WO2010014978A1

WO2010014978A1 - Determination of circulating 1alpha, 25 (oh) 2d3 by enzyme electrode

Info

Publication number: WO2010014978A1
Application number: PCT/US2009/052564
Authority: WO
Inventors: Daniel Ludwig; Steven Rhieu; Tayhas Palmore
Original assignee: Brown University
Current assignee: Brown University
Priority date: 2008-07-31
Filing date: 2009-08-03
Publication date: 2010-02-04
Anticipated expiration: 2011-01-31

Abstract

This invention includes a method of determining the level of 1α,25 (OH)₂D₃ by the steps of (a) bringing a bio-electrode bearing an immobilized recombinant 1α-hydroxylase into electrical contact with a biological material; (b) determining the current generated at said bio-electrode caused by catalytic turnover of immobilized said recombinant 1α-hydroxylase; and, (c) relating said current to a level of 1α,25(OH)₂D₃ in said biological material

Description

DETERMINATION OF CIRCULATING 1ALPHA, 25 (OH) 2D3 BY ENZYME ELECTRODE

This application claims US provisional application Serial No. 61/137,480 filed August 1, 2008, the disclosure of which is incorporated herein by reference.

Field of the Invention

This invention includes a method of determining the level of 101,25(OH)₂D₃ by the steps of

(a) bringing a bio-electrode bearing an immobilized recombinant lα-hydroxylase into electrical contact with a biological material;

(b) determining the current generated at said bio-electrode caused by catalytic turnover of immobilized said recombinant lα-hydroxylase; and,

(c) relating said current to a level of lα,25(OH)₂D₃ in said biological material_.

Background

Vitamin D is metabolized by sequential hydroxylations in the liver and kidney to a family of secosteroids. There are two types of vitamin D compounds: cholecaliciferol (vitamin D₃), which is formed in the skin by photolysis of 7-dehydrocholesterol by UV radiation from sunlight and ergocalciferol (vitamin D₂), which is derived solely from plant sources. Overall vitamin status in humans is thus dependant on endogenous and exogenous sources. Vitamin D₃ is transported in the circulation via vitamin D binding protein and is hydroxylated in the liver to 25(OH)D₃. Another hydroxylation reaction occurs in the kidney to form the active hormone lα,25(OH)D₃. This reaction is tightly regulated by induction of a cytochrome P450 enzyme CYP27B1. Clinically, low vitamin D status is associated with a variety of diseases and conditions, including secondary hyperparathyroidism, impaired absorption of intestinal calcium, disturbed muscle metabolism, congestive heart failure, and prostate, breast and colon cancer. Severe deficiency has long been associated with rickets in children and osteomalacia in adults.

Although D and the active hormone can be measured directly in the blood, measurement of 25(OH)D would provide the best estimate of D levels because of the long half-like of the compound (around 3 weeks) and because the hydroxylation step is unregulated. Many assays have been developed for measuring 25(OH)D concentration in blood. However, these assays require chromatographic purification and this leads to variable co-precipitation of the analyte. A chemiluminsecence based assay exists which requires incubation of the serum with anti-vitamin D coated microparticles and an isoluminol derivative-conjugated 25(OH)D. The disadvantage of this approach is the lack of selectivity for 25(OH)D₂ and 25(OH)D₃ and its high costs. HPLC based assays are capable of resolving 25(OH)D₂ and 25(OH)D₃ but these assays are labor intensive and require costly equipment, complex sample preparation and large sample volumes.

Recent research on the electrochemistry of cytochrome P450s (CYPs) has focused on elucidating their electron transport pathways and their development for use in biosensors and bioreactors [1-8].

All documents cited herein are incorporated in their entirety by reference. (1) Shinki, T.; Shimada, H.; Wakino, S.; Anazawa, H.; Hayashi, M.; Saruta, T.; DeLuca, H.

F.; Suda, T. Proc. Natl. ScL U.S.A. 1997, 94, 12920-12925. (2) (a) Fraser, D. R.; Kodicek, E. Nature 1970, 228, 164-166. (b) Ghazarian, J. G.; Jefcoate, C. R.; Knutson, J. C; Orme-Johnson, W. H.; Deluca, H. F. J. Biol. Chem. 1974, 249, 3026- 3033.

(3) (a) Kazlauskaite, J.; Westlake, A. C. G.; Wong, L. L.; Hill, H. A. O. Chem. Commun. 1996, 18, 2189-2190. (b) Fleming, B. D.; Tian, Y.; Bell, S. G.; Wong, L. L.; Urlacher, V.; Hill, H. A. O. Eur. J. Biochem. 2003, 270, 4082-4088. (c) Aguey-Zinsou, K. F.; Bernhardt, P.

V.; De Voss, J. J.; Slessor, K. E. Chem. Commun. 2003, 3, 418-419. (d) Fantuzzi, A.; Fairhead, M.; Gilardi, G. J. Am. Chem. Soc. 2004, 126, 5040-5041. (e) Iwuoha, E. I.; Joseph, S.; Zhang, Z.; Smyth, M. R.; Fuhr, U.; Ortiz de Montellano, P. R. J. Pharm. Biomed. Anal. 1998, 17, 1101-1110. (f) Zhang, Z.; Nassar, A. E. F.; Lu, Z. Q.; Schenkman, J. B.; Rusling, J. F. J. Chem. Soc. Faraday Trans. 1997, 93, 1769-1774. (g) Shumyantseva, V. V.; Ivanov, Y.

D.; Bistolas, N.; Scheller, F. W.; Archakov, A. L; Wollenberger, U. Anal. Chem. 2004, 76, 6046-6052. (h) Johnson, D. L.; Conley, A. J.; Martin, L. L. J. MoI. Endocrinol. 2006, 36, 5188-5200. (4) (a) Gelo-Pujic, M.; Kim, H. H.; Butlin, N. G.; Palmore, G. T. R. Appl. Environ. Microbiol. 1999, 65, 5515-5521. (b) Fei, J. F.; Song, H. K.; Palmore, G. T. R. Chem. Mater. 2007, 19, 1565-1570.

(5) (a) Hamachi, L; Noda, S.; Kunitake, T. /. Am. Chem. Soc. 1991, 113, 9625-9630. (b) Rusling, J. F.; Nassar, A. E. F. J. Am. Chem. Soc. 1993, 115, 11891-11897.

(6) Uchida, E.; Kagawa, N.; Sakaki, T.; Urushino, N.; Sawada, N.; Kamakura, M.; Ohta, M.; Kato, S.; Inouye, K. Biochem. Biophys. Res. Commun. 2004, 323, 505-511.

(7) Rusling, J. F.; Zhang, H. Langmuir 1991, 7, 1791-1796.

(8) Joseph, S.; Rusling, J. F.; Lvov, Y. M.; Friedberg, T.; Fuhr, U. Biochem. Pharmacol. 2003, 65, 1817-1826.

(9) Laviron, E. J. Electroanal. Chem. 1979, 101, 19-28.

(10) McMurry, T. J.; Groves, J. T. in Cytochrome P450: Structure, Mechanism, and Biochemistry; Ortiz de Montellano, P. R., Ed.; Plenum Press: New York, 1986; pp 1-28.

(11) Yamamoto, K.; Uchida, E.; Urushino, N.; Sakaki, T.; Kagawa, N.; Sawada, N.; Kamakura, M.; Kato, S.; Inouye, K.; Yamada, S. /. Biol. Chem. 2005, 280, 30511-30516.

(12) Gunsalus, I. C; Meeks, J. R.; Lipscomb, J. D.; Debrunner, P.; Mϋnck, E. in Molecular Mechanisms Of Oxygen Activation, Hayaishi, O., Ed.; Academic Press: New York, 1974; pp 559-613.

(13) Daff, S. N.; Chapman, S. K.; Turner, K. L.; Holt, R. A.; Govindaraj, S.; Poulos, T. L.; Munro, A. W. Biochemistry 1997, 36, 13816-13823.

(14) Born, M. Z. phys. 1920, 1, 45-48.

(15) Kassner, R. J. Proc. Natl. ScL U.S.A. 1972, 69, 2263-2267. (b) Kassner, R. J. /. Am. Chem. Soc. 1973, 95, 2674-2677. (c) Salamon, Z.; Tollin, G. J. Bioenerg. Biomembr. 1997, 29, 211-221. (16) Sakaki, T.; Sawada, N.; Takeyama, S.; Kato, S.; Inouye, K. Eur. J. Biochem. 1999, 259,

731-738. (17) Laviron, E. /. Electroanal. Chem. 1979, 101, 19-28.

Accordingly, there is a need for an improved method to accurately measure vitamin D levels. Summary of the Invention

Disclosed is a method of determining the level of lα,25(OH)2D3 by the steps of (a) bringing a bio-electrode bearing an immobilized recombinant lα-hydroxylase such as CYP27B 1 into electrical contact with a biological material; (b) determining the current generated at an electrode caused by catalytic turnover of immobilized CYP27B1 (c) relating said current to a level of lα,25(OH)2D3.

This determination is typically made with reference to a second electrode bearing the same immobilized recombinant lα-hydroxylase into electrical contact with a reference material

Brief Description of the Drawings

FIG. 1 shows, from left to right, an exploded view of an exemplary embodiment of an apparatus for measuring the vitamin D level concentration, a schematic diagram of the electrode according to the invention, and schematically a process of electro- catalytic conversion of 25(OH)D₃ to 1(1,25(OH)₂D₃ using the electrode according to the invention; and FIG. 2 shows, schematically, a circuit for measuring electrolytic current.

Summary of the Invention Disclosed is a method of determining the level of lα,25(OH)₂D₃ by the steps of

(a) bringing a bio-electrode bearing an immobilized recombinant lα-hydroxylase such as CYP27B1 into electrical contact with a biological material;

(b) determining the current generated at an electrode caused by catalytic turnover of immobilized CYP27B1 (c) relating said current to a level of 10,25(OH)₂D_3.

This determination is typically made with reference to a second electrode bearing the same immobilized recombinant lα-hydroxylase into electrical contact with a reference material Detailed Description of the Invention

To overcome the foregoing problems and disadvantages, it is provided herein a method and apparatus based on electrochemical detection.

The instant device and method in a specific embodiment addresses use of CYP27B1 to determine levels of hydroxyvitamin D3, or other enzymes in the vitamin D pathway. The invention will be better understood with resort to the following definitions.

"Bio-electrode" is, broadly, an electrode that functions as an interface between biological structures and electronic systems, hi the present system the electrode comprises recombinant lα- hydroxylase (i.e., CYP27B1). CYP27B1 catalyzes the conversion of 25-hydroxy Vitamin D. During the catalysis, an electrical signal so generated is measured and calibrated against known concentrations. An lα-hydroxylase (CYP27B1) is a heme-containing monooxygenase that accepts two electrons and reduces on atom of 02 to water, inserting the other oxygen atom onto the substrate. Electrons can be transferred directly to CYP27B1, eliminating the need for components of the in vivo electron transport system. In vitro electrons can be supplied catalytically, with the catalytic current directly proportional to the amount of 25(OH)D.

"Biological material" shall be broadly understood to mean blood, serum, tears whether in vivo or in vitro.

Referring now in particular to FIG. 1, the invention relates to a biosensor comprising a device for measuring vitamin D concentration comprising an enzyme based working electrode and a means of measuring an electrical signal generated at the electrode. The electrode (which in one exemplary embodiment represents a working electrode WE shown in FIG. 2) includes recombinant lα-hydroxylase (CYP27B1). This enzyme catalyzes the conversion of 25-hydroxy Vitamin D to its active form 1,25-dihydroxy Vitamin D. During the catalysis, the electrical signal generated will be measured by the measuring means and calibrated against known concentrations. CYP27B1 is a heme-containing monooxygenase that accepts two electrons and reduces one atom of 02 to water, inserting the other oxygen atom onto the substrate. In vitro, electrons can be supplied catalytically, with the catalytic current directly proportional to the amount of 25(OH)D.

Several investigators have shown that an enzyme can be integrated with electrodes to form a biosensor, and with the development of microfabrication technology biosensors have become inexpensive, miniaturized, and reproducible. Various methods of immobilizing P450s such as CYP27B1 onto the electrodes have been described in the following publications..

CYP450 Immobilization Techniques: 1) Multilayer Polyionic Films [Cfiembiochem 2003 4: 82-89.]

Films of CYP27B1 with polyions such as PSS (polystyrene sulfonate) and PDDA (poly(diallyldimethylammonium chloride) are used to immobilize CYP27B 1 "sandwiching" the enzyme between two polyionic layers. The formation of films can be denoted as (protein/polyion)_n, where n is the number of protein/polyion bilayers. Construction of the films is monitored using quartz crystal microbalance (QCM) resonators to determine the thickness of the films. The Sauerbrey equation gives the relation between adsorbed mass and frequency shift ΔF(Hz) of the quartz resonator.

2) Tethering onto chemically modified electrode surface [Expert Opin. Drug Toxicol. 2006 2: 581-589.]

The use of metal affinity surface/histidine-tag technology (HisChip™, Taenia Pty) to immobilize proteins on electrode surfaces was recently reported (/. Am. Chem. Soc. 2005 127: 2018-2019). Features of this protocol are increased flexibility, homogeneity and stability of the attached P450 enzymes. CYP27B1 has a C-terminal tetra histidine tag, which is suitable for employing such immobilization method. The tethering process is designed such that conformational changes that occur during electron transfer or substrate binding can occur freely and unhindered at the site or location of the P450 enzyme. The HisChip method of immobilization results in electron transfer rates for thioredoxin 80-times faster compared with covalent attachment of the protein to the electrode under similar experimental conditions. This improved electronic coupling may be the result of a more efficient orientation of the protein.

3) Phospholipid Bilayer/ Synthetic Lipid Layer [J. Chem. Soc, Faraday Trans. 1997 93: 1769-1774.1 Vesicle dispersion of the lipids such as DDAB (didodecyldimethylammonium bromide) and DMPC (Dimyristoyl-L-a-phosphatidylcholine) is accomplished by sonication as described J. Chem. Soc, Faraday Trans. 1997 93: 1769-1774. CYP27B1 is subsequently added to make vesicle-enzyme dispersions for casting onto electrodes. Films are prepared from 2 mM aqueous vesicle dispersions of DMPC or DDAB containing 40 μ M CYP27B 1.

4) Conducting Polymer [Biosensors and Bioelectronics 2008 23: 1733-1737.] CYP27B 1 is immobilized onto the working electrode surface by electropolymerization.

CYP27B1 has been immobilized onto a gold working electrode by pyrrole (py) electropolymerization. During electropolymerization, the oxidized form of the polymer is positively charged and requires the incorporation of doping anions in order to maintain its electroneutrality. The association between polymer cations and dopant anions depends on the electrochemical conditions applied during the process of polymerization. The electrode is immersed in a solution containing anionic dopant, pyrrole, and CYP27B 1 at room temperature. The potential domain is scanned between OV and +0.9V vs. Ag/AgCl. As a result, CYP27B1- polypyrrole films grow onto the gold electrode.

5) Clay nanoparticles [Anal. Chem. 2004 76: 6046-6052.]

The clay colloid is prepared from sodium montmorillonite colloid, colloidal Platinum, and deionized water. Subsequently, electrodes are prepared with aliquots of 1 : 1 (v/v) mixtures of the clay colloid with either water or 0.1% CHAPS followed by incubation in CYP27B 1 solution. Alternatively, the clay colloid is mixed 1 : 1 (v/v) with protein stock solution or monomerized protein solution, and 5 μL aliquots of the final mixtures are spread on a freshly polished carbon electrodes. For film formation, the electrodes are put in a refrigerator overnight followed by a 30-min final drying under low-pressure atmosphere.

Calibration options may include chronoamperometry, voltammetry, and linear sweep voltammetry. The physiological levels of 25(OH)D₃ in human serum range between 10-40 ng/ml (25- 125 nM). As for an amperomeric sensor, the output current based on catalytic reaction of immobilized CYP27B1 is generally between nano to micro amperes. Based on our preliminary data depending on the method of immobilization, we observed a redox potential of immobilized CYP27B1 ranging between -180 and -450 mV vs. Ag/ AgCl. In order to achieve the electrocatalytic conversion of 25(OH)D₃, it is useful to poise a potential at a value more negative than -450 mV vs. Ag/AgCl.

FIG. 2 shows a block circuit diagram of an apparatus 20 for measuring electrolytic currents with a control amplifier (CA) to control the potential of the working electrode (WE) with respect to the reference electrode (RE) by injecting current through the counter electrode (CE), with a current follower (IV), which outputs a voltage proportional to the current. The microelectronic circuit was designed with the goal of simplicity and the minimization of noise in order to measure small currents. Although the working electrode (WE) is no longer grounded, it is held at virtual ground potential by the current follower (IV). On one embodiment the control amplifier (CA) has high open-loop gain and a high input impedance to faithfully control the cell. In such embodiment the current follower (IV) uses an op-amp with low offset current, high input impedance, while introducing very little noise. The resistor (R) used in the feedback loop is selected based on the desired current range. A capacitor (C) is placed in the feedback loop of the current follower (IV) to serve as the first stage of filtering. A stronger low-pass filter (LPF) is employed after the current follower (IV) to remove all frequencies above the Nyquist frequency of the analog-to-digital converter (ADC). A microcontroller is used to generate the control waveform for the experiment, acquire the resulting data, determine the catalytic current, compare this current to a preset calibration current, and output the result to a liquid crystal display (LCD). The microcontroller will also perform additional levels of digital filtering as necessary. A digital- to-analog converter (DAC) and the ADC are used to translate signals between the digital realm of the microcontroller and the analog realm of the potentiostat circuit. The potentiostat circuit design in this embodiment is essentially conventional.

The design was tested by attaching the circuit to an electrochemical cell. A standard 2,2'- azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) was used as the redox active molecule. Experiments were performed with a platinum working electrode (WE), a platinum mesh counter electrode (CE), and a Ag/AgCl reference electrode (RE). Current- voltage measurements were performed in at a scan rate of 20 mV/s in a 0.2 M HCl electrolyte. The concentration of ABTS was varied between 1 mM and 33.8 μM, with the peak current varying from over 10 μA to almost no peak current. The measured data confirm that the potentiostat operates as expected. Surprisingly little noise, on the order of 200 nA, was observed in the absence of strong analog or digital filtering or special cable shielding. When using LabVIEW in a Windows® environment, the maximum data exchange rate was about 300 Hz.

CYP27B1/DDAB/EPG film preparation

DDAB films were fabricated as described previously³'⁴ with minor modifications. A freshly polished EPG disk electrode was coated with 15 μL of 10 mM DDAB in chloroform, which was evaporated at room temperature for Ih. DDAB/EPG electrodes were placed into 50 μL of 6.85 μM purified CYP27B1 for 1 h at 4 ⁰C. The modified C YP27B 1 /DDAB/EPG electrode was dried under a stream of nitrogen prior to the experiments.

Electrochemistry experiments

A three-neck round-bottom glass was utilized as the electrochemical cell containing an EPG disk (working, geometric area, A = 0.126 cm²), a platinum gauze (counter), and a Ag/AgCl (reference, 0.197 V vs. NHE). All potentials in this study were reported with reference to Ag/AgCl. An EG&G Potentiostat/Galvanostat, model 263 A, was used for cyclic voltammetry (CV) experiments. All CV experiments were performed at room temperature (23 ⁰C) with 4 mL of 50 mM potassium phosphate buffer (pH 7.4) containing 0.1% CHAPS and 50 mM NaBr. Prior to performing CV, the working solution was fully deoxygenated (< 1.5 ppm) by purging with argon for at least 30 min. An argon atmosphere was also maintained over the solution throughout the experiments. For electrocatalytic studies, the same conditions were applied except the working solution was fully oxygenated by purging with oxygen for at least 10 min. An oxygen atmosphere was also maintained over the solution. After adding 25-hydroxyvitamin D₃ to a final concentration of 0.4 μM, the solution was allowed to equilibrate for at least 5 min prior to running the CV experiment. For HPLC analysis, CV conditions were kept the same. Five cycles were performed with an interval of at least 1 min between each cycle to allow the solution to re- equilibrate.

High-performance Liquid Chromatography

High-performance liquid chromatography (HPLC) analysis was performed with a Waters System Controller (Millennium 3.2) equipped with a photodiode array detector (Model 996) to monitor the ultraviolet (UV) absorbing material at 265 nm. A straight phase HPLC utilized a Zorbax-SIL column (9 x 250 mm) (Dupont, Wilmington, DE) eluted with 20% (v/v) isopropanol in hexane at a flow rate of 2 niL/min. The synthetic 25(OH)D₃ and 101,25(OH)₂D₃ standards in pure ethanol were dried under nitrogen and dissolved in 300 μL of the mobile phase solvent prior to injection. To prepare the electrocatalysis solution for HPLC, 24 niL of a mixture of methanol/methylene chloride (1:2, v/v) was added for extraction. The organic phase was collected and dried under nitrogen. Prior to injection, the solutes were re-dissolved in 300 μL of the mobile phase solvent. Referring back to FIG. 1, and more particularly to the left side of FIG. 1, the exemplary device 10, which may be made portable, battery powered and hand-held, includes a bottom case 11, a battery to supply electric power to the electronic circuits (on microelectronics board 13) of device 10, and a top case 14 with a sample port 15. The device 10 may also include a liquid crystal display 16 for displaying measurement data, such as the vitamin D level. Optionally, a desiccant 17 may be incorporated in the device. A test strip 18, which includes the working electrode with the CYP27B1 coating, is inserted into sample port for the measurement.

In a particular embodiment the device applies a fixed potential to the counter electrode to determine the steady-state amperometric current, and uses the calibration curve to determine vitamin D concentration. Particular embodiments minimize noise and allow measurement of very small currents. In a specific embodiment the circuit was interfaced with a PC using either a National Instalments data acquisition card or PC interface. In certain applications (virtual instrument) was written using LabVIEW. This Virtual Instrument was designed to perform a cyclic voltammogram, allowing the user to set experiment parameters and view the data in real time. In particular embodiments, a CYP27B 1 biosensor is sensitive and accurate, allowing for rapid and inexpensive determination of Vitamin D status a clinical or home setting.

Claims

We Claim

1. A method of determining the level of 101,25(OH)₂D₃ by the steps of

(a) bringing a bio-electrode bearing an immobilized recombinant lα-hydroxylase into electrical contact with a biological material; (b) determining the current generated at said bio-electrode caused by catalytic turnover of immobilized said recombinant lα-hydroxylase; and, (c) relating said current to a level of 1(1,25(OH)₂D₃ in said biological material_.

2. A method of determining the level of lα,25(OH)₂D₃ by the steps of (a) bringing a bio-electrode bearing immobilized CYP27B 1 into electrical contact with a biological material; (b) determining the current generated at said bio-electrode caused by catalytic turnover of immobilized CYP27B1; and,

(c) relating said current to a level of 10,,25(OH)₂D₃ in said biological material_.