Patent Application
20130172705
The present invention is broadly concerned with the crafting and manufacturability of an implantable enzymatic-based sensor characterized by a small size, optimum geometry, linearity of response over the concentration range of interest, extended shelf-life, selectivity for the analyte in question, and the ability to exclude bioactive interferents. More particularly, it is preferably concerned with a general approach to optimize the performance of the biorecognition elements required to produce biosensors of the type designed to provide, and in conjunction with a suitable signal processing unit, a current which is proportional to the concentration of the analyte of interest. The biosensors described herein may be implanted in vivo, including intra-cerebral, sub-cutaneous, intra-muscular, inter-peritoneal, oral, serum, and vascular implantation, the majority of which may act as a surrogate for systemic monitoring and used to monitor analytes of interest in real-time. Multiple biosensors can be joined together to allow for the simultaneous recording of multiple analytes of interest. In addition to the in vivo applications, sensors of the design described herein may also find use in medical monitoring, industrial processes, fermentation, environmental monitoring, and waste water stream monitoring. The present invention offers co-factor enhancement of the biorecognition element, providing access to a range of biorecognition elements heretofore difficult to incorporate into a manufacturing process for the large-scale production of biosensors.
The present invention broadly provides a method of forming a sensor, where the method comprises incubating an enzyme in the presence of a co-factor for the enzyme, and immobilizing the enzyme in its active form.
In another embodiment, a sensing element is provided. The sensing element comprises a support having a surface. The element further comprises a layer on the surface, where the layer comprises an enzyme and a co-factor for the enzyme in a matrix. The enzyme is predominantly in its active form and the matrix retains the enzyme in the active form.
In a further embodiment, the invention is concerned with an amperometric biosensor comprising a working electrode and a reference electrode. The working electrode comprises a sensing element comprising a support having a surface, and a layer on the surface. The layer comprises an enzyme and a co-factor for the enzyme in a matrix, with the enzyme being predominantly in its active form and the matrix retaining the enzyme in the active form.
In yet a further embodiment, an assembly for oral, intra-cerebral, intramuscular, intravascular, vascular, inter-peritoneal, or sub-cutaneous placement and anchoring is provided. The assembly comprises an amperometric biosensor and a device selected from the group consisting of a cannula, a cannula headpiece, a patch, an implant, and a trocar, with the biosensor being attached to the device. The amperometric biosensor comprises:
This invention provides a general approach to yield properly conditioned biorecognition elements that provide the foundation for the construction of biosensors. By definition, biosensors require a biorecognition element to provide activity, specificity, and selectivity to the analyte(s) of interest. The conditioning of a biorecognition element as a discreet step in the fabrication of a properly functioning biosensor is of critical importance and has not been widely recognized or generalized. The biosensors disclosed herein, including those for in vivo biological applications, enjoy a wide range of use, and may be deployed via intra-cerebral, sub-cutaneous, intra-muscular, inter-peritoneal, oral, serum, and vascular implantation, the majority of which may act as a surrogate for systemic monitoring. Applications for biosensors are broad and include monitoring of disease states and infectious diseases (e.g., West Nile Virus and SARS), pathogens e.g., listeria and salmonella), various E. coli contaminations, analytes for drug control and interdiction (e.g., alcohol, cannabis and THC), physiological states (e.g., pregnancy), cholesterol levels, and heart health (e.g. cardiac biomarkers, coagulation PT, coagulation ACT). Biosensors may provide timely data needed to treat trauma and traumatic brain injuries and battlefield insults. A single biosensor may be fabricated for the sensing of multiple analytes of interest. Multiple biosensors can be joined together to allow for the simultaneous recording of multiple analytes of interest. In addition to the in vivo applications, sensors of the design described herein may also find use in medical monitoring, industrial processes, fermentation, environmental monitoring, and waste water stream monitoring. These biosensors are possible because of the conditioning of the biorecognition element prior to its introduction into the sensing matrix.
All enzymes are identified and classified according to the Enzyme Commission number (EC number) classification system (Webb, Edwin C., 1992, Enzyme nomenclature 1992: Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the nomenclature and classification of enzymes. San Diego: Published for the International Union of Biochemistry and Molecular Biology by Academic Press). The EC system is a numerical classification scheme for enzymes, based on the chemical reaction that is catalyzed. As a system of enzyme nomenclature, every EC number is associated with a recommended name for each enzyme.
Some enzymes do not need any additional components to show full activity. If an enzyme requires one or more non-protein molecules to be bound to the enzyme for activity, these molecules are collectively referred to as co-factors (de Bolster, M. W. G., 1997, “Glossary of Terms Used in Bioinorganic Chemistry: Cofactor”, International Union of Pure and Applied Chemistry). These non-covalently bound molecules are usually found proximal to or part of the active site of the enzyme and are involved in catalysis. For example, flavin and heme co-factors are often involved in redox reactions and the former is often found in oxidoreductases.
Co-factors can be either inorganic compounds (e.g., iron-sulfur clusters and metal ions selected from the group consisting of iron, zinc, selenium, cobalt, magnesium, molybdenum, vanadium, manganese, copper, tungsten, cadmium, and nickel) or organic compounds (e.g., heme, flavin, flavin adenine dinucleotide—FAD, flavin mononucleotide—FMN). Organic co-factors can be either prosthetic groups, which are non-covalently bound to an enzyme, or co-enzymes, which are released from the enzyme's active site during the reaction. Co-enzymes include NAHD, NADPH, and adenosine triphosphate—ATP. The term co-enzyme refers specifically to enzymes and, as such, to the functional properties of a protein. Most co-factors are not covalently attached to an enzyme. In some instances, organic prosthetic groups such as FAD and FMN can be covalently bound.
The term prosthetic group emphasizes the nature of the binding of a co-factor to a protein (tight or covalent) and, thus, refers to a structural property. Different sources give slightly different definitions of co-enzymes, co-factors, and prosthetic groups. Some consider non-covalently bound organic molecules as prosthetic groups and not as co-enzymes, while others define all non-protein organic molecules needed for enzyme activity as co-enzymes and classify those that are non-covalently bound as co-enzyme prosthetic groups. It should be noted that these terms are often used loosely. As used here, the terms “co-factor,” “prosthetic group,” and “co-enzyme prosthetic group” refer to a molecule that is not a protein, that is needed for enzyme activity, and that is non-covalently bound to the enzyme of interest.
The co-factors are not limited to those only found in human iso-forms of a particular enzyme, but also embrace all iso-forms of said enzyme found in all organisms. It is well accepted and understood that different iso-forms of the same enzyme from different organisms will possess different amino acid sequences and may rely on different co-factors for activity. For example, the human iso-form of diamine oxidase (EC 1.4.3.22), sometimes referred to as histamine oxidase, does not require a co-factor for activity. Instead, human diamine oxidase relies on a post-translational hydroxylation of an active site tyrosine residue to a trihydroxyphenylalanine residue and then finally to a topaquinone (TPQ) residue (McGrath, A. P. et al. Biochemistry, 2009, 48(41), 9810-9822). By contrast, the diamine oxidase found in rice seedlings from the species Oryza sativa requires FAD as a co-factor for activity (Chaudhuri, M. M.; Ghosh, B. Phytochemistry, 1984, 23, 241-243).
Enzymes that require a co-factor but do not have one bound are called apo-enzymes or apo-proteins. An apo-enzyme together with its co-factor(s) is called a holo-enzyme or holo-protein. By definition, the holo-enzyme is the competent, active form of the enzyme and is capable of supporting catalysis without the need for other co-factors. The term holo-enzyme can also be applied to enzymes that contain multiple protein subunits, where this collection of subunits is also in a competent active form capable of function.
An example of an enzyme that requires a co-factor for activity is lactate oxidase (Streitenberger, S. A.; et al. J. Enzyme Inhib. Med. Chem. 2003, 18, 285-288), which can use either FAD or FMN to form the holo-enzyme and promote catalysis. In the absence of either co-factor, the apo-form of the enzyme is catalytically inactive and cannot promote catalysis.
The construction of biosensors has been reviewed (Wilson, G. S.; Johnson, M. A.; Chem Rev., 2008, 108(7), 2462-81. Johnson, D. A.; Wilson, G. S.; In: Michael A C, Borland L M, editors. Electrochemical Methods for Neuroscience. Boca Raton (FL): CRC Press; 2007. Chapter 20. Wilson, G. S.; Gifford, R.; Biosens Bioelectron., 2005, 20(12), 2388-403. Matsumoto, N.; Chen, X.; Wilson G. S.; Anal Chem., 2002, 74(2), 362-7. Ward W. K.; Jansen, L. B.; Anderson, E.; Reach, G.; Klein, J. C.; Wilson, G. S.; Biosens Bioelectron., 2002, 17(3), 181-9). In general, biosensors are composed of four components: (1) a sensing electrode (the transducing element or component) typically fashioned from platinum, a platinum/iridium alloy, gold, silver, other precious metals, alloys or glasses, and where said sensing element can support an electrochemical or optical measurement; (2) an inner-membrane that provides a separation layer between the sensing electrode and the enzyme layer and which helps to enhance the selectivity of the biosensor; (3) an enzyme layer or sensing matrix that is minimally composed of one or more biorecognition element(s) and which may also have other proteins and non-protein molecules as part of its composition; and (4) an outer-membrane layer which may help regulate the flux of the analyte of interest to the other layers. For the purposes of this invention, the sensing layer (1), the inner-membrane (2) and the outer-membrane (4) can be considered generic layers in that this invention will be equally applicable to all sensing layers, all inner-membranes, and all outer-membranes capable of supporting biosensor construction. These layers do not necessarily need to be present as discreet distinct layers and may in fact be intermingled. One or more of the generic layers may be absent in the final biosensor design and fabrication. However, if an enzyme layer or sensing matrix exists as part of a biosensor, and said enzyme layer has a co-factor dependent enzyme as part of said layer, then this invention will find utility.
By definition, the term biosensor specifically refers to a sensor that employs a biorecognition element to provide analyte recognition and analyte specificity (Wilson G. S.; Ammam, M. FEBS J. 2007, 274(21), 5452-61). The biorecognition element(s) used as part of the biosensor construction are typically oxidoreductases, which are a sub-class of flavoproteins (Macheroux, P. et al. FEBS J. 2011, 278, 2625-2634). Oxidoreductases often catalyze the conversion of a substrate with the generation of electroactive hydrogen peroxide as a by-product. The amperometric measure of the hydrogen peroxide generated thus provides the means by which the presence of the substrate analyte is determined. In principle, any oxidoreductase that produces an electroactive by-product and which requires a co-factor(s) for activity can be utilized as part of this invention.
By definition, the term sensitivity used herein refers to the electrical response as a function of concentration and refers to the biosensor composite. Activity refers to a measure of the performance of an enzyme (biorecognition element), which may be co-factor driven, and is typically defined in terms of units, wherein one unit is defined as the amount of enzyme which generates 1 μmole of H2O2 per minute at 37° C. under a set of standard conditions, and where standard condition may vary from enzyme to enzyme.
Flavoproteins are proteins that contain a nucleic acid derivative of riboflavin: tFAD or FMN (Dym, O.; Eisenberg, D.; Protein Sci., 2001, 10(9), 1712-1728). Flavoproteins are involved in a wide array of biological processes, including, but by no means limited to, bioluminescence, removal of radicals contributing to oxidative stress, photosynthesis, DNA repair, and apoptosis flavoproteins (Macheroux, P. et al. FEBS J. 2011, 278, 2625-2634). The spectroscopic properties of the flavin co-factor make it a natural reporter for changes occurring within the active site; this makes flavoproteins one of the most-studied enzyme families (Massey, V.; Biochem. Soc. Trans. 2000, 28, 283-296). The first evidence for the requirement of flavin as an enzyme co-factor came in 1935 (Theorell, H.; Biochemische Zeitschrift 1935, 275, 344-346). Hugo Theorell and coworkers showed that a bright-yellow-colored yeast protein, identified previously as essential for cellular respiration, could be separated into apoprotein and a bright-yellow pigment. Neither apo-protein nor pigment alone could catalyse the oxidation of NADH, but mixing of the two restored the enzyme activity. However, replacing the isolated pigment with riboflavin did not restore enzyme activity, despite their being indistinguishable under spectroscopy. This led to the discovery that the protein studied required not riboflavin but FMN to be catalytically active.
Oxidoreductases generally require FAD or FMN as co-factors, but are not limited to just these groups. Some members of the family are known to require one or more of the following co-factors for activity: thiamine pyrophosphate (TPP), quinone, heme, copper, and other heavy metals ions selected from the group consisting of iron, zinc, selenium, cobalt, magnesium, molybdenum, vanadium, manganese, copper, tungsten, cadmium, and nickel. Some family members rely upon post-translation modification(s) of active site amino acid residues, which results in a covalently modified protein where the co-factor(s) is an integral part of the enzyme. Enzyme preparations that do not include the addition of the co-factor(s) universally result in loss of enzymatic activity, which may be restored by the addition of co-factor(s) in solution. The flavoprotein family displays a wide range of activities and stabilities, with loss of activity directly linked to loss of co-factor(s). As a general rule, the co-factors are not covalently bound, although there are exceptions such as glucose oxidase (EC 1.1.3.4). Temperature plays an important role in the loss of co-factor, as higher temperatures lead to faster loss of co-factor. Considerable effort has been described in developing temperature stable mutants of flavoproteins, most especially for those flavoproteins with direct applications in human analyte monitoring (e.g., glucose, glutamate, alcohol, lactate, histamine, etc). While modern molecular biology techniques allow for the directed evolution of temperature stable flavoprotein mutants, such mutants can often affect the fidelity of the catalytic process and lead to a protein that is less active than the wild-type.
Enzyme preparations, produced via recombinant methods or obtained by isolation from natural sources, may contain high proportions of apo-protein. The functional stability of flavoproteins is known to directly relate to an enzyme's ability to retain co-factor(s). Restoration of the activity by the addition of co-factor(s) will likely reflect conditions that help drive the equilibrium of the system to produce the fully activated form of the enzyme (based on Le Chatelier's principle). Factors that can positively contribute to the restoration of enzyme activity include the co-factor to enzyme ratio, incubation temperature of the co-factor/enzyme mixture, and the incubation time of said mixture. The ratio of co-factor(s) to enzyme can range from about 1:1 to about 100,000,000:1 or larger, preferably from about 1:1 to about 100,000:1, and more preferably from about 1:1 to about 10,000:1. The incubation temperature can range from about −10° C. to about 100° C., preferably from about −10° C. to about 50° C., and more preferably from about -10 ° C. to about 25° C. The incubation time can range from about zero seconds (immediate use) to about 72 hours, preferably from about 1 minute to about 12 hours, and more preferably from about 1 minute to about 3 hours. A good set of starting conditions is to provide a 10,000:1 ratio of co-factor(s) to enzyme and to allow the mixture to incubate at 0° C. for 90 minutes. The optimal ratio of co-factor to enzyme, the length of incubation and the temperature of incubation is enzyme dependent and may be determined and optimized for each enzyme to be employed as biorecognition elements. Further, if multiple co-factors are required for activity, the order of addition of the co-factors may be important.
Enzyme-based biorecognition elements may benefit from the incubation with co-factor(s) prior to immobilization in a solid matrix. Incubation of the biorecognition element with known co-factor(s) enhances the activity of the enzyme and coverts all apo-protein to active, fully functional holo-protein (the limiting case). This fulfills the catalytic potential for activity of the mass of the enzyme in the sensing matrix used in the biosensor construction. The importance of incubation of the biorecognition element with co-factors prior to immobilization has not been generally recognized and is key to the manufacturability of biosensors, most especially those biosensors whose biorecognition element is co-factor driven and whose overall stability as a function of time and temperature is not robust. The oxidoreductase enzymes in Table 1 benefit from conditioning prior to introduction of the enzyme, as the biorecognition element, into the enzyme layer of each biosensor. The following oxidoreductases dramatically benefit from incubation: Lactate oxidase (EC 1.1.3.15), D-amino acid oxidase (EC 1.4.3.3), (S)-6-Hydroxynicotine oxidase (EC 1.5.3.5), (R)-6-Hydroxynicotine oxidase (EC 1.5.3.6), Alcohol oxidase (EC 1.1.3.13), Pyruvate oxidase(EC 1.2.3.3), Glucose oxidase (EC 1.1.3.4), Glutamate oxidase (EC 1.4.3.11), Acyl coenzyme A oxidase (EC 1.3.3.6), Choline oxidase (EC 1.1.3.17), Glutathione Sulfhydryl oxidase (EC 1.8.3.3), Glycerolphosphate oxidase (EC 1.1.3.21), Sarcosine oxidase (EC 1.5.3.1), Xanthine oxidase (EC 1.1.3.22), Oxalate oxidase (EC 1.2.3.4), Cholesterol oxidase (EC 1.1.3.6), Gamma-glutamyl-putrescine oxidase (EC undefined), GABA oxidase (EC undefined), Histamine oxidase (Diamine oxidase, EC 1.4.3.22), Nucleoside oxidase (EC 1.1.3.39), L-Lysine oxidase (EC 1.4.3.14), L-Aspartate oxidase (EC 1.4.3.16), Glycine oxidase (EC 1.4.3.19), and Galactose oxidase (EC 1.1.3.9). GABA is defined as gamma alpha-butyric acid.
Biosensor response (also referred to as the biosensor performance and biosensor sensitivity) is monitored in vitro by testing the sensor against a known concentration of the analyte of interest in a buffered solution. When the analyte of interest comes into contact with the enzyme layer of the biosensor, the enzyme catalytically processes the analyte to produce H2O2 as a by-product. The amount of H2O2 produced is directly proportional to the concentration of the analyte. The response of the biosensor results from the oxidation, at the transducing element, of the enzymatically produced H2O2 and is recorded as a current, commonly referred to as an oxidation current. The oxidation current is typically reported in nanoamps, a convention that will be preserved herein.
Oxidoreductases possess a range of stabilities, which usually differ between the apo- and holo-forms of each enzyme. Key stability parameters that are usually not experimentally determined are the kinetics and thermodynamics of co-factor exchange. Unless the co-factor(s) is covalently attached to the oxidoreductase, said co-factor(s) may be lost from the enzyme as a function of time and temperature. Most preparations of oxidoreductases exist as a mixture of apo- and holo-enzyme. Lactate oxidase (EC 1.1.3.15 or EC 1.13.12.10) is one of the more robust oxidase enzymes, and preparations of the purified enzyme often achieve activities that are suitable for direct use.
The biosensor in Example 1 (Table 1) showed the response to lactate analyte where said biosensor was fabricated from a typical commercial preparation of lactate oxidase. Even though biosensor response was observed, the overall sensitivity of the biosensor to lactate analyte could be significantly increased by pre-incubating, prior to immobilization, the lactate oxidase used to fabricate the biosensor. Co-factors FMN co-(Example 2, the natural co-factor), FAD (Example 3), and riboflavin (Example 4) all result in improved biosensor response to lactate analyte when the co-factors are individually incubated with the lactate oxidase enzyme prior to immobilization into the sensing cavity of the biosensor. This is shown in
The biosensors in Examples 6, 7, and 8 further demonstrated and supported this concept, where apo-lactate oxidase was made into a biosensor without the benefit of pre-incubation with co-factor prior to immobilization. Consistent with this invention, the biosensor fabricated with no pre-incubation of the enzyme with co-factor prior to immobilization failed to respond to a lactate analyte bolus (Example 6). If such a biosensor from Example 6 was allowed to incubate with FMN (Example 7) or FAD (Example 8), at either 25° C. or 37° C., rescue of the enzyme activity and therefore the biosensor response did not occur and the biosensor was still unable to respond to a lactate analyte bolus to any appreciable extent.
Rescue of a biosensor response is specifically defined as the post-immobilization incubation of said finished biosensor in a concentrated solution of the appropriate co-factor(s) at either 25° C. or 37° C. for at least 30 minutes to reactivate the enzyme prior to measurement of the biosensor's response. Note that these conditions are typically suitable for the reactivation of an oxidoreductase in solution.
The order of addition of the co-factor is of critical importance for the purposes of fashioning a functioning biosensor, and this invention has not been articulated nor would it be obvious to one skilled in the art. The apo-lactate oxidase that gave no discernible signal in the absence of co-factor pre-incubation prior to immobilization (when integrated into a biosensor, Examples 6, 7, and 8) was converted into a competent enzyme suitable for biosensor construction by pre-incubating the apo-lactate oxidase with FMN co-factor prior to immobilization as shown in Example 9. This was also shown in
The biosensors in Examples 10-12 and
The difference in biosensor response (to a lactate analyte bolus) for biosensors fabricated from the same apo-lactate oxidase enzyme that was pre-incubated with co-factor prior to immobilization (Example 9) versus those biosensors that were incubated with co-factor only after enzyme immobilization (Examples 7 and 8) was profound and indicative of the importance of this invention (
Examples 13, 14, and 15 showed biosensors fabricated from D-amino acid oxidase enzyme. Functional biosensors (as determined by the biosensor response to D-serine analyte) were best achieved when said biosensors were fabricated from D-amino acid oxidase enzyme that was pre-incubated with FAD co-factor prior to immobilization into the enzyme matrix. The enzyme used to fabricate the biosensor in Example 13 was pre-incubated with FAD prior to immobilization and said biosensor responded to D-serine analyte. If rescue (Example 15) of a biosensor's response was attempted by co-factor incubation after the enzyme was immobilized in the matrix but without conditioning (by pre-incubation with co-factor prior to immobilization, Example 14), the biosensor response was not restored versus that seen for Example 13. Prior to the application of a generic outer-membrane, the biosensor in Example 13 had a linear response range (>95% linear) of over 40 μM of D-serine analyte.
The biosensor in Example 16 was fabricated from D-amino acid oxidase enzyme where the activity of the D-amino acid oxidase enzyme and the function of the biosensor were enabled by pre-incubating the enzyme with FAD co-factor prior to immobilization. At least 73% of the biosensor response was preserved after four months (Example 17) relative to the starting biosensor response (Example 16).
Examples 18, 19, 20, and 21 showed biosensors fabricated from L-6-hydroxynicotine oxidase enzyme. Functional biosensors (as determined by the biosensor response to analyte) were best achieved when said biosensors were fabricated from L-6-hydroxynicotine oxidase enzyme that was pre-incubated with FAD co-factor prior to immobilization into the enzyme matrix. These biosensors responded to nicotine analyte (Example 18), anabasine analyte (Example 20) and nor-nicotine analyte (Example 21). If a biosensor was fabricated with L-6-hydroxynicotine oxidase enzyme that was not conditioned by pre-incubation with FAD co-factor prior to immobilization (Example 19), said biosensor failed to respond to nicotine analyte when tested at the same concentration as Example 18. Prior to the application of a generic outer-membrane, the biosensors in Example 18, 20 and 21 had linear response ranges (>95% linear) of over 60 μM of nicotine, anabasine and nor-nicotine analytes respectively.
Examples 22 and 23 showed biosensors fabricated from alcohol oxidase enzyme. Functional biosensors (as determined by the biosensor response to ethanol analyte) were best achieved when said biosensors were fabricated from alcohol oxidase enzyme that was pre-incubated with FAD co-factor prior to immobilization into the enzyme matrix. The biosensor in Example 22 was pre-incubated with FAD prior to immobilization and responded to ethanol analyte. The enzyme used to fabricate the biosensor in Example 23 was not pre-incubated with FAD prior to immobilization and said biosensor failed to respond to ethanol analyte when tested at the same concentration as Example 22. After the application of a generic outer-membrane, biosensors fashioned as Example 22 had a linear response range (>95% linear) of over 50 mM of ethanol analyte in oxygen enriched media.
Examples 24, 25, 26, 27 and 28 showed biosensors fabricated from pyruvate oxidase enzyme. Functional biosensors (as determined by the biosensor response to pyruvate analyte) were best achieved when said biosensors were fabricated from pyruvate oxidase enzyme that was pre-incubated with both FAD and TPP co-factors prior to immobilization into the enzyme matrix (Example 24). If a biosensor (Example 25) was fabricated from pyruvate oxidase that was not pre-incubated with either co-factor prior to immobilization no response to pyruvate analyte was observed. If a biosensor was fabricated from pyruvate oxidase that was pre-incubated with only FAD co-factor prior to immobilization, no response to pyruvate analyte was observed (Example 26). If a biosensor is fabricated from pyruvate oxidase that was pre-incubated with only TPP co-factor prior to immobilization, no response to pyruvate analyte is observed (Example 27). If a biosensor was fabricated from pyruvate oxidase that was pre-incubated with only FAD co-factor prior to immobilization, no response to pyruvate analyte was observed even if the amount of enzyme used was increased five-fold (Example 28).
Examples 24-28 also demonstrated the utility when more than one co-factor was needed for enzyme activation. Pyruvate oxidase enzyme requires both FAD and TPP to support the catalytic cycle of the enzyme. If both co-factors were present (Example 24), then activity of the biosensor towards pyruvate analyte was observed. If both co-factors were absent (Example 25) or if either FAD was used alone (Example 26) or TPP is used alone (Example 27), then no activity towards pyruvate was observed. An increase in the enzyme concentration five-fold over the standard conditions but with just FAD present (TPP was absent) also resulted in a biosensor that failed to sense pyruvate (Example 28).
Examples 29 and 30 showed biosensors fabricated from glucose oxidase enzyme. Functional biosensors (as determined by the biosensor response to glucose analyte) were best achieved when said biosensors were fabricated from glucose oxidase enzyme that was pre-incubated with FAD co-factor prior to immobilization into the enzyme matrix. The biosensor in Example 29 was pre-incubated with FAD prior to immobilization and responded best to glucose analyte. The enzyme used to fabricate the biosensor in Example 30 was not pre-incubated with FAD prior to immobilization and the said biosensor showed over a 10-fold reduction in response to glucose analyte when tested at the same concentration as Example 29. Prior to the application of a generic outer-membrane, the biosensor in Examples 29 showed a linear response range (>72% linear) up to 4 mM of glucose analyte. In the presence of a generic outer-membrane, this linear response range (>95% linear) improved to over 5 mM of glucose analyte.
Examples 31 and 32 showed biosensors fabricated from glutamate oxidase enzyme. Functional biosensors (as determined by the biosensor response to glutamate analyte) were best achieved when said biosensors were fabricated from glutamate oxidase enzyme that was pre-incubated with FAD co-factor prior to immobilization into the enzyme matrix. The biosensor in Example 31 was pre-incubated with FAD prior to immobilization and responded to glutamate analyte. The enzyme used to fabricate the biosensor in Example 32 was not pre-incubated with FAD prior to immobilization and the said biosensor failed to respond to glutamate analyte when tested at the same concentration as Example 31. Prior to the application of a generic outer-membrane, the biosensor in Example 31 had a linear response range (>99% linear) of over 40 μM of glutamate analyte.
Example 33 showed a biosensor fabricated from acyl Co-A oxidase enzyme. The functional biosensor (as determined by the biosensor response to N-butyrl Co-A analyte) was best achieved when said biosensor was fabricated from acyl Co-A oxidase enzyme that was pre-incubated with FAD co-factor prior to immobilization into the enzyme matrix. The enzyme used to fabricate the biosensor in Example 33 was pre-incubated with FAD prior to immobilization and said biosensor responded to N-butyrl Co-A analyte. Prior to the application of a generic outer-membrane, the biosensor in Example 31 had a linear response range (>98% linear) of over 40 μM of N-butyrl Co-A analyte.
Example 34 showed a biosensor fabricated from choline enzyme. The functional biosensor (as determined by the biosensor response to choline analyte) was best achieved when said biosensor was fabricated from choline oxidase enzyme that was pre-incubated with FAD co-factor prior to immobilization into the enzyme matrix. The enzyme used to fabricate the biosensor in Example 34 was pre-incubated with FAD prior to immobilization and said biosensor responded to choline analyte. Prior to the application of a generic outer-membrane, the biosensor in Example 34 had a linear response range (>99% linear) of over 40 μM of choline analyte.
Example 35 showed a biosensor fabricated from glutathione sulfhydryl oxidase enzyme. The functional biosensor (as determined by the biosensor response to reduced glutathione analyte) was best achieved when said biosensor was fabricated from glutathione sulfhydryl oxidase enzyme that was pre-incubated with FAD co-factor prior to immobilization into the enzyme matrix. Prior to the application of a generic outer-membrane, the biosensor in Example 35 had a linear response range (>98% linear) of over 5 mM of reduced glutathione analyte.
Example 36 showed a biosensor fabricated from glycerolphosphate oxidase enzyme. The functional biosensor (as determined by the biosensor response to glycerol-3-phosphate analyte) was best achieved when said biosensor was fabricated from glycerolphosphate oxidase enzyme that was pre-incubated with FAD co-factor prior to immobilization into the enzyme matrix. The enzyme used to fabricate the biosensor in Example 36 was pre-incubated with FAD prior to immobilization and said biosensor responded to glycerol-3-phosphate analyte. Prior to the application of a generic outer-membrane, the biosensor in Example 36 had a linear response range (>99% linear) of over 300 μM of glycerol-3-phosphate analyte.
Example 37 showed a biosensor fabricated from sarcosine oxidase enzyme. The functional biosensor (as determined by the biosensor response to sarcosine analyte) was best achieved when said biosensor was fabricated from sarcosine oxidase enzyme that was pre-incubated with FAD co-factor prior to immobilization into the enzyme matrix. The enzyme used to fabricate the biosensor in Example 37 was pre-incubated with FAD prior to immobilization and said biosensor responded to sarcosine analyte. Prior to the application of a generic outer-membrane, the biosensor in Example 37 had a linear response range (>98% linear) of over 1 mM of sarcosine analyte.
Example 38 showed a biosensor fabricated from xanthine oxidase enzyme. The functional biosensor (as determined by the biosensor response to xanthine analyte) was best achieved when said biosensor was fabricated from xanthine oxidase enzyme that was pre-incubated with FAD co-factor prior to immobilization into the enzyme matrix. The enzyme used to fabricate the biosensor in Example 38 was pre-incubated with FAD prior to immobilization and said biosensor responded to xanthine analyte. Prior to the application of a generic outer-membrane, the biosensor in Example 38 had a linear response range (>98% linear) of over 50 μM of xanthine analyte.
Examples 30, 40 and 41 showed biosensors fabricated from oxalate oxidase enzyme. Functional biosensors (as determined by the biosensor response to oxalate analyte) were best achieved when said biosensors were fabricated from oxalate oxidase enzyme that was pre-incubated with Mn2+ co-factor prior to immobilization into the enzyme matrix. The enzyme used to fabricate the biosensor in Example 39 was pre-incubated with Mn2+ prior to immobilization and said biosensor responded to oxalate analyte. If the enzyme used to fabricate the biosensor in Example 41 was not pre-incubated with Mn2+ prior to immobilization and the said biosensor failed to respond to oxalate analyte when tested at the same concentration as Example 39. Prior to the application of a generic outer-membrane, the biosensor in Example 39 had a linear response range (>97% linear) of over 5 mM of oxalate analyte.
Example 39, 40, and 41 also demonstrated that this invention is useful for co-factors that are heavy metals. Entry 40 also demonstrated the importance of using the proper co-factor for complete conditioning and enhancement of enzyme activity. Here, FAD was used instead of Mn2+ as the co-factor. Even though a discernible signal was observed, the signal was still five-fold less than that observed for the properly Mn2+ conditioned enzyme (Example 39). Example 41 demonstrated that in the absence of any co-factor, the enzyme is once again unable to support catalysis as part of a functioning biosensor.
Example 42 showed a biosensor fabricated from cholesterol oxidase enzyme. The functional biosensor (as determined by the biosensor response to cholesterol analyte) was best achieved when said biosensor was fabricated from cholesterol oxidase enzyme that was pre-incubated with FAD co-factor prior to immobilization into the enzyme matrix. The enzyme used to fabricate the biosensor in Example 42 was pre-incubated with FAD prior to immobilization and said biosensor responds to cholesterol analyte.
Examples 43, 44 and 45 showed biosensors fabricated from gamma-glutamyl-putrascine oxidase enzyme. Functional biosensors (as determined by the biosensor response to GABA analyte) were best achieved when said biosensors were fabricated from gamma-glutamyl-putrascine oxidase enzyme that was pre-incubated with FAD co-factor prior to immobilization into the enzyme matrix. The enzyme used to fabricate the biosensor in Example 43 was pre-incubated with FAD prior to immobilization and said biosensor responded to GABA analyte. The enzyme used to fabricate the biosensor in Example 44 was pre-incubated with FMN prior to immobilization and said biosensor responded to GABA analyte. The enzyme used to fabricate the biosensor in Example 45 was not pre-incubated with co-factor prior to immobilization and said biosensor did not respond to GABA analyte.
Example 46 shows a biosensor fabricated from GABA oxidase enzyme. The functional biosensor (as determined by the biosensor response to GABA analyte) is best achieved when said biosensor is fabricated from GABA oxidase enzyme that is pre-incubated with FAD co-factor prior to immobilization into the enzyme matrix. The enzyme that is used to fabricate the biosensor in Example 46 is pre-incubated with FAD prior to immobilization and said biosensor responds to GABA analyte.
Example 47 shows a biosensor fabricated from histamine oxidase enzyme (diamine oxidase). The functional biosensor (as determined by the biosensor response to histamine analyte) is best achieved when said biosensor is fabricated from histamine oxidase enzyme that is pre-incubated with FAD co-factor prior to immobilization into the enzyme matrix. The enzyme that is used to fabricate the biosensor in Example 47 is pre-incubated with FAD prior to immobilization and said biosensor responds to histamine analyte.
Example 48 shows a biosensor fabricated from nucleoside oxidase enzyme. The functional biosensor (as determined by the biosensor response to adenosine analyte) is best achieved when said biosensor is fabricated from nucleoside oxidase enzyme that is pre-incubated with FAD co-factor prior to immobilization into the enzyme matrix. The enzyme that is used to fabricate the biosensor in Example 48 is pre-incubated with FAD prior to immobilization and said biosensor responds to adenosine analyte.
Example 49 shows a biosensor fabricated from L-lysine oxidase enzyme. The functional biosensor (as determined by the biosensor response to L-lysine analyte) is best achieved when said biosensor is fabricated from L-lysine oxidase enzyme that is pre-incubated with FAD co-factor prior to immobilization into the enzyme matrix. The enzyme that is used to fabricate the biosensor in Example 49 is pre-incubated with FAD prior to immobilization and said biosensor responds to L-lysine analyte.
Example 50 shows a biosensor fabricated from L-aspartate oxidase enzyme. The functional biosensor (as determined by the biosensor response to L-aspartate analyte) is best achieved when said biosensor is fabricated from L-aspartate oxidase enzyme that is pre-incubated with FAD co-factor prior to immobilization into the enzyme matrix. The enzyme that is used to fabricate the biosensor in Example 50 is pre-incubated with FAD prior to immobilization and said biosensor responds to L-aspartate analyte.
Example 51 shows a biosensor fabricated from glycine oxidase enzyme. The functional biosensor (as determined by the biosensor response to glycine analyte) is best achieved when said biosensor is fabricated from glycine oxidase enzyme that is pre-incubated with FAD co-factor prior to immobilization into the enzyme matrix. The enzyme that is used to fabricate the biosensor in Example 51 is pre-incubated with FAD prior to immobilization and said biosensor responds to glycine analyte.
Example 52 shows a biosensor fabricated from galactose oxidase enzyme. The functional biosensor (as determined by the biosensor response to galactose analyte) is best achieved when said biosensor is fabricated from galactose oxidase enzyme that is pre-incubated with pyrroloquinoline and quinone co-factor prior to immobilization into the enzyme matrix. The enzyme that is used to fabricate the biosensor in Example 52 is pre-incubated with pyrroloquinoline and quinone prior to immobilization and said biosensor responds to galactose analyte.
The biosensors in Examples 53, 56, 57, 59, 61, and 63, were fabricated on polyphenol film that formed the inner-membrane of the sensing cavity. In each instance, the indicated oxidase enzyme was pre-incubated with the indicated co-factor prior to immobilization into the sensing matrix. These biosensors all responded to analyte as indicated.
The biosensors in Examples 54, 58, 60, 62, and 64 were fabricated on poly-o-cresol film that formed the inner-membrane of the sensing cavity. In each instance, the indicated oxidase enzyme was pre-incubated with the indicated co-factor prior to immobilization into the sensing matrix. These biosensors all responded to analyte as indicated.
For Example 55, the biosensor was fabricated on polypyrrol film that formed the inner-membrane of the sensing cavity. For this biosensor, the lactate oxidase enzyme was pre-incubated with the FMN co-factor prior to immobilization into the sensing matrix. This biosensor responded to lactate analyte as indicated.
It is important to note that this invention is applicable regardless of the range of analyte tested. For example, a biosensor fabricated from lactate oxidase showed improvement when tested with 10 μM, 100 μM, or 1 mM boluses of lactate analyte. D-amino acid oxidase, glutamate oxidase, and L-6-hydroxynicotine oxidase showed improvement when tested with 10 μM boluses of D-serine, glutamate, and nicotine analytes respectively. Alcohol oxidase shows an improvement when tested with 1 mM boluses of ethanol ethanol. The actual range one might use for any particular biosensor will reflect the expected range that needs to be monitored in vivo for the analyte of interest. The other enzyme/analyte combinations in Table 1 further demonstrated that this invention is applicable for a range of analyte concentrations.
In another embodiment of the invention, biosensors fashioned from enzymes that were conditioned by pre-incubation with the appropriate co-factor(s) showed an increase in shelf stability relative to those biosensors whose biorecognition elements were not conditioned or pre-incubated. This is shown in
Another embodiment of the invention involves its use for enzymes that can be directed to the measurement of analytes that are not the natural substrate of said enzyme. For example, Entries 18, 20, and 21 demonstrated that L-6-hydroxynicotine oxidase could be used to fabricate a biosensor that would measure nicotine, anabasine, and nor-nicotine even though the natural substrate for the enzyme is 6-hydroxynicotine. This invention is also concerned with the discovery of new co-factor dependent oxidoreductase enzymes that have yet to be described. These new enzymes could be derived from natural sources, by single or multiple mutagenesis changes to an oxidoreductase gene, or by directed evolution using molecular biology techniques of an existing oxidoreductase. These new enzymes may find utility for the sensing of new analytes. According to the invention, these new enzymes may benefit from conditioning with co-factor(s) prior to use as the biorecognition element in a biosensor.
In another embodiment, the invention is useful for co-factor dependent enzymes even if only one species produces such an enzyme, while all other species produce iso-forms that are not co-factor dependent. For example, the human iso-form of diamine oxidase (EC 1.4.3.22), sometimes referred to as histamine oxidase, does not require a co-factor for activity. Instead, human diamine oxidase relies on a post-translational hydroxylation of an active site tyrosine residue to a trihydroxyphenylalanine residue and then finally to a topaquinone (TPQ) residue. By contrast, the diamine oxidase found in rice seedlings from the species Oryza saliva requires FAD as a co-factor for activity (Chaudhuri, M. M.; Ghosh, B. Phytochemistry, 1984, 23, 241-243). Thus, this invention is applicable even if only one species produces an iso-form of a particular oxidoreductase that is co-factor dependent.
In another embodiment of the invention, the utilization of co-factors are not limited to only human iso-forms of a particular enzyme, but also embrace all iso-forms of said enzyme found in all organisms so long as the iso-form is co-factor dependent. It is well accepted and understood that different iso-forms of the same enzyme from different organisms will possess different amino acid sequences and may rely on different co-factors for activity.
In another embodiment, the invention finds utility in activating enzymes where the natural co-factor is normally covalently bonded to the protein, but where the co-factor(s) may have been absent during expression in a natural setting or within a laboratory. This invention may also be used to rescue enzymes whose co-factor(s) was lost during purification of the enzyme, and would be independent of whether the co-factors(s) is non-covalently or covalently bonded.
Enzyme-based biorecognition elements that may benefit from the incubation with co-factor(s) prior to immobilization in a solid matrix include (but are not limited to) malate oxidase, EC 1.1.3.3, co-factor(s)=FAD; hexose oxidase, EC 1.1.3.5, co-factor(s)=Cu; aryl-alcohol oxidase, EC 1.1.3.7, co-factor(s)=FAD; L-gulonolactone oxidase, EC 1.1.3.8, co-factor(s)=FAD; pyranose oxidase, EC 1.1.3.10, co-factor(s)=FAD; L-sorbose oxidase, EC 1.1.3.11, co-factor(s)=FAD(?)(?); pyridoxine 4-oxidase, EC 1.1.3.12, co-factor(s)=FAD; (S)-2-hydroxy-acid oxidase, EC 1.1.3.15, co-factor(s)=FAD, FMN; ecdysone oxidase, EC 1.1.3.16, co-factor(s)=NADPH; secondary-alcohol oxidase, EC 1.1.3.18, co-factor(s)=FAD(?)(?); 4-hydroxymandelate oxidase, EC 1.1.3.19, co-factor(s)=co-factor(s)=FAD(?)(?), Mn2+; long-chain-alcohol oxidase, EC 1.1.3.20, co-factor(s)=FAD; thiamine oxidase, EC 1.1.3.23, co-factor(s)=FAD; hydroxyphytanate oxidase, EC 1.1.3.27. co-factor(s)=FAD(?)(?); N-acylhexosamine oxidase, EC 1.1.3.29, co-factor(s)=FAD(?)(?); polyvinyl-alcohol oxidase, EC 1.1.3.30, co-factor(s)=FAD(?)(?); D-Arabinono-1,4-lactone oxidase, EC 1.1.3.37, co-factor(s)=FAD; vanillyl-alcohol oxidase, EC 1.1.3.38, co-factor(s)=FAD; D-mannitol oxidase, EC 1.1.3.40, co-factor(s)=FAD(?)(?); alditol oxidase, EC 1.1.3.41, co-factor(s)=FAD; choline dehydrogenase, EC 1.1.99.1, co-factor(s)=Coenzyme Q; gluconate 2-dehydrogenase EC 1.1.99.3, co-factor(s)=FAD; glucooligosaccharide oxidase, EC 1.1.99.133, co-factor(s)=FAD; alcohol dehydrogenase, EC 1.1.99.8, co-factor(s)==PQQ; cellobiose dehydrogenase, EC 1.1.99.18, co-factor(s)=FAD; aldehyde oxidase, EC 1.2.3.1, co-factor(s)=FAD; glyoxylate oxidase, EC 1.2.3.5, co-factor(s)=FAD(?)(?); indole-3-acetaldehyde oxidase, EC 1.2.3.7, co-factor(s)==FAD; aryl-aldehyde oxidase, EC 1.2.3.9, co-factor(s)==ATP, NADPH; retinal oxidase, EC 1.2.3.11, co-factor(s)=FAD; abscisic-aldehyde oxidase, EC 1.2.3.14, co-factor(s)==MoCo; aldehyde ferredoxin oxidoreductase, EC 1.2.7.5, co-factor(s)==Ferredoxin tungsten co-factor; indolepyruvate ferredoxin oxidoreductase, EC 1.2.7.8, co-factor(s)=TPP; aldehyde dehydrogenase, EC 1.2.99.7, co-factor(s)=FE-molybdenum; dihydroorotate oxidase, EC 1.3.3.1, co-factor(s)==FAD-FMN; acyl-CoA oxidase, EC 1.3.3.6, co-factor(s)=FAD; dihydrouracil oxidase, EC 1.3.3.7, co-factor(s)==FMN; tetrahydroberberine oxidase, EC 1.3.3.8, co-factor(s)==FAD; tryptophan alpha,beta-oxidase, EC 1.3.3.10, co-factor(s)=(?); L-galactonolactone oxidase, EC 1.3.3.12, co-factor(s)=FAD; acyl-CoA dehydrogenase, EC 1.3.99.3, co-factor(s)=FAD; Isoquinoline-1-oxidoreductase, EC 1.3.99.16, co-factor(s)=iron-sulfur centre or molybdopterin cytosine dinucleotide; quinaldate 4-oxidoreductase, EC 1.3.99.18, co-factor(s)=FAD or pterin molybdenum co-factor; D-aspartate oxidase, EC 1.4.3.1, co-factor(s)=FAD; L-amino-acid oxidase, EC 1.4.3.2, co-factor(s)=FAD; monoamine oxidase, EC 1.4.3.4, co-factor(s)==FAD; pyridoxal 5′-phosphate synthase, EC 1.4.3.5, co-factor(s)==FMN; D-glutamate oxidase, EC 1.4.3.7, co-factor(s)=FAD; ethanolamine oxidase, EC 1.4.3.8; putrescine oxidase, EC 1.4.3.10, co-factor(s)=FAD; cyclohexylamine oxidase, EC 1.4.3.12, co-factor(s)=FAD; protein-lysine 6-oxidase, EC 1.4.3.13, co-factor(s)==lysyl-tyrosyl quinone; D-glutamate(D-aspartate) oxidase, EC 1.4.3.15, co-factor(s)=FAD; L-lysine 6-oxidase, EC 1.4.3.20, co-factor(s)=(?); primary-amine oxidase, EC 1.4.3.21, co-factor(s)==2,4,5-trihydroxyphenylalanine quinone; 7-chloro-L-tryptophan oxidase, EC 1.4.3.23, co-factor(s)=FAD; N-methyl-L-amino-acid oxidase, EC 1.5.3.2, co-factor(s)=FAD; non-specific polyamine oxidase, EC 1.5.3.B2, co-factor(s)=FAD; N8-acetylspermidine oxidase (propane-1,3-diamine-forming), EC 1.5.3.B3, co-factor(s)=FAD; N6-methyl-lysine oxidase, EC 1.5.3.4, co-factor(s)=FAD; polyamine oxidase (propane-1,3-diamine-forming), EC 1.5.3.B4, co-factor(s)=FAD; NI-acetylpolyamine oxidase, EC 1.5.3.B5, co-factor(s)=FAD; spermine oxidase, EC 1.5.3.B6, co-factor(s)=FAD; pipecolate oxidase, EC 1.5.3.7, co-factor(s) =FAD; dimethylglycine oxidase, EC 1.5.3.10, co-factor(s)=FAD; polyamine oxidase, EC 1.5.3.11, co-factor(s)=FAD+Fe; Dihydrobenzophenanthridine oxidase, EC 1.5.3.12, co-factor(s)==FAD+Cu; NAD(P)H oxidase, EC 1.6.3.1, co-factor(s)==FAD+HEME+Ca; urate oxidase, EC 1.7.3.3; 3-aci-nitropropanoate oxidase, EC 1.7.3.5, co-factor(s)==FMN; sulfite oxidase, EC 1.83.1, co-factor(s)==HEME, OLYBDOPTERIN; methanethiol oxidase, EC 1.8.3.4; prenylcysteine oxidase, EC 1.8.3.5, co-factor(s)=FAD; L-ascorbate oxidase, EC 1.10.3.3, co-factor(s)==HEME+CU; 3-hydroxyanthranilate oxidase, EC 1.10.3.5, co-factor(s)==Fe; rifamycin-B oxidase, EC 1.10.3.6, co-factor(s)=(?); superoxide dismutase, EC 1.15.1.1, co-factor(s)=(?); reticuline oxidase, EC 1.21.3.3, co-factor(s)=FAD; lactate oxidase, L-EC 1.1.3.15, co-factor(s)=FAD, FMN; D-amino acid oxidase, EC 1.4.3.3, co-factor(s)=FAD; (S)-6-hydroxynicotine oxidase, EC 1.5.3.5, co-factor(s)=FAD; (R)-6-hydroxynicotine oxidase, EC 1.5.3.6, co-factor(s)=FAD; alcohol oxidase, EC 1.1.3.13, co-factor(s)=FAD; pyruvate oxidase, EC 1.2.3.3, co-factor(s)=FAD, TPP; glucose oxidase, EC 1.1.3.4), co-factor(s)=FAD; L-glutamate oxidase, EC 1.4.3.11, co-factor(s)=FAD; acyl coenzyme A oxidase, EC 1.3.3.6, co-factor(s)=FAD; choline Oxidase, EC 1.1.3.17, co-factor(s)=FAD; glutathione sulfhydryl oxidase, EC 1.8.3.3, co-factor(s)=FAD; glycerolphosphate oxidase, EC 1.1.3.21, co-factor(s)=FAD; sarcosine oxidase, EC 1.5.3.1, co-factor(s)=FAD; xanthine oxidase, EC 1.1.3.22, co-factor(s)==FAD; oxalate oxidase, EC 1.2.3.4, co-factor(s)=Mn2+; cholesterol oxidase, EC 1.1.3.6, co-factor(s)=FAD; gamma-glutamyl-putrescine oxidase, EC undefined, obtained from Escherichia coli K12, co-factor(s)=FAD—capable of oxidizing GABA; GABA oxidase, EC undefined, obtained from: Penicillium sp. KAIT-M-117, co-factor(s)=FAD(?)(?); histamine oxidase (diamine oxidase), EC 1.4.3.22, co-factor(s)=pyrroloquinoline, quinone, FAD, others; nucleoside oxidase, EC 1.1.3.39, co-factor(s)=FAD; L-lysine oxidase, EC 1.4.3.14, co-factor(s)=FAD; L-aspartate oxidase, EC 1.4.3.16, co-factor(s)=FAD; glycine oxidase, EC 1.4.3.19, co-factor(s)=FAD; galactose oxidase, EC 1.1.3.9, co-factor(s)=pyrroloquinoline, quinone. The (?) entry indicates that the co-factor is either completely unknown, or that the indicated co-factor has not been verified.
A list of preferred members of this group of enzymes include:
The invention also encompasses the discovery of new co-factor dependent oxidoreductases that may find utility in measuring analytes for which no oxidoreductase presently exists. New oxidoreductases with new or altered analyte activities, specificities, and selectivities may be derived from natural sources, derived by the conversion of an existing oxidoreductase via standard molecular biology mutagenesis techniques or derived by the directed evolution of an existing enzyme. The new co-factor dependent oxidoreductase would have activity, selectivity, and specificity for an analyte not presently available. Such analytes that would benefit from the discovery of a new co-factor dependent oxidoreductase are selected from the list consisting of nicotine, caffeine, cocaine, amphetamine, cortisol, corticosterone, dopamine, serotonin, norepinephrine, L-DOPA, GABA, ATP, and acetylcholine. While some of these analytes may be the substrate of an existing oxidoreductase (e.g., (S)-6-hydroxynicotine oxidase has some activity for nicotine; gamma-glutamyl putrisciene oxidase has some activity for GABA), said analytes are not the primary substrate for said oxidoreductase. Furthermore, said activity, selectivity, and specificity do not necessarily mean that the enzyme is suitable for use in biosensor production. The monitoring of these analytes may benefit by the alternation of said enzyme for the purposes of biosensor fabrication.
In another embodiment, the invention is completely compatible with all aspects of biosensor fabrication. The fabrication and manufacture of biosensors for commercial applications requires that the biosensor be compatible with the in vivo environment into which it will be placed and must reject endogenous in vivo electroactive interferents that may confound an in vivo measurement. This invention is compatible with existing methods to promote in vivo compatibility, as well as existing methods for excluding potential electroactive interferents. Any process designed to improve the function of the enzyme layer or sensing matrix must be compatible with a biosensor that is fully functional in vivo. This invention, by conditioning an enzyme that will be used as a biosensor's biorecognition element with natural co-factors, is completely compatible with in vivo monitoring of analytes (e.g. glucose and lactate as shown in
In another embodiment of the invention, biosensors that are fabricated with pre-incubation of the enzyme with the appropriate co-factor(s) prior to immobilization perform at a stable level in vivo for extended periods of time from days to a week or more. This positive effect on biosensor performance is shown in
All enzymes are constantly in motion, and even the most rigid of enzymes have motional and vibrational states that cause atomic level movement within the protein. In general, all oxidoreductases undergo conformation changes as a function of co-factor presence. For oxidoreductases, this process is understood. There exists two limiting conformations, which are reflective of the apo-form of the protein (co-factor(s) absent from the enzyme) and active, holo-form of the protein (co-factor(s) present in the enzyme). The conformations of these two limiting states are distinctly different and can, when immobilized as part of a solid matrix, interact with said matrix differentially.
The act of immobilization establishes the global minimum of the enzyme-matrix system, which is reflective of the predominant state of the enzyme at the time of immobilization. If an enzyme is immobilized as part of a matrix, the form of the enzyme will determine the global thermodynamic minimum of the enzyme-matrix (referred to hereafter as “the system”). If the enzyme is predominantly apo when immobilized as part of the matrix, the thermodynamic minimum for the system reflects the fact that the enzyme is of an apo conformation and an apo shape, and, therefore, the solid matrix surrounding the enzyme interacts and reinforces said conformation. This is reflected in the results from Entries 6, 7, and 8 where the apo enzyme could not be rescued post-immobilization by the addition of co-factor or Entries 10, 11 and 12 where the rescue of the partially holo-enzyme is limited in activity and time.
Other elements of the enzyme layer that may be present further contribute to the stabilization of the apo-enzyme conformation. An energetic price must be paid to force the apo-enzyme into an active, holo-conformation because of the interactions of the enzyme with the surrounding matrix. Thus, while the apo-enzyme can be forced into an active conformation by incubating the system with co-factor(s), the resulting activated form of the enzyme is now at energetic odds with the matrix. Consequently, the system will facilitate the loss of co-factor and revert the enzyme back to the corresponding apo-conformation to re-establish the thermodynamic minimum of the enzyme-matrix system. This is supported with the data from Entries 6, 7 and 8 as well as Entries 14 and 15, Entries 43 and 45and Entries 10, 11, and 12.
Immobilization of enzyme in its active conformation (co-factor(s) present) will result in an active-enzyme/matrix system that reflects the global thermodynamic minimum for the holo-enzyme or active-enzyme conformation. This matrix, built around the active enzyme conformation, promotes efficient enzyme activity. Other elements of the enzyme layer that may be present further contribute to the stabilization of the holo-enzyme conformation. Thus, in comparison to the enzyme-matrix system where the enzyme was predominantly in the apo form, this system is more energetically compatible with the state of the enzyme that is required for catalytic activity and use as the biorecognition element in a biosensor. Note that the enzyme can still lose co-factor even when the thermodynamics of the system are reflective of the active form of the enzyme. It is known that oxidoreductase can readily lose co-factor and that this loss is temperature driven but is reversible. In contrast to the situation in which the system reflects the energetics of the apo conformation, the system that reflects the energetics of the active conformation is more readily reactivated, and the reactivated system is more stable.
An important component of the above, especially with respect to the manufacturability of biosensors is that difficult to handle enzyme preparations can be immobilized en mass for long-term shelf storage and then reactivated prior to use. The importance of the incubation process is independent of the actual biosensor design and is applicable to many designs presently disclosed, including (but not limited to) nano-biosensors, lab-on-chip biosensors, and ceramic biosensors.
Enzymes to be immobilized as biorecognition elements in biosensor construction that are part of this invention are incubated with their co-factor(s) prior to immobilization. Incubation of an enzyme is not limited to a single co-factor, but rather should reflect the optimum interaction of co-factors. Optimization of the enzyme incubation can be determined for each instance of an enzyme and will include the temperature of the incubation, the co-factor(s) concentration, the length of time for incubation, the order of addition of reagents, the ratio of co-factors, and the order of addition and time between additions if multiple co-factors are required for enzyme activity. Even in the absence of optimization of the incubation process, it is expected that any incubation with co-factor(s) will result in an enhancement of the activity of the enzyme which, in turn, will result in an enhancement of the biosensor performance relative to a biosensor whose biorecognition element was not incubated with co-factor(s). The determination of the optimum co-factor enhancement strategy can occur in solution and may be monitored using standard techniques.
This invention allows for the co-immobilization of multiple enzymes simultaneously, which may occur together as part of a single layer or as separate discreet layers that comprise a composite biosensor. In principle, there is no limit to the number of enzymes that function as biorecognition elements that may be co-immobilized. In practical terms, the limit will be determined by the ability of the panoply of enzymes to work in concert with each other. Further, the immobilization of said enzymes could take place on separate regions within a larger array, thereby providing the ability to simultaneously monitor multiple analytes with a single probe. Finally, the mixing of multiple biorecognition elements is not limited to just those that benefit from co-factor enhancement, but rather can reflect a composite group of enzymes that work in concert, at least one of which benefits from said co-factor enhancement. For example, the placement of acetylcholine esterase and choline oxidase as part of the same layer or different layers within a biosensor would allow for the monitoring of acetylcholine levels by the action of (1) conversion of acetylcholine to choline and acetate and (2) the conversion of choline to betaine aldehyde and H2O2, the latter of which would be monitored.
This invention is compatible with any number of enzymes, despite their nascent activity prior to immobilization, so long as each enzyme is incubated with the co-factors necessary to insure said enzyme's conversion to the holo-enzyme.
This invention would find utility in a wide range of biosensor designs. Commercially available biosensors that make use of immobilized enzyme(s) as the biorecognition element include the biosensors based on oxidoreductases from Pinnacle Technology Inc. (www.pinnaclet.com) such as those for L-glutamate, D-glucose, L-lactate, and alcohol; sensors based on oxidoreductases from Sarrisa Biomedical (www.sarissa-biomedical.com) such as those for L-lactate, L-glutamate, choline, acetylcholine/choline; sensors based on oxidoreductases from Bluebox Sensors (www.blueboxsensors.com) such as the sensor for D-glucose; and sensors based on oxidoreductases from Quanteon LLC (quanteon.cc) such as those for L-glutamate and choline; sensors based on oxidoreductases from YSI (www.ysilifesciences.com) such as the sensor for D-glucose, sucrose, lactose, L-Lactate, D-galactose, L-glutamate, L-glutamine, choline, ethanol and methanol. Furthermore, the invention of co-factor enhancing the biorecognition element prior to immobilization can work for essentially any biosensor probe design (e.g., planar, cylindrical, disc) or size (about 2 microns to 1000 microns, although in principle there is no lower or upper size limit for which this would apply).
In another embodiment of this invention, the use of co-factor incubation to optimize the performance of the biorecognition element is coupled with an electropolymerization strategy utilizing a phenol precursor to produce a polyphenol film that could serve as either the second layer of a biosensor, the fourth layer of a biosensor, or some combination thereof. In one manifestation of this approach, said film can take the place of polyurethane, providing a more uniform layer that may possess better selectivity properties, better thermal properties, and better mechanical properties. In another manifestation of this approach, the electropolymerization strategy can be utilized to assist in the immobilization of the enzyme. This simultaneously provides the matrix for localization of the biosensing element within the sensing cavity and further provides a film layer that increases the ability of the biosensor to exclude potential interferents. In the extreme case, the polyphenol film layer may preclude the need for other layers as part of the biosensor construction process.
Electropolymerization of phenol precursors to provide a polyphenol film that simultaneously results in the immobilization of the biosensing element can now take complete advantage of the co-factor enhancement process as described. The film will now fully reinforce the shape and consequently the state and conformation of the enzyme by virtue of the nature of the film. For many enzymes that could be utilized as biosensing elements, the stability of the enzyme/co-factor complex in a polyphenol film will far exceed that seen for the same enzyme/co-factor complex in solution. Thus, to maximally exploit the innate nature of a fully activated enzyme, the enzyme should be placed within an environment that helps preserve the active shape, state, and form of the enzyme. Even if loss of co-factor occurs over time, the regeneration of the enzyme's activity, when the enzyme is part of a polyphenol matrix, would be readily accomplished by soaking the biosensor in a solution of co-factor(s) that is consistent with that used in the original biosensor construction.
Polyphenol films may also confer exceptional stability to the biosensor, providing a superior method of crafting amperometric biosensors, most especially those for use in humans. The development of biosensors safe for acute and chronic human applications continues to be an unsolved problem. The utilization of the co-factor enhancement of the biosensing element in conjunction with a polyphenol mediated immobilization of said sensing element now provides for the sensing of a range of analytes that heretofore have been inaccessible.
Examples of biosensors whose biorecognition elements were conditioned by co-factor enhancement prior to immobilization are shown in Entries 46-57 (Table 2). Three different electropolymerized films were tested—polyphenol, poly-o-cresol, and polypyrole—and all three electropolymerized films proved compatible with this invention.
The ability to readily determine lactate levels in a new patient presenting with trauma is now recognized as one of the most important diagnostic parameters to help determine the most appropriate course of treatment. Traumas, including brain injuries, shock, sepsis, and haemodynamical instabilities, have all been identified as producing elevated levels of serum lactate. The lactate levels often coincide with the severity of the trauma, and in the case of brain injuries, the magnitude of the insult. Trauma patient morbidity and mortality is known to track with serum lactate levels. Further, decreases in serum lactate levels, as a function of time, can be an early indicator of improved prognoses.
In another embodiment of this invention, a system is developed that provides the ability to determine, in real-time, in vivo lactate levels, including intra-cerebral, sub-cutaneous, intra-muscular, inter-peritoneal, oral, serum, and vascular, the majority of which may act as a surrogate for systemic lactate levels. Such a system would provide a number of distinct improvements to the treatment of soldiers in theater, and the foundation of such a system is a lactate biosensor that has optimal performance characteristics including sensitivity and shelf life. In broad terms, the adaptation of a lactate biosensor of a design described herein for use in humans would provide battlefield medics the ability to appropriately triage soldiers for treatment, most especially those suffering from traumatic brain injuries (TBIs). In the case of TBIs, the speed with which a diagnosis and subsequent treatment options can be made available to medical personnel is often a key mitigant in defining a soldier's long-term prognosis.
The system provides a first responder the ability to monitor, in real-time, the lactate levels of a patient by using a biosensor coupled to recording system (which may be a telemetry system). Telemetry data will be monitorable on essentially any device with wireless capabilities. Long-term accuracy of the sensor, while important, is less critical than delivering acute lactate levels shortly after implantation with a clear indication of how the lactate levels are changing (fast up, fast down). For this application, the biosensor is “pre-charged” to stabilize prior to implantation. Variations (based on biosensor pre-calibration values that are stored in non-volatile memory in the system) are handled via numerical methods embedded within the monitoring software. This system allows for the unprecedented ability to quickly and continuously determine lactate levels as the first step in trauma response and provide a complete history from time of application. A biosensor strategy for monitoring lactate levels offers the following advantages: (1) a highly portable, cost-effective system that would be compatible with complex trauma and battlefield situations, (2) no need for a blood draw with subsequent lab work-up, (3) the elimination of complex blood analysis equipment, and (4) continuous, real-time determination of lactate levels that provide higher temporal resolution than can be provided by traditional draws and work-up. The system also provides for the monitoring of two or more analytes at the same time. For example, in addition to lactate, pyruvate levels are also known to spike in some traumas. Glucose and histamine are also key diagnostic measurements that are known to be useful in defining early treatment strategies and could be measured and monitored in conjunction with lactate levels.
Lactate oxidase (EC 1.1.3.15 or EC1.13.12.4) was obtained from Genzyme Diagnostics. D-Amino acid oxidase (EC1.4.3.3) was obtained from Calzyme Laboratories or BBI Enzymes. Apo-L-lactate oxidase (EC 1.1,3.15 or EC 1.13.12.4) was obtained as a gift from Professor Mark Richter, University of Kansas. (S)-6-hydroxynicotine oxidase was obtained as a gift from Professor Mark Richter, University of Kansas. Alcohol oxidase (EC 1.1.3.13) was obtained from MP Biomedical. Pyruvate oxidase (EC1.2.3.3) was obtained from Genzyme Diagnostics. L-Glucose oxidase (EC 1.1.3.4) was obtained from BBI Enzymes. L-Glutamate oxidase (EC1.4.3.11) was obtained from Yamasa. Acyl Co-A oxidase (EC 1.3.3.6) was obtained from Genzyme Diagnostics. Choline oxidase (EC 1.1.3.17) was obtained from Asahi Kasei Pharma Corporation. Glutathione Sulthydryl oxidase (EC 1.8.3.3) was obtained from Yamasa. Glycerolphosphate oxidase (EC 1.1.3.21) was obtained from Genzyme Diagnostics. Sarcosine oxidase (EC1.5.3.1) was obtained from Genzyme Diagnostics. Xanthine oxidase (EC 1.17.3.2) was obtained from Diazyme Laboratories. Oxalate oxidase (EC 1.2.3.4) was obtained from Roche Diagnostics. Cholesterol oxidase (EC 1.1.3.6) was obtained from Genzyme Diagnostics. Gamma-Glutaminyl-Putrascine oxidase (EC To be determined) was obtained as a gift from Professor Mark Richter, University of Kansas. Ascorbate oxidase (EC 1.10.3.3) was obtained from Calzyme Laboratories.
Co-factors FAD, FMN, Riboflavin, TPP, and MnCl2 were all obtained from Sigma-Aldrich. All analytes in Tables 1 and 2 were obtained from commercial sources.
For Examples 1-45 (Table 1) and Examples 53-64 (Table 2), the enzyme was dissolved in a 0.1 M solution of the indicated co-factor (in the case of no co-factor, enzymes were dissolved in water) to a nominal concentration of 220 mg/mL.
Each enzyme solution was allowed to incubate at 0° C. for ninety minutes.
Following the incubation period the immobilization mixture was formed by combining (i) 0.35 μL of each enzyme solution, (ii) 0.2 μL of ice cold purified (iii) 0.2 μL of ice cold L-ascorbate oxidase solution, (iv) 0.15 μL of ice cold bovine serum albumin (BSA) solution ([9048-46-8], Sigma) made from 0.4 mg of BSA in 20 μL purified H2O, and (v) 0.2 μL of ice cold glutaraldehyde solution (0.6 μL glutaraldehyde ([111-30-8], Sigma-Aldrich) in 39.4 μL purified H2O). The immobilization mixture was then applied to the surface of the sensing cavity. For examples 1-45, a commercially available Pinnacle Technology Inc. #7004-BLANK bare electrode pre-charged with a selective membrane was used. For Examples 53-64, a commercially available Pinnacle Technology Inc. #7004-BLANK bare electrode pre-charged with a selective membrane consisting of the indicated electropolymerized film. When the entire immobilization mixture was consumed, the biosensor was allowed to cure at room temperature. In the case of electrodes made with alcohol oxidase (EC 1.1.3.13), ascorbate oxidase was not included as part of the immobilization mixture.
For Examples 5, 7, 8, 11, 12, and 15, the post-immobilization activation of the enzyme, activation was carried out by incubation of the sensor with the co-factor as described in Table 1 at either 25° C. or 37° C. for a minimum of 30 minutes. This process of post-immobilization activation was usually performed no earlier than 1 day after the enzyme immobilization process as described above.
The biosensors in Examples 1-41, 43-45 and 53-64 were tested in 20 mL of 0.1 M phosphate buffered saline solution. Testing was performed at either ambient or at 37° C. A magnetic stir bar was added to the solution and the container was placed above a magnetic stirrer set to an adequate stirring speed. Biosensors were immersed in the buffer solution and connected to a potentiostat. A bias of 0.6 V with respect to a silver/silver chloride reference electrode (also immersed in the buffered solution) was applied to all electrodes. Once the bias was applied, the sensors were allowed to stabilize (approximately 10 to 20 minutes) and then the analyte for each Example was added to the stirred solution to achieve the indicated concentration within the beaker. All recordings were made on a commercially available Pinnacle Technology Inc. 8400 systems and Pinnacle Technology Inc, 8102 systems.
For Examples 46-52, the enzyme is dissolved in a 0.1 M solution of appropriate co-factor to a nominal concentration of 220 mg/mL.
Each enzyme solution is allowed to incubate at 0° C. for ninety minutes.
Following the incubation period the immobilization mixture is formed by combining (i) 0.35 μL of each enzyme solution, (ii) 0.2 μL of ice cold purified H2O, (iii) 0.2 μL of ice cold L-ascorbate oxidase solution, (iv) 0.15 μL of ice cold bovine serum albumin (BSA) solution ([9048-46-8], Sigma) made from 0.4 mg of BSA in 20 μL purified H2O, and (v) 0.2 μL of ice cold glutaraldehyde solution (0.6 μL glutaraldehyde ([111-30-8], Sigma-Aldrich) in 39.4 μL purified H2O). The immobilization mixture is then applied to the surface of the sensing cavity (e.g, of a commercially available Pinnacle Technology Inc. #7004-BLANK bare electrode pre-charged with a selective membrane or a commercially available Pinnacle Technology Inc. #7004-BLANK bare electrode pre-charged with a selective membrane consisting of an electropolymerized film). When the entire immobilization mixture is consumed, the biosensor is allowed to cure at room temperature. Ascorbate oxidase is included as part of the immobilization mixture as appropriate
The biosensors in Examples 42, 46, 47, 48, 49, 50, 51, and 52 are tested in 20 mL of 0.1 M phosphate buffered saline solution. Testing is performed at either ambient or at 37° C. A magnetic stir bar is added to the solution and the container is placed above a magnetic stirrer set to an adequate stirring speed. Biosensors are immersed in the buffer solution and connected to a potentiostat. A bias of 0.6 V with respect to a silver/silver chloride reference electrode (which is also immersed in the buffered solution) is applied to all electrodes. Once the bias is applied, the sensors are allowed to stabilize (approximately 10 to 20 minutes) and then the analyte for each Example to be tested is added to the stirred solution to achieve the indicated concentration within the beaker. All recordings are made on a commercially available Pinnacle Technology Inc. 8400 systems and Pinnacle Technology Inc. 8102 systems.
For Examples 53, 56, 57, 59, 61 and 63, the polyphenol film was electropolymerized according to the method of Chen (Chen, X. et al.;Biosensors Bioelect., 2002, 17, 1005-1013) onto a commercially available blank sensor (e.g., Pinnacle Technology Inc. #7004-BLANK bare electrode). For Example 55, the polypyrrole film was electropolymerized according to the method of Wassum (Wassum, K. M. et al.; Sensors, 2008, 8, 5023-5036) onto a commercially available blank sensor (e.g., Pinnacle Technology Inc. #7004-BLANK bare electrode). For Examples 54, 58, 60, 62, and 64, the poly-o-cresol film adapted the method of Chen (Chen, X. et al.; Biosensors Bioelect., 2002, 17, 1005-1013) for o-cresol, which was electropolymerized onto a commercially available blank sensor (e.g., Pinnacle Technology Inc. #7004-BLANK bare electrode). All recordings were made on a commercially available Pinnacle Technology Inc. 8400 systems and Pinnacle Technology Inc. 8102 systems.
In vivo bioscnsor recordings were performed on C57B16 male mice obtained from commercial sources (3.9±0.2 months of age, average weight of 29.8±0.9 g). Surgery and biosensors recordings were done according to the published procedure of Naylor et al. (Naylor et al. J. Electroanal, Chem. 2011, 656, 106-113).
This application claims the priority benefit of a provisional application entitled ENZYME CO-FACTOR ENHANCEMENT OF BIOSENSOR PERFORMANCE, U.S. Patent Application No. 61/381,695, filed Sep. 10, 2010, incorporated by reference herein,
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US11/51193 | 9/12/2011 | WO | 00 | 3/8/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61381695 | Sep 2010 | US |
