biosensors and Insulin Delivery Pumps

In one aspect of the invention, one pair of bioluminescent reporters can be used which are present in series and which specifically respond to variations in glucose concentration. One bioreporter uses the luxAB and luxCDE genes from X. luminescens incorporated into the plasmid-based system designated p.LPK.Luc _FF , which contains eukaryotic luc genes that can respond to glucose concentrations ( Increased bioluminescence corresponds to increased glucose concentration). In addition, the second bioreporter uses a plasmid construct containing a promoter for the phosphoenolpyruvate carboxylase gene (PEPCK) that responds to glucose concentration, with the increased bioluminescence corresponding to reduced glucose levels. . The incorporation of the luxAB and luxCDE genes into each construct allows bioluminescence measurements to occur using variations in glucose concentration in real time, thus denying the requirement for cell destruction and substrate addition.

In this aspect, the integrated circuit includes a separate photodetector unit for each bioreporter (FIGS. 9A, 9B and 9C). The bioluminescent response from each construct can be monitored independently, allowing the signal processing circuit to distinguish between the bioreporter's response to increased glucose concentration and the second bioreporter's response to reduced glucose concentration. The signal processing circuit processes the signal from the photodetector, converts it to digital form, and relays the information to the implanted insulin pump (FIG. 10). The serial set of bioreporters allows for more accurate signals as well as repeatability in the detector. In addition, due to the frequent-fatal consequences of hypoglycemia, this serial system allows for more careful monitoring and warning of the onset of hypoglycemia.

Cells used in the serial bioreporter system can be attached to each photodetector either directly by attachment or encapsulated in a hydrogel (Prevost et al., 1997). It may be necessary to separate bioreporters using semipermeable membranes to allow transmembrane transport of small molecules (eg glucose and insulin) and to prohibit the influx of immune effector cells and antibodies (Monaco et al., 1993, Suzuki et. al., 1998). However, small molecules (eg cytokines) can still enter the selective membrane and interfere with the bioluminescent reporter cell line. This approach is widely used by those skilled in the art.

If applicable, bioluminescent reporter cell lines can be constructed from cells taken directly from a patient receiving the transplant. This method is particularly desirable in the case of long term transplantation (eg implantable insulin delivery). Cells can be obtained from a patient, genetically generated for appropriate surveillance functions, grown in cell media, assessed and then preserved for long term storage. The use of cell lines generated from the patient's own cells is particularly desirable because it reduces the chance of host rejection and the generation of an immune response to the implanted device. Preferably, hepatocytes (if possible, hepatocytes which do not die) are used if necessary, which can be maintained and grown in a suitable culture medium. Such pluripotent, omnipotent, or other non-dying cell lines offer particular advantages in the formation of suitable long-term implantable devices.

Prior to implantation, the biosensor can be assayed and injected into the chamber containing the cells using different concentrations of glucose delivered from the auxiliary pump and the insulin delivery pump (FIG. 10). This allows the determination of suitable parameters that allow delivery of an appropriate dose of insulin. Once the parameters are set, the pump can evaluate for insulin delivery. Systemically, the glucose biosensor is retested in the patient using the glucose standard contained in the delivery pump.

In the case of drug delivery systems, the glucose biosensor can be suitably connected to the delivery pump via wiring or wireless connection. Biochips provide digital data that can be put directly into the signal processing circuit of a pump that proportionally distributes insulin. Alternatively, the digital data can be converted into analog data and used to control the pump. When wireless capability is added to the bioreporter device, remote monitoring of the sensor is possible. For example, in this architecture, the patient can place the wireless transmitter / receiver outside the body close to the implanted device to communicate data remotely from the implanted device. In certain applications, the wireless transmitter / receiver may connect to a computer that is intended to send data over a network (eg, local area network, wide area network, or even the Internet) to a remote state. This wireless application allows for remote monitoring and maintenance of the patient. There are currently many pumps for markers, which are candidates for connecting with biosensors. In one aspect of the invention, a Medtronic Synchronized fusion system can be used because it is widely used for drug delivery and uses a portable computer that allows programming of the pump from outside the body (www.asri.edu/neuro /brochure/pain6.htm). The pump can also be refilled through the skin across the self-sealing septum. The pump is 1 inch thick, 3 inches in diameter, and weighs about 6 ounces. Biosensors can be integrated into existing electrical circuits first to take advantage of out-of-body programming by portable computers. The chip may use a rechargeable battery to move the delivery pump.

The biosensor / insulin pump device can be surgically implanted using local anesthesia in the abdominal cavity. The sensor and pump can be implanted into the peritoneal space of the embryo to simplify both and to prevent complications of catheter placement directly into the bloodstream. Glucose concentrations are monitored and insulin required by the patient is delivered to the peritoneum (FIG. 10).

Example 6

Bioluminescent Reporter Construction and Transfection of Rat β-Islet Cells and H4IlE Liver Cancer Cells

Modulation of the PEPCK gene is used in the construction of bioluminescent reporters to detect reduced glucose concentrations. This system is highly regulated because phosphoenolpyruvate carboxylase is a rate-limiting enzyme in grapevine life. PEPCK gene expression increases in the presence of glucocorticoids and cAMP and decreases in the presence of insulin (Sasaki et al., 1984; Short et al., 1986). Insulin effects are prominent in both rat liver and H4IE liver cells, with the addition of glucocorticoids and cAMP. The promoter region of PEPCK is cloned in front of the fused luxAB. The construct obtained then produces increased bioluminescence in the presence of low glucose concentrations.

Bioluminescent reporter plasmids for detecting increased glucose concentrations can be constructed by removing the luc gene encoding firefly luciferase from p.LPK.Luc _FF and replacing it with the fused luxAB gene. This is done by separating the luc gene from p.LPK.Luc _FF and cloning in the luxAB gene. Bioluminescent reporter plasmids for detecting low glucose concentrations were substituted for the luxAB gene by replacing the CAT gene in the previously constructed PEPCK promoter chloramphenicol transferase (CAT) fusion (Petersen et al., 1988; Quinn et al., 1988). To construct. The resulting plasmid is amplified in E. coli, plasmid DNA is extracted and purified twice with CsCl gradient.

Islet cells and liver cancer cells are prepared as previously described (German et al., 1990; Petersen et al., 1988) and contain the constantly expressed luxCDE gene constructed in bioluminescent reporter constructs and targets. The plasmid may be co-transfected. This structure maintains a pool of aldehyde substrates available for the reporter gene (luxAB). Cells are selected for light production in the glucose concentration range of 3 mM to 30 mM. The transfected cells are washed, concentrated, placed in microwells in a slightly dense chamber and then attached to the integrated circuit. Different concentrations of glucose and assay medium (Kennedy et al., 1997) are added to the cells to examine the sensitivity and response time of glucose BBIC. After initial characterization, bioluminescent glucose reporters can also be tested in flow cells. Cells are placed in inclusion medium above the integrated circuit, and then media containing glucose at different concentrations (3-30 mM) are perfused across the cells to examine the dynamic response.

Example 7

BBIC in the Diagnosis and Detection of Cancer

Colon cancer is the second leading cause of cancer deaths after lung cancer in the United States, and the incidence increases with age, with 97% of colon cancers occurring in people over 40 (Coppola and Karl, 1998). Most cases of colon cancer are sporadic, but in 15% of patients there is a potent family history of similar tumors in the highest relative relationship (Coppola and Karl, 1998). These familial cancers (eg hereditary non-polyposis colon cancer (HNPCC)) and familial adenocarcinoma polyposis (FAP) are allegedly derived from autosomal predominant genetic genetic mutations in the tumor suppressor gene, with the spectrum of the lesion proliferating -Is dysplastic-adeno-carcinoma (Coppola and Karl, 1998). Since many early molecular lesions are known for inherited colon cancer, they represent a useful model for the development of new biosensor methods for early clinical detection. Biosensors are hybrid devices that combine biological components and automated measuring transducers.

This example describes the adaptation of an implantable biosensor device that allows for early detection of cancer and means for monitoring the alleviation and recurrence of cancer. Since the small biosensors of the present invention are small enough to be implantable and can be combined with the resulting reporter system to generate light without the need for cell lysis or additional substrates, tools currently available for the early diagnosis of colon cancer in the form of implantable devices are currently available. It is possible for the first time.

As described above for glucose and other metabolite biosensors, the inducible reporter system used is based on the luxAB and luxCDE genes from X. luminescens placed in eukaryotic reporter cells, so that the expression of a specific gene or product thereof is BBIC. Detection by expression of bioluminescence by the device. Eukaryotic reporter cells are treated with mitomycin C and thus cannot be isolated, but can still respond to metabolism and produce quantitative bioluminescent signals.

Colon cancer is the second leading cause of cancer death in the United States, with over 50% of the population showing colorectal tumors at age 70 (Kinzler and Vogelstein, 1996). Most cases of colorectal cancer are sporadic, but 15% are hereditary cancer syndrome, familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC) (Kinzler and Vogelstein, 1996). Familial adenocarcinoma polyposis is a syndrome characterized by the development of hundreds to thousands of adenomas or polyps in the colon and rectum, with only a few of these invasive cancers (Kinzler and Vogelstein, 1996). The loss of function of both alleles of the adenomatous polyposis coll (APC) tumor suppressor gene gives human predisposition to 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 non-polyposis colorectal cancer, there is a significant minor pituitary instability secondary to mutations in the DNA misfit repair genes (eg, hMSH2 and hMSH1); Single advanced tumors develop in adolescence and are generally limited to the right colon (Coppola and Karl, 1996; Smyrk, 1994). Cells with mutations in the gene APC are generally not euploid from loss of the entire segment of the chromosome, whereas cells with mutations in hMSH2 and hMSH1 are euploid (Lengauer et al., 1998).

Molecular events leading to the expression of colon tumorigenesis are well understood (Kinzler and Vogelstein, 1996). Persons with complete loss of APC show lesions in the colon called dysplastic abnormalities that progress to early adenoma (Kinzler and Vogelstein, 1996). As in K-Ras or p53, other mutations begin to accumulate and the tumor progresses to terminal adenomas, carcinomas and metastatic carcinomas (Kinzler and Vogelstein, 1996). Similar progress is shown in HNPCC as well. Since the sequence of genetic events is very well understood for these types of cancers, they represent an excellent model for the development of sensitive and specific diagnostic tests that can be used to detect one or more altered cells in vitro.

The APC gene, localized in the lesion attachment complex to the end of the microtubule, encodes a cellular protein (Kinzler and Vogelstein, 1996). As cells enter through the bovine, the expression of APC increases until finally differentiated and localized colonic epithelial cells undergo apoptosis (Kinzler and Vogelstein, 1996). Catherine is a dural protein local to most epithelial cells with lesion attachment spots (Aplin et al., 1998). The carboxy terminus of each katherine interacts with cytoplasmic structural proteins known as catenin (Aplin et al., 1998). There are three kinds of catenin: β-catenin binds to the cytoplasmic region of catenin; -Catenin binds via -actinine to β-catenin and actin cytoskeleton; Catenin acts on specific cell types instead of β-catenin (Aplin et al., 1998). In addition, β-catenin is part of a signal transduction pathway including the secreted glycoprotein Wnt and glycogen synthase kinase 3 (GSK3) (Aplin et al., 1998). APC interacts with a number of components of the Wnt-β-catenin-GSK3 pathway, including β- and γ-catenin, GSK3, and tubulin (Aplin et al., 1998). Most of the mutations in colorectal cancer are present in the carboxy terminal region of APC so that they no longer bind β-catenin (Aplin et al., 1998). In fact, catenin is below APC and is important for acting as a tumor suppressor gene (Aplin et al., 1998). When the Wnt pathway is inactivated, GSK3 phosphorylates the N-terminus of β-catenin and targets degradation by the ubiquitin pathway (Munemitsu et al., 1996). When β-catenin accumulates, it activates gene transcription via the transcription factor Lef-1 / TCF (Morin et al., 1997). APC acts on GSK3 to inhibit catenin-mediated transcriptional activity (Kinzler and Vogelstein, 1996).

In hereditary non-polyposis colon cancer, subploid instability is the result of mutation in one or more DNA-incompatible repair genes (Jiricny 1998; Nicholaides et al., 1994). More than 90% of HNPCC tumors show minor pituitary instability (Karran 1996; Smyrk 1994). One possible label for small pituitary instability in colorectal tumors is the inactivation of type II receptors for TGF-β (Markowitz et al., 1995). Loss of function of βRII is associated with loss of growth regulation and tumor progression of colorectal adenoma in HNPCC (Wang et al., 1995). Other signaling components of the TGF-β pathway involved in colorectal tumorigenesis include mutations in Smad 3 and Smad 4, both of which cause colorectal adenocarcinoma in mice (Zu et al., 1998; Takaku et al. , 1998). Malfunction of βRII is a useful marker for early lesions in HNPCC (Markowitz et al., 1995).

Since mutations in APCs are the most common mutations in colorectal cancer, reporter constructs for T cell transcription factor (Tcf) are designed to select multiple colon cancer cell lines for activation of transcriptional activity. Mutations in APC or β-catenin activate Tcf-reactive transcription through the accumulation of insufficiently phosphorylated cytoplasmic β-catenin (Morin et al., 1997), and activation detection of reporter constructs results in mutations in any of these genes. Useful as a marker for Vector pDISPLAY (Invitrogen) allows expression of a promoter for Tcf on the surface of bioreporter cells; This construct consists of a direct set of Tcf promoters: one top of the gene for luxAB, the other top of luxCDE. In the presence of excess β-catenin the promoter construct stimulates reporter activity and bioluminescence is produced.

Once HepG2 and HeLa cells are transfected with pcDNA3, which encodes the luxAB gene, the cells attach to the biosensor chip. It is necessary to ensure that these cells are not isolated, so after transfection and selection, the cells are irradiated with 6,000 rad γ-radiation from a ⁶⁰ Co source (UT College of Veterinary Medicine). In certain embodiments it may be necessary to attach the cells to the biochip prior to irradiation so that efficient attachment occurs. An alternative to this is to treat the cells with mitomycin C to prevent further mitosis. Biochips can be coated with Matrigel, the lowest membrane material that promotes adhesion of epithelial cells. In an alternative approach, cells are suspended in Matrigel and allow it to form a gel on the surface of the biochip. Next, the cells are fixed to the lowermost membrane material and do not apply to frictional movement. Optionally, the surface of the chip can be altered by adding pure filler (e.g. poly-L-lysine) and coating the surface with surgical tissue glue, or by adding other surface modifications that allow the biopolymer to adhere closely to the surface. Can be. Since mutations in APC are the most common mutations in colorectal cancer, reporter constructs for T cell transcription factor (Tcf) can be designed to select multiple colon cancer cell lines for activation of transcriptional activity.

The present invention also provides a biosensor that can be used for endoscopic screening of colonic mucosa to detect the presence of mutated cells before the initiation of an overall morphology change. It may be necessary to attempt the detection of one or more abnormalities to the extent of sensitivity required to detect small lesions of malignant transformation at a particular point in time. For example, having mutations in many colon tumors, particularly APCs, overexpresses cyclooxygenase-2 (COX-2) and secretes large amounts of prostaglandins (Kutchera et al., 1996; Scheng et al. , 1997; Coffey et al., 1997; Kinzler and Vogelstein, 1996). Cyclooxygenase-2 is an early response gene that is not essentially expressed, but is converted by growth factors and tumor promoters in colonic epithelial cells (Kutchera et al., 1996; Schen et al., 1997; Coffey et al., 1997). Biotech reporter cells may be bioluminescent in the presence of increased levels of prostaglandins in the intestinal lumen. Prostaglandins pass freely through the cell membrane and can enter the cytoplasm of reporter cells to activate reporter constructs. The production of reporter cells that detect increased levels of prostaglandin through the use of a cyclooxygenase-2 promoter fused to the luxAB gene may also be beneficial for early detection of colon cancer. Since levels of prostaglandins can be elevated in tumors as well as inflammation, this approach lacks suitable specificity for diagnosing cancer. However, it is useful in determining whether a patient benefits from treatment with a specific cyclooxygenase inhibitor.

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All compositions, methods, instruments, devices and systems described and claimed herein can be made and performed without undue experimentation in light of the techniques of the present invention. The methods, instruments, devices and systems of the present invention are described by the preferred embodiments, but to those skilled in the art, the methods, instruments, devices and systems, and in the order of or in the order of the methods described herein, the present invention It is clear that modifications can be applied without departing from the concept, spirit and scope of the In particular, it is evident that certain chemically and physiologically relevant agents may be substituted for the agents described herein, while the same or similar results are obtained. All similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope and concept of the invention as defined by the appended claims. Accordingly, the exclusive right to patent is as set forth in the claims below.

Claims (45)

(a) a bioreporter on a substrate on an integrated circuit, capable of metabolizing the analyte when in contact with the analyte and emitting light as a result of this metabolism; And (b) a sensor disposed proximate to the integrated circuit that generates an electrical signal in response to receiving the emitted light, Implantable monolithic bioelectronic device for detecting an analyte in vivo in an animal contained in a biocompatible container implanted in vivo in the animal. The implantable monolithic bioelectronic device of claim 1, wherein the biocompatible container comprises a polymer matrix. The implantable monolithic bioelectronic device of claim 2, wherein the polymer matrix comprises polyvinyl alcohol, poly-L-lysine or alginate. The implantable monolithic bioelectronic device of claim 2, wherein the polymer matrix further comprises a microporous, mesh-reinforced or filter-supported hydrogel. 2. The implantable monolithic bioelectronic device of claim 1, wherein said integrated circuit comprises an optical transducer. 6. The implantable monolithic bioelectronic device of claim 5 further comprising a transparent biocompatible bio-resistant separator operably disposed between the light transducer and the bio reporter. The implantable monolithic bioelectronic device of claim 1, wherein the bioreporter comprises a plurality of eukaryotic or prokaryotic cells that produce a bioluminescent reporter polypeptide in response to the presence of an analyte. 8. The implantable monolithic bioelectronic device of claim 7, wherein said plurality of prokaryotic cells comprise bacteria. 8. The implantable monolithic bioelectronic device according to claim 7, wherein said plurality of eukaryotic cells comprise mammalian cells. 10. The implantable monolithic bioelectronic device of claim 9, wherein said plurality of eukaryotic cells comprise β-islet cells, intact stem cells, or hepatocytes. 11. The implantable monolithic bioelectronic device of claim 10, wherein said plurality of eukaryotic cells comprise human intact recombinant stem cells. 8. The transplant of claim 7, wherein said plurality of cells comprises a nucleic acid fragment encoding a luciferase polypeptide or green fluorescent protein produced by said cells in response to the presence of said analyte. Possible monolithic bioelectronic devices. 13. The implantable monolithic bioelectronic device of claim 12, wherein said nucleic acid fragment encodes Aqueorea victoria or Renilla reniformis green fluorescent protein. 13. The implantable monolithic bioelectronic device of claim 12, wherein said nucleic acid fragment encodes a humanized green fluorescent protein. 13. The implantable monolithic bioelectronic device of claim 12, wherein said nucleic acid fragment encodes a bacterial Lux polypeptide. The implantable monolithic bioelectronic device of claim 15, wherein the nucleic acid fragment is a bacterial LuxA, LuxB, LuxC, LuxD or LuxE polypeptide, or encodes a LuxAB or LuxCDE fused polypeptide. The implantable monolithic bioelectronic of claim 16, wherein the nucleic acid fragment is Vibrio fischerii or Xenorhabdus luminescens LuxA, LuxB, LuxC, LuxD or LuxE polypeptide, or encodes a LuxAB or LuxCDE fused polypeptide. Device. 18. The implantable monolithic bioelectronic device of claim 17, wherein the nucleic acid fragment is a Xenorhabdus luminescens LuxA, LuxB, LuxC, LuxD or LuxE polypeptide, or encodes a LuxAB or LuxCDE fused polypeptide. 19. The implantable monolithic bioelectronic device of claim 18, wherein said polypeptide is encoded by a sequence comprising at least 25 contiguous nucleotides derived from SEQ ID NO: 1. 20. The implantable monolithic bioelectronic device of claim 19, wherein said polypeptide is encoded by a sequence comprising at least 30 consecutive nucleotides derived from SEQ ID NO: 1. The implantable monolithic bioelectronic device of claim 20, wherein said polypeptide is encoded by a sequence comprising at least 35 contiguous nucleotides derived from SEQ ID NO: 1. 17. The implantable monolithic bioelectronic device of claim 16, wherein the expression of said nucleic acid fragment is regulated by a nucleic acid sequence comprising a cis-functional element that is reactive to the presence of said analyte. 23. The method of claim 22, wherein said cis-acting response element comprises: S14 gene sequence, hepatic L-pyruvate kinase gene sequence, hepatic 6-phosphofructo-2-kinase gene sequence, β islet insulin gene sequence, glomerular epilepsy An implantable monolithic bioelectronic device, characterized in that the nucleotide sequence is selected from the group consisting of the transforming growth factor-β gene sequence and the acetyl-coenzyme-A carboxylase gene sequence. 24. The implantable monolithic bioelectronic of claim 23, wherein said cis-functional response element comprises a continuous nucleotide sequence derived from a β-islet insulin gene sequence or a liver L-pyruvate kinase gene sequence. Device. 13. The implantable monolithic bioelectronic device of claim 12, wherein the expression of said nucleic acid sequence is regulated by a promoter sequence derived from an L-pyruvate kinase coding gene. The implantable monolithic bioelectronic device of claim 1, wherein said analyte is glucose, glucagon or insulin. 8. The implantable monolithic bioelectronic device of claim 7, further comprising a source of nutrients capable of maintaining said cells. The implantable monolithic bioelectronic device of claim 1, further comprising a wireless transmitter. 10. The implantable monolithic bioelectronic device of claim 1, further comprising an antenna. The implantable monolithic bioelectronic device of claim 1, further comprising an implantable, drug delivery pump that is controlled by the device and capable of delivering the drug to a living body of the animal. The implantable monolithic bioelectronic device of claim 1, wherein the biocompatible container further comprises a membrane that is permeable to the analyte but impermeable to the bioreporter. The implantable monolithic bioelectronic device of claim 1, wherein the bioreporter expresses the luminescent polypeptide after metabolism of the analyte by the bioreporter. 3. The implantable monolithic bioelectronic device of claim 2, wherein the biocompatible container comprises silicon nitride or silicon oxide. The implantable monolithic bioelectronic device of claim 1, wherein the integrated circuit is a complementary metal oxide semiconductor (CMOS) integrated circuit. 6. The implantable monolithic bioelectronic device of claim 5 wherein said photoconverter comprises a photodiode. 2. The implantable monolithic bioelectronic device of claim 1, wherein said integrated circuit further comprises a photodiode and a current-frequency converter. 2. The implantable monolithic bioelectronic device of claim 1, wherein said integrated circuit further comprises a current-frequency converter and a digital counter. The implantable monolithic bioelectronic device of claim 1, further comprising a transmitter. 39. The implantable monolithic bioelectronic device of claim 38, wherein said transmitter is capable of transmitting digital data. An implantable controlled drug delivery system comprising an apparatus of claim 1 and an implantable drug delivery pump operably controlled by the device of claim 1. A method of delivering a drug to a patient in a controlled manner, the method comprising implanting the controlled drug delivery system of claim 40 in the patient's body. Measuring the amount of drug required by the patient, including implanting the device of claim 1 in the patient's body and measuring the amount of drug needed by the patient based on the results calculated from the device of claim 1 Way. A kit for detecting an analyte, comprising instructions for using the device of claim 1 and the device of claim 1. 44. The kit of claim 43, further comprising a standardized reference solution. Using the device of claim 1 or the kit of claim 43 to monitor glucose levels in a patient's blood or interstitial fluid and administering to the patient an effective amount of an insulin composition sufficient to control blood glucose levels, How to adjust.