text:
|Publication number
|Publication type
|Publication date
|Jul 22, 1975
|Filing date
|Dec 20, 1971
|Priority date
|Oct 28, 1971
|Publication number
|Melvin H Keyes
|Original Assignee
|Owens Illinois Inc
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (6), Referenced by (12), Classifications (12), Legal Events (1)
|External Links: USPTO, USPTO Assignment, Espacenet
United States Patent 1191 Keyes 1 1 ELECTROCHEMICAL POTENTIOMETRIC METHOD FOR SELECTIVELY DETERMINING ALKALINE PHOSPHATASE CONTENT IN AQUEOUS FLUIDS [75] Inventor: Melvin H. Keyes, Sylvania, Ohio [73] Assignee: Owens-Illinois, lnc., Toledo, Ohio [22] Filed: Dec. 20, 1971 [21] Appl, No.: 209,741
Related US. Application Data [63] Continuation-impart of Ser. No. 193,468, Oct. 28,
1971, abandoned.
OTHER PUBLICATIONS Montalvo et a1., Anal. Chem.," Vol. 41, No. 13, Nov. 1969, pp. 1897-1899. Mosbach, "Scientific America," V01. 224, No. 3, March 1971, pp. 26-33. Guilbault et a1., Anal. Chim. Acta," 52, (1970), pp. 287-294. Guilbault et a1., JACS," April 22, 2533-2538. Guilbault et a1., Anal. Chem, Vol. 41, No. 4, April 1969, pp. 600 605. White et a1., Principles of Biochemistry," 1959. 3rd ed., p. 501.
[ 1 July 22, 1975 Primary Examiner-T. Tung Attorney, Agent, or FirmRichard D. Heberling; E. J.
Holler [57] ABSTRACT Electrochemical apparatus and potentiometric method for rapidly, accurately and selectively determining alkaline phosphatase content in aqueous fluids. Electrochemical apparatus of this invention can comprise a D-serine deaminase electrode (hereinafter also referred to as a D-serine specific or enzyme electrode) referenced to either a constant or variable potential reference electrode. According to the method of this invention, a known quantity of D-phosphoserine is added to an aqueous fluid sample containing an unknown concentration of alkaline phosphatase. The D- phosphoserine is hydrolyzed by the alkaline phosphatase to inorganic phosphate and D-serine at a rate proportional to the concentration of alkaline phosphatase in the sample, while the sample is continuously monitored with the apparatus of this invention at regular intervals for changes in D-serine content, The incremental increases in D-serine content in the fluid sam ple are reflected in increases in electrical potential as a result of the deamination of D-serine at the enzyme electrode. The particular combination of enzyme electrode, together with the ionic composition of the aqueous fluid sample to be subjected to such assay, will directly affect the selection and sequence of steps in conducting such analysis. For example, where the aqueous fluid sample contains sodium, potassium and /or ammonium ions in concentrations sufficient to generate an electrical potential at the ammonium ion membrane of the enzyme electrode, the sample need be contacted with cation-exchange resin either prior to or concurrent with such analysis. The apparatus and method of this invention are readily adaptable for clinical assay of clinical samples of biologic fluids for alkaline phosphatase content in the diagnosis of various forms of obstructive jaundice and other hepatic diseases.
6 Claims, 6 Drawing Figures PATENTEDJUL22 ms 3.896.008
FIG. 2 fi INVENTOR. (sumo H. kevcs PATENTED JUL 2 2 I975 SHEET FIG. 4
FIG. 5 4" INVENTOR. MELVIN H. LZEVES BY QHQ H RRO 8 E weblfiousfi Wows ELECTROCHEMICAL POTENTIOMETRIC METHOD FOR SELECTIVELY DETERMINING ALKALINE PHOSPHATASE CONTENT IN AQUEOUS FLUIDS CROSS REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of my copending application of the same Title, Ser. No. 193,468, filed Oct. 28, 1971, now abandoned.
BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to an improved electrochemical apparatus and a potentiometric method for selectively determining alkaline phosphatase content of aqueous fluids. More specifically, the apparatus and method of this invention enable selective determination of alkaline phosphatase content of industrial and biologic fluids also containing ionic and proteinaceous materials ordinarily considered incompatible with this type analysis. Analysis of alkaline phosphatase content is achieved by determining its rate of hydrolysis of D- phosphoserine. As D-phosphoserine is hydrolyzed by the alkaline phosphatase, D-serine is produced at a rate and in concentrations proportional to alkaline phosphatase in the sample. The rate of change in D-serine concentration in the sample is subsequently monitored at regular intervals by selective deamination of this compound at a D-serine specific enzyme electrode and the recording of the potential generated as a result of change in ammonium ion concentration in the sample with an electrode sensitive to such changes.
2. Description of the Prior Art Traditionally, analytical electrochemical apparatus have had a sensing electrode and a constant potentialtype reference electrode. The sensing electrode of this type of apparatus is ordinarily designed to respond to ionic activity of a compound in solution whose concentration is sought to be determined or from whose activity the concentration of another compound can be determined; whereas, the constant potential-type reference electrode (e.g. a saturated Calomel electrode) only puts out a constant electrical signal irrespective of the ionic activity of the background materials in solution.
With the development of various types of cationsensitive glass electrode membranes by Eisenman (U.S. Pats. Nos. 2,829,090 and 3,041,252) and other researchers has come a resurgence in interest in the field of analytical potentiometry. Electrodes are now currently available that directly measure ionic activity, that are nondestructive, easy to use and relatively inexpensive. These ion-selective electrodes generally fall into three major categories: (a) glass electrodes; (b) solid-state or precipitate electrodes; and (c) liquidliquid membrane electrodes.
Paralleling the development of the ion-sensitive glass membrane has been the increasing interest in the use of enzymes in analytical chemistry as a result of the ability of researchers to effectively insolubilize these biocatalysts in a form which permits subsequent recovery and reuse without substantial loss of the enzyme's activity. Early attempts to combine these two technologies achieved only marginal success because of the inability of the ion-sensitive membranes of the so-called enzyme electrodes to effectively discriminate between products of the enzyme-substrate reaction and those materials indigenous to the aqueous sample which were also capable of generating an electrical potential at the enzyme electrode. Initial attempts to reduce the effect these indigenous materials have on enzyme electrode response through the pre-treatment of the aqueous fluid sample with ion-exchange resins has resulted in substantial improvement in accuracy in enzyme electrode response, although such pre-treatment dramatically increases the time required to complete such analysis.
Even after separate pre-treatment of the sample solution with cation-exchange resin, those apparatus wherein the enzyme electrode is referenced to a traditional type of constant potential reference electrode are still unable to accurately detennine low concentrations (-10" to 10 M) of substrate in test samples without substantial error. The deficiency in this type of system is believed attributable, in part, to the fact that all traditional types of constant potential reference electrodes have a narrow aperture in their immersion tip which provides for the controlled flow of an electrolyte, ordinarily a potassium salt, from the internal chamber of the immersion tip into the sample being analyzed. This constant low-volume flow of potassium ions into the sample solution can result in the independent generation of an electrical potential at the enzyme electrode reflecting this potassium ion leakage.
Attempts to compensate for this leakage by the addition of cation-exchange resin directly to the sample during analysis have not proven very practical or successful. Cation-exchange resin, when added directly to the solution being assayed, reportedly results in the generation of an electrical potential independent of the concentration of other cations also present in the sample, Guilbault et al., Anal. Chim. Acta, 52, 287 1970). This change in potential has been attributed by Eisenman to a change in liquid junction potential in solutions of colloids or in suspensions, G. Eisenman, Glass Electrades for Hydrogen and Other Cations, Marcel Dekker, New York, (1967). The independent generation of potential by the ion-exchange resin can, however, be controlled by limiting the quantities of these resins that are present in the sample during analysis and by rigorous buffering of the test sample. This limited addition of cation-exchange resin, however, appears to be inadequate in controlling both the interfering cations indigenous to the sample and the electrolyte seepage from the traditional type of reference electrode.
The development of an analytical system apparently free from the problems associated with reference electrode contamination of the sample has recently been reported in the literature, Guilbault et al., Anal. Chim. Acta, 52, 287 (1970). Guilbault observed that by the use of reference electrode having a continuous cationsensitive glass membrane at its immersion tip in place of a standard saturated calomel electrode, urea content of test samples could be potentiometrically determined relatively free from the problems discussed previously.
The Guilbault system is, however, subject to error where successive samples, having variable concentrations of ionic contaminants (Na", K", NH, etc.), are analyzed. The variable potential reference electrode of his system will vary in its response with those variations in ionic composition and, therefore, result in an artificial shift in the magnitude of the potential unless a series of calibration curves are also made reflecting the shifting concentrations of these so-called ionic contaminants.
In order to avoid the problems encountered by electrolyte contamination from the reference electrode and yet provide a system capable of measuring successive samples without recalibration of the apparatus for each and every ionic contaminant. a system is needed wherein the reference electrode has a continuous nonporous membrane and is sensitive to an ion in solution whose concentration is constant from sample to sample or whose concentration in such samples can be readily maintained at a constant level by buffering. The referencing of an enzyme electrode to a pH electrode, for example, provides one possible solution to this problem.
SUMMARY OF THE INVENTION This invention is an electrochemical apparatus comprising an enzyme electrode and a reference electrode electrically connected in series to a potentiometer, wherein the enzyme electrode is provided with an ammonium ion-sensitive membrane in contact with and substantially encapsulated by a coating of immobilized D-serine deaminase, said coating containing a catalytically effective amount of D-serine deaminase and being permeable to both the aqueous fluid sample and reaction products of the aqueous fluid sample and the D- serine deaminase. The D-serine specific electrode of this apparatus can be referenced to either a constant potential or variable potential electrode.
In the preferred apparatus of this invention, the D- serine specific electrode is referenced to either a pH or a cation-sensitive variable potential electrode.
Accordingly, the potentiometric method of this invention for selectively determining alkaline phosphatase content of an aqueous fluid comprises initially diluting the sample in an appropriate buffer having a pH in the range of from about 8.5 to about 9. The degree of dilution of the sample with buffer should be sufficient to reduce the native concentration of alkaline phosphatase in a typical sample of, for example, blood serum, from about 60 to 100 m Units (International Units) per milliliter at 25C. to about 30 to about 50 m Units. Subsequent to dilution, the sample is contacted with an enzyme electrode and reference electrode of an apparatus of the type described above and the background activity of the sample recorded. Once an activity base-line for the sample has been ascertained, D-
'phosphoserine is added to the diluted sample in concentrations ranging anywhere from about 5X10 to about 5X l moles per liter for each 50 m Units of alkaline phosphatase activity. After the D-phosphoserine is added, incremental changes in electrical potential are recorded at regular intervals for a period ranging anywhere from about to about minutes or until sufficient data is available for determination of the rate of hydrolysis of D-phosphoserine by the alkaline phosphatase present into the sample.
BRIEF DESCRIPTION OF THE DRAWING FIG. 1 is a side-elevational view of the electrochemical apparatus of the invention;
FIG. 2 is an elevational view, partly in section, of the constant potential reference electrode of FIG. 1;
FIG. 3 is an elevational view, partly in section, of the enzyme electrode of FIG. 1;
FIG. 4 is a side-elevational view of the preferred embodiment of the electrochemical apparatus of the invention;
FIG. 5 is an elevational view, partly in section, of the variable potential reference electrode of FIG. 4;
FIG. 6 is an elevational view, partly in section, of the enzyme electrode of FIG. 4 (substantially identical to FIG. 3).
DESCRIPTION OF THE INVENTION INCLUDING PREFERRED EMBODIMENTS Electrodes An electrode which can be used in the preparation of the D-serine specific electrode and as a variable potential reference electrode of the apparatus of this invention can be any ion-sensitive device capable of detecting changes in ammonium ion activity in aqueous solution and converting it to an electrical signal; e.g. cationsensitive glass electrode, Model No. 39l37, and ammonium ion-sensitive solid-state electrode, Model No. 39626, both manufactured by Beckman Instruments, Inc., Fullerton, Calif.
structurally, the D-serine specific enzyme electrodes 1, 1', shown in FIGS. 3 and 6, each comprise a hollow glass stem 2, 2' having an opening at each end thereof. One end of each glass stem is tightly capped with a lid 3, 3 which acts both as a closure and as a support for an electrically conductive lead 4, 4' which forms part of the internal reference electrode 5, 5'. The opposite end of each glass stem, the immersion tip 9, 9 of the enzyme electrodes is sealed with a continuous ammonium ion-sensitive membrane 6, 6', which is itself encapsulated by a coating of insolubilized enzyme 7, 7. A mesh material 8, 8' anchored to the immersion tip of each electrode by means of an elastic O-ring 10, I0 provides additional mechanical support for the insolubilized enzyme coating. The interior chamber 11, ll of each immersion tip is partially filled with an electrolytic medium 12, 12' capable of transmitting the electrical potential, generated by the presence of ammonium ions at the outer surface of the glass membrane, from the interior wall of the glass membrane to the internal reference electrode.
The variable potential reference electrode 25 illustrated in partial section in FIG. 5 is of substantially identical physical structure to those D-serine specific enzyme electrodes 1, 1' illustrated in partial section in FIGS. 3 and 6 respectively; its physical structure, however, differing, of course, in that the ion-sensitive membrane 6" of its immersion tip 9" remains fully exposed to the aqueous test sample 20.
The ion-sensitive membrane of these electrodes can be bulbous in shape, that is substantially spherical and of a diameter equal to or larger than the terminal end of the glass stem of the electrode, or blunt ended. The ionic sensitivity and selectivity of this membrane will vary directly with its permselectivity, which in turn is determined by the membrane's chemical composition. The ion-sensitive membranes of these variable potential electrodes can be continuous non-porous diaphragms selectively permeable to one or more cations and/0r anions. For example, the glass composition of the ion-sensitive membrane of Beckman's cationsensitive glass electrode (Model No. 39137) is believed to be based on an alkali-alumina-silica composition in the nature of that disclosed in US. Pat. No. 3,278,399. The cation-sensitive electrode referred to above has been reported by its manufacturer to be equipped with an ion-sensitive membrane having the following ionic selectively H Ag* K NH, Na Li Mg Ca The variable potential electrode of the apparatus of this invention can be alternatively equipped with a highly selective membrane sensitive only to hydrogen ions and in some instances, sodium ions, but only at higher pH levels). These electrodes, commonly referred to as pH electrodes, are readily available commercially in a variety of shapes and sensitivities, e.g. Model No. 39000, (general purpose electrode) and 39004 (Type E-2 electrode having low-sodium ion error), Beckman Instruments, Inc., Fullerton, Calif.
A second type of electrode which can be used as a variable potential electrode of the apparatus and in the method of this invention is the highly selective solidstate ammonium ion-sensitive electrode briefly referred to previously. This particular type of solid-state electrode is not, however, as durable as the glass electrode, the organic sensor material of its immersion tip being gradually chemically and physically eroded over relatively brief periods of use. Other solid-state variable potential electrodes which, for example, can also be referenced to the enzyme electrode of this invention are selective for the detection of activity of calcium ions; chloride ions; or fluoride ions.
The constant potential electrode that can be referenced to the D-serine specific electrode of the apparatus of this invention can be any device which has a chemically-inert junction, a low electrical resistance, a low-volume flow rate of electrolytic solution and, of course, enables measurements of the potential developed at the enzyme electrode by completion of the electrical circuit across the test sample, e.g. Quartz Junction reference electrode (Calomel Internal) Model No. 39400 or Fiber Junction reference electrode (Calomel Internal) Model No. 39170, Beckman Instruments, Inc., Fullerton, Calif.
The constant potential reference electrode 13 illustrated in partial section in FIG. 2 comprises a hollow glass stem 14 tapered to a narrow aperture 15 at the immersion tip end 16 and open at the opposite end thereto. The open end of the glass stem is capped by a lid 17, serving both as a closure and as a supportive means for an electrically conductive lead 18 which forms part of the internal reference electrode 19. The aperture at the immersion tip of the constant potential reference electrode permits communication between the solution 20 being assayed and the electrolyte 21, normally a saturated potassium chloride solution, occupying the internal chamber 22 of the immersion tip of the electrode. An asbestos fiber wad 23 in the lower portion of the internal reference electrode allows a controlled flow of Calomel 24 through the aperture 25 at the base of the internal reference electrode into the internal chamber of the immersion tip, thereby establishing electrical contact between the test solution and the internal reference electrode.
The D-serine specific electrode used in the apparatus of this invention is prepared by encapsulation of the ammonium ion-sensitive portion of the immersion tip of an electrode with a substrate permeable layer of insolubilized D-serine deaminase. The manner of insolubilization of the D-serine deaminase does not appear to be of critical importance to the apparatus and method of this invention, to the extent that the D-serine deaminase retains substantial catalytic activity and can be intimately affixed to the sensing portion of the immersion tip, either by physical or chemical means.
Several of the standard methods currently available for the insolubilization of enzymes can be used in the preparation of the encapsulating layer of the enzyme electrode of the apparatus of this invention. Among those methods which have been found suitable in this regard are entrapment of the enzyme in a polymer hydrogel, covalent bonding of the enzyme to the functional groups of both natural and synthetic polymeric substances or absorption of a solution of the enzyme on a netting and then covering the netting with a dialysis membrane permeable to substrate trasport but impermeable to the passage of the enzyme. The insolubilization of enzymes by entrapment in a highly crosslinked polymer hydrogel, especially polyacrylamide, can often adversely affect the conformation of the enzyme macromolecule resulting in denaturation and loss of biocatalytic activity, Degani and Miron, Biochim. Biophys. Acta 212,362-364 (1970). Therefore, adjustment of the concentration of cross-linking agents should be carefully controlled, as suggested in the above article, in order to permit retention of maximum biocatalytic activity of the enzyme. Such adjustment in the degree of cross-linking of the polymer hydrogel need also take into full account the permeability requirements of the system. An excellent review of the techniques and mechanisms involved in enzyme insolubilization has recently been published by Klaus Mosbach', his article appearing in the Mar. l97l issue of Scientific American, Vol. 224:3, 26 (1971); see also generally Katchalskis article, appearing in Structure Function Relationship of Proleolyric Enzymes (Desnuelle, Neurath and Ottesen, Eds.) Munksgaard, Copenhagen, Denmark, pp. l98-220 (l970); and lnman and Dintzis, Biochemistry 8, 4074 1969).
During insolubilization of the D-serine deaminase, especially during entrapment in a polymer hydrogel, the co-enzyme, pyridoxal S-phosphate, may become dissociated from the enzyme protein and dialyzed away, rendering the insolubilized enzyme partially inactive. ln order to provide for this contingency, it is advisable to add this coenzyme to the polymerization medium during insolubilization in the hydrogel. Ordinarily, 5X10 mole per liter of coenzyme added directly to the polymerization medium will minimize such activity loss. As a further precaution, a like amount of the coenzyme can also be added directly to the sample during assay.
The physical form of the insolubilized enzyme may, however, determine the form of the encapsulating layer, and the means required to achieve encapsulation of the sensing tip of the electrode. The physical shape of the sensing tip of the electrode can also influence the form of the encapsulating layer, and the technique required to achieve such encapsulation. Encapsulation of the ammonium ion-sensitive membrane of the immersion tip of the electrode need not be complete, nor need the enzyme itself be in intimate contact with the ammonium ion-sensitive portion of the electrode. AS long as the insolubilized D-serine deaminase substantially and intimately covers the ammonium ion-sensitive tip of the electrode and the insolubilized enzyme is itself permeable to both the substrate and ionic transport of ammonium ions, hydrolysis of D- serine by D-serine deaminase either occurring within the insolubilized enzyme matrix or on the surface of the encapsulating layer will generate an increase in electrical potential at the sensing membrane surface. Encapsulation of the ammonium ion-sensitive membrane of the immersion tip of the enzyme electrode should, however, be as complete as possible; especially in those instances where the test sample has not been pre-treated with ion-exchange resin for the complete removal of ionic interferants which are also capable of generating a potential at the enzyme electrode. If this ammonium ion-sensitive membrane is not totally encampsulated, only that portion of the membrane so enveloped should be exposed to the aqueous fluid sample.
The physical and chemical nature of the insolubilizing medium selected will, to a certain extent, be determined by the operating conditions to which the enzy me electrode is exposed. For example, if the insolubilizing medium is sensitive to degradation upon exposure to alkali, it would probably not prove suitable where potentiometric measurement is to be made with the enzyme electrode in a highly caustic solution. Similarly, an insolubilizing medium that did not have sufficient physical durability would probably not be suitable in the preparation of an enzyme electrode for a continuous or a semicontinuous monitoring system because of consequent hydrodynamic erosion of the encapsulating layer from the continuous flow-through of the solution being analyzed. The problems encountered by hy drodynamic erosion of the encapsulating layer can be ameliorated to a limited degree by covering the encapsulating layer with a dialysis membrane anchored to the stem of the electrode by an elastic O-ring. Of course, the dialysis membrane used to shield the enzyme layer should itself be permeable to substrate molecules.
Because physical and chemical durability of the enzyme layer is essential in a semicontinuous or continuous monitoring system, the method of choice for enzyme insolubilization in the preparation of the enzyme electrode favors covalent bonding of the enzyme to the insolubilizing medium.
in some instances, it may be advisable and necessary to provide a mesh material to lend additional physical support to the enzyme layer or as a means of attaching the insolubilized enzyme to the ion-sensitive membrane of the enzyme electrode.
Although the nature and physical parameters with respect to the mesh size of such supporting materials do not appear to be of critical importance, the thickness of such supporting materials can, to a limited degree,
I affect the response time of the enzyme electrode by indirectly affecting the thickness of the encapsulating enzyme layer. Mesh materials are also available that can be used as an absorption medium or carrier for the soluble enzyme; however, unless the mesh material is itself subsequently covered, after attachment to the electrode tip, with a layer of dialysis type material impermeable to diffusion of these larger proteinaceous molecules, the soluble enzyme will be readily stripped from the mesh upon immersion of the enzyme electrode into an aqueous medium. Mesh materials which have very fine sizes (-60 micron pores or less) can be used to attach the insolubilized enzyme (ordinarily a powder or granular material) to the electrode, provided the pore size of the mesh is sufficiently retentive of the insolubilized enzyme. The thickness of the encapsulating enzyme layer should not exceed one-tenth of a millimeter and preferably should be about half that thickness in order for the system's response times to be within the range of from about to about 120 seconds.
The quantity of enzyme that need be affixed to the ammonium ion-sensitive portion of the enzyme electrode is determined ultimately by the enzymes activity. If, for example, the activity of a deaminase enzyme, such as urease, is 375 Sumner Units per gram, the concentration of urease needed to effectively and yet economically catalyze the deamination of urea in sufficient quantities to generate a reproducible Nernstian response which accurately reflects (to the extent practically possible) the concentration of urea in the sample, will generally not exceed about 20 milligrams enzyme per cubic centimeter of polymeric insolubilizing medium.
Although higher concentrations of urease on the enzyme electrode have reportedly been used to generate higher potentials and therefore a more accurate measurement of the samples urea content, this slight increase in accuracy, except for special limited situations, does not justify the added increase in cost expended on the additional enzyme in extensive clinical use, Guilbault and Montalvo, J. Am. Chem. Soc. 90, 2533 1970). This level of optimum enzyme concentration and point of diminishing returns can be determined for the D-serine specific electrodes of this invention in the same manner described in the Guilbault article.
D-serine deaminase isolated from any source could conceivably be used (provided it is specific for D- amino acids) in the preparation of the D-serine specific electrode of the apparatus of this invention. Two of the better-known sources of this enzyme and techniques for their isolation can be found in the technical literature in separate articles by Dupourque et al. and Labow et al. appearing in J. Biolog. Chem., Vol. 241:5, 1233 (1966) and J. Biolog. Chem., Vol. 241:5, 1239 (1966) respectively.
For most practical clinical applications of the apparatus of this invention, anywhere from about 5 to about 50 milligrams of highly purified D-serine deaminase (prepared according to the Dupourque article 300 Units of activity per milligram of enzyme) per cubic centimeter of polymeric insolubilizing medium should prove adequate to effectively catalyze the selective deamination of the D-serine produced as a result of the hydrolysis of D-phosphoserine by the alkaline phosphatase present in the sample. Because of the relative thin ness of the encapsulating enzyme layer on the enzyme electrode, usually less than 0.1 millimeter, the total amount of active enzyme present in such layer will generally not exceed about 1 to about 2 milligrams.
METHOD The electrochemical apparatus of this invention enables the accurate and selective determination of alkaline phosphatase content in aqueous fluids. Those aqueous fluids which can be subjected to potentiometric assay according to the method of this invention are industrial fluids and biologic fluids whose liquid content comprises in excess of water by volume. Biologic fluids which can be assayed according to the method of this invention are those aqueous fluids extracted from any animal (vertebrate or invertebrate), vegetable or bacterial source and excretions from such sources.
According to the method of this invention, an aqueous fluid sample having an unknown concentration of alkaline phosphatase is diluted with an appropriate buffer; contacted with a pair of electrodes of an apparatus of this invention; a base-line potential for the sample recorded; about SXlO" to about SXlO mole per liter of D-phosphoserine for each 50 m Units of activity of alkaline phosphatase added to the sample; and incremental changes in electrical potential recorded at regular intervals at the enzyme electrode.
The aqueous fluid sample is ordinarily prepared for assay by the method of this invention by dilution with an aqueous buffer such that the approximate concentration of alkaline phosphatase in the resultant solution will not generally exceed approximately 30 to 50 m Units of activity per milliliter at 25C. as monitored by p-nitrophenyl phosphate, Rich and Hausamen, Zeitschr. Anal. Chem. 212, 267 (1965 Ordinarily, dilution of a biologic fluid, such as blood serum with an equivolume amount of buffer provides this sufficient degree dilution of the sample.
Often during extraction and dilution of the aqueous fluid sample prior to actual assay, some of the metal activator groups of alkaline phosphatase can be washed away or bound to ion-exchange resin, thereby rendering the enzyme partially inactive. This can be readily remedied by the addition of, for example, selected divalent metal salts of Mg, Ca and Sr to the sample during assay in concentrations of up to l moles per liter of sample. In the event that selected Zn salts are used to restore the alkaline phosphatase to full potency, the concentration fo the Zn should not exceed to -10 moles per liter of sample or inhibition of the alkaline phosphatase may occur.
The above sequence of steps, of course, assumes that the sample does not contain any ionic interferants, e.g. monovalent of divalent cations, having sufficient ionic activity to independently generate an electrical potential at the D-serine specific electrode of sufficient magnitude to significantly alter or distort the potential generated by the increase in ammonium ions resulting from the deamination of D-serine.
In the event that ionic interferants are present in the sample, the sample need be pre-treated with cationexchange resin prior to potentiometric assay; or, alternatively, the sample assayed with an apparatus that can be selectively discriminate between these interferants and the ammonium ions generated by deamination of the D-serine. In most instances, the selectivity of such apparatus cannot help but be enhanced by some pretreatment of the sample, either prior to or during analysis with similar cation-exchange resins. Whether or not pre-treatment of the sample, known to contain ionic interferants, with cation-exchange resin is required is largely dependent upon the type of apparatus being used to conduct the assay of the sample. For example, if the apparatus is provided with a D-serine specific solid-state enzyme electrode, referenced to a constant potential electrode, the presence of sodium, potassium and/or ammonium ions in the sample should have little, if any, effect on the highly selective ammonium ionsensitive membrane of the D-serine specific electrode. Where, however, the more durable ammonium ionsensitive glass electrode is used in place of a solid-state ammonium ion-sensitive device in preparation of the D-serine specific electrode, the aqueous fluid sample need be contacted with ion-exchange resin prior to or during potentiometric assay, depending upon whether or not the D-serine glass electrode is referenced to a constant or variable potential reference electrode. When the apparatus is provided with a constant poten tial or pH reference electrode, the sample should be contacted with cation-exchange resin prior to potentiometric assay. lf, on the other hand, the D-serine specific glass electrode is referenced to a variable potential electrode having substantially the same ionic sensitivity and selectivity as the enzyme electrode, the sample can be contacted with-cation-exchange resin during analysis.
Ordinarily, one to two grams of strongly acidic cation-ion exchange resin present in about 50 milliliters of buffered sample solution during analysis is sufficient to remove approximately of the ionic interference caused by the presence of sodium, potassium and/or ammonium ions in a typical sample of blood serum. The addition of ion-exchange resin in excess of the above concentration to the sample during analysis has been found to reduce the sensitivity of the enzyme electrode and often results in fluctuation of the sensing electrodes signal.
The cation-exchange resins which can be added to the test solution to further reduce the effect of sodium, potassium and/or ammonium ion interferants on enzyme electrode response generally have a high degree of sulfonic acid functionality. Among those strongly acidic resins which can be effectively used to improve enzyme electrode sensitivity are Amberlite CGSO (Rohm and Haas, Philadelphia, Pa), Dowex SOW-X (Dow Chemical Co., Midland, Mich), and Dowex 5OW-X (Dow Chemical Co., Midland, Mich.).
The strongly acidic cation-exchange resin marketed by Dow Chemical Company under the brand name Dowex SOW-Xf is preferred over other similar materials presently commercially available because of the selectivity, exchange capacity and compatiblity of these resins with the equipment and materials being analyzed.
Because the resins used in the reduction in concentration of ionic interferants are highly acidic, they tend to substantially lower the pH of the system. Since the activity of both alkaline phosphatase and D-serine deaminase, as is true for all enzymes, is pH dependent and the ammonium ion-sensitive membranes of D-serine specific electrodes and pH reference electrode are sensitive to hydrogen ion activity, the pH of the sample need be adjusted and maintained within a pH range which both favors maximum enzyme activity and yet minimizes hydrogen ion activity. The maintenance of pH stability is achieved through the use of any of a number well-known buffering systems, see generally Good, N.E., et al., Biochem. 5, 467-477 (I966). For the purpose of this invention, the pH range of the buffered test sample during analysis should be maintained within about 8.5 to about 9. Representative of those buffering systems which can be used in the dilution and maintenance of pH stability of the aqueous fluids being assayed according to the method of this invention are Bicene (N, N-bis [2-hydroxyethyl] glycine); tricene (N-tris-[hydroxymethyl]lmethyl glycine; and TAPS (tris [hydroxymethyl] methylaminopropane sulfonic acid). The use of a TRIS buffering system should generally be avoided because of the tendency of this buffer to cause inhibition of the D-serine deaminase on the enzyme electrode.
Once the buffered solution containing the D- phosphoserine has been added to the sample, and contacted with the unknown quantity of alkaline phosphatase present therein, the D-phosphoserine will undergo enzymatic hydrolysis to inorganic phosphate and D- serine. Either concurrent with or immediately after this contacting of the D-phosphoserine with the sample, this sample should be subjected to semicontinuous potentiometric assay for detection of changes in D-serine concentration; the potential at the D-serine specific electrode being noted at regular intervals for a period ranging from about I to minutes. The rate of change in D-serine concentration in the sample, as determined by such analysis, is proportional to the log of the concentration of alkaline phosphatase in the sample. Of course, the temperature of the test solution during such analysis must be maintained at a constant value.
The temperature of the test solution during assay should be kept below about 37C. in order to prevent denaturation of either the alkaline phosphatase in solution or D-serine deaminase on the enzyme electrode. As a practical matter, the temperature of the test solution should be maintained in excess of l5C. in order to facilitate the enzymatic hydrolysis of both D- phosphoserine in the test solution and the D-serine at the enzyme electrode. Contacting the electrodes and the sample at room temperature generally provides the preferred mode of conducting such analysis both from the standpoint of speed of electrode response times and simplicity.
In order to further improve the response times of both the enzyme and reference electrodes, it is often advisable to hydrate their ion-sensitive membranes overnight in either distilled water, 0.1 M Bicene buffer, pH 8.5 or other suitable buffer. After analysis of each successive sample, the electrodes, especially the enzyme electrode, need be thoroughly washed in buffer or distilled water to remove traces of substrate and ionic materials that may be adjacent to the ammonium ion-sensitive membrane of the electrode or dispersed within the insolubilization medium encapsulating the electrode. The enzyme electrode should be stored in buffer when not in use. Permitting the encapsulating enzyme coating of the enzyme electrode to dry out often results in loss of biocatalytic activity as a result of denaturation of the enzyme. The enzyme electrode employed in such method can be reused to preform numerous analyses, often having a useful life of about 21 days.
Where the enzyme electrode is intended to be used over such extended periods of time to conduct numerous analyses of several samples or in a continuous analysis routine, it is advisable to add -5 l0' moles per liter sample of the D-serine deaminase cofactor, pyridoxal S-phosphate directly to the sample during analysis in order to restore that original complement of this compound associated with the enzyme protein which may have been washed out during insolubilization andfor potentiometric analysis.
The examples which follow further illustrate the preparation and operation of the apparatus and the method of this invention. The parts and percentages appearing in these examples are by weight unless otherwise stipulated.
EXAMPLE I An enzyme immobilizing medium is prepared by dissolving 4.0 grams of acrylamide monomer and 0.2 grams of N, N'-methylene-bisacrylamide (Eastman Kodak Company, Rochester, NY.) in 25 milliliters 0.1 M Bicene buffer, pH 7.0. Once the monomer and crosslinking agent have been substantially dissolved in the buffer, 0.0027 gr'ams riboflavin and 0.0027 grams potassium persulfate are added to the buffered solution. This buffered monomer stock solution can be used immediately or stored in the dark at room temperature for up to two days.
One milliliter of monomer stock solution is pipetted into a 5 milliliter centrifuge tube containing 0.100 grams D-serine deaminase (Mutant C E. Coli W. activity 300 international Units) and 5 l0 M pyridoxal phosphate. The resulting enzyme-monomer suspension is stirred for about two minutes and then set aside for about 20 minutes at room temperature to permit more complete dissolution of the enzyme. After the enzyme has dissolved in the monomer solution to the extent permitted under the above conditions, the tube and its contents are refrigerated at 2C. for 10 minutes, centrifuged for l0 minutes at 3000 rpm (1470 gs), and the supernatant drawn off in a pipette.
Immediately preceding the preparation of the enzyme-monomer solution, the immersion tip of an ammonium ion-sensitive glass electrode, (Model 39l37, Beckman Instruments, Inc., Fullerton, Calif.) is washed in distilled water, wiped dry with lintless tissue paper and mounted on a supporting bracket in an inverted position. A two-inch square of nylon netting, 350p pore size (from a sheer nylon stocking, J .C. Penney, lnc.) is draped over the cation-sensitive membrane of the immersion tip of the electrode, where it is anchored in place by means of an elastic O-ring.
Two drops (-0.l milliliters) of monomer-D-serine deaminase is applied to the cation-sensitive membrane and nylon netting covering the immersion tip of the electrode. The electrode, still in the inverted position, is placed in a water-jacketed tube provided with a nitrogen gas inlet. The air in the space between the interior wall of the water-jacketed tube and the inverted electrode is displaced by purging with nitrogen for about 15 minutes. Once the oxidizing environment has been replaced with nitrogen, the immersion tip of the electrode, still under a blanket of nitrogen, is irradiated with a General Electric BBA photoflood lamp equipped with a reflector. The temperature of the photochemical polymerization is carefully monitored with a mercury bulb thermometer that is strapped to the bracket supporting the electrode. During this exposure to the photoflood lamp, nitrogen is continually passed over the immersion tip of the electrode, for the purpose of both maintaining an oxygen-free polymerization environment and controlling the temperature of the polymerization which should be maintained between about 22 and 28C. Ordinarily, the D-serine deaminase is entrapped and monomer fully polymerized within the first 10 to 15 minutes of exposure to the photoflood lamp; however, irradiation of the monomer-enzyme coating for at least 60 minutes is recommended.
After the D-serine specific electrode has been prepared in the manner described above, it, together with a Quartz Junction constant potential reference electrode (Model 39400, Beckman Instruments, Inc., Fullerton, Calif), are electrically connected to a Beckman Zeromatic Model ll pH meter which in turn is electrically connected to a Brown potentiometric recorder.
The cation-sensitive glass membrane of the D-serine specific electrode is then hydrated prior to use by immersion in distilled water for about 24 hours. After hydration of the ion-sensitive membrane of the electrode, the cell is calibrated by measuring the D-serine concentratrion of a number of solutions having known and varying concentrations of D-serine in 0.5 M Bicene buffer containing 2.0 grams Dowex 50W-X cationexchange resin.
Ten milliliters of a sample of human blood serum is contacted with 1 gram Dowex OW-X cationexchange resin for the removal of ionic interferants. Subsequent to this exposure to ion-exchange resin, the sample is placed in a thermostated chamber (22C. i 0.lC.), and 8.5, with an equivolume amount of Bicene buffer, pH 8.5, containing 5 X M MgCl per liter of sample. The solution is mildly agitated with a magnetic stiring bar while a base-line potential is recorded for the sample. After the ionic background activity of the sample is recorded, D-phosphoserine is added to the diluted solution in an amount such that its concentration in the sample is about 5X10 moles per liter of solution. With the addition of D-phosphoserine to the solution, the D-serine content of solution immediately begins to rise. As the D-serine content of the solutions in crease, so too does the potential at the enzyme electrode. This change in potential of the D-serine specific electrode is continually monitored for a period of 10 minutes as the solution is agitated periodically; the results being recorded on the Brown potentiometric recorder.
EXAMPLE 1] The analytical rountine described in Example I is repeated except for the referencing of the D-serine specific electrode to a pH electrode (Model No. 39000, Beckman Instruments, Inc., Fullerton, Calif.
EXAMPLE Ill The analytical routine described in Example I is repeated except for: (a) the addition of one gram of Dowex SOW-X cation-exchange resin directly to the diluted sample immediately preceding analysis instead of the separate pretreatment step used previously and, (b) the referencing of the D-serine specific electrode to a variable potential electrode of substantially the ionic sensitivity and selectivity as the enzyme electrode (Model No. 39137, Beckman Instruments, Inc., Fullerton, Calif).
EXAMPLE [V 0.l grams agarose, reagent grade, is added to 50 milliliters distilled water. and allowed to swell for 2 hours with constant agitation. The suspension is permitted to settle for two minutes, the supernatant decanted off and the swollen agarose resuspended in sufficient quantities of distilled water such that total volume of the suspension is 5 milliliters. The pH of the agarose suspension is then adjusted to a pH ll.0 with 0.1 M NaOH. One-tenth of a gram of finely divided cyanogen bromide, reagent grade, is then added to the suspension while the pH is maintained at l 1.0 by constant titration with 2.0 N NaOl-l, and the temperature held at about C. (room temperature) by the addition of ice to the reaction mass as necessary. The reaction is deemed complete when there is no additional base consumed. The suspension is then quickly transferred to a Buchner funnel and washed with equal portions of cold (-4C.) distilled water and cold (-4C.) 0.1 M Bicene buffer. pH 8.0. Washing of the filtrate is performed by aspiration and ordinarily is complete within two minutes.
The activated agarose is transferred from the funnel to a beaker containing five milliliters 0.1 M Bicene buffer, pH 8.0, 0. I00 grams D serine deaminase (same activity as in Example I) and 5 l0"" M pyridoxal phosphate. The resulting monomer-enzyme suspension is stirred for l6-20 hours at 0 to 3C. Once formation of the polymer-enzyme conjugate is complete, the suspension is thoroughly washed with alternating solutions of Bicene buffer and distilled water.
Immediately preceding the preparation of the polymer-enzyme conjugate, the immersion tip of a cationsensitive electrode, Model 39137 (Beckman lnstruments, lnc., Fullerton, Calif.) is washed in distilled water and wiped dry with a lintless tissue paper. Approximately one milliliter of cyanogen bromide modified agarose-D-serine deaminase conjugate is placed in the center of a two-inch square of nylon netting, 60p pore size (Pharmacia Fine Chemicals Inc., Piscataway, N..l.). The immersion tip of the electrode is then centered in the polymer-enzyme conjugate mass, the nylon netting folded up along the barrel of the electrode and anchored in place by means of an elastic O-ring. The netting is then manually manipulated so that an even layer of conjugate is distributed over the cationsensitive bulb of the electrode.
The D-serine specific electrode prepared in the manner described above can be referenced to either a constant or variable potential electrode and potentiometric assay of an aqueous fluid sample conducted according to the procedures described in the foregoing Examples.
EXAMPLE V Ten grams of polyacrylamide beads (Bio-Gel P-300, Bio Rad Laboratories, Richmond, Calif.) together with 15 milliliters distilled water are placed in a siliconized flask, the flask stopped and beads allowed to swell overnight (-l2 hours). The swollen polyacrylamide hydrogel is then crushed in a Waring blender and the hydrogel separated into major fractions of like particle size by standard gravity sedimentation techniques. Two milliliters polyacrylamide hydrogel is added to a flask containing a stoichiometric amount of anhydrous hydrazine, based on polyacrylamide hydrogel, reagent grade, (Matheson, Coleman and Bell, Norwood, Ohio, assayed 99-l00%, 20.4 M); the flask containing the anhydrous hydrazine being pre-heated in a constant temperature oil bath at 49C. for 45 minutes prior to the addition of the polyacrylamide hydrogel. The hydrogelhydrazine mixture is reacted at 49C. for about 20 hours, during which time the mixture is continually agitated by an immersible magnetic stirring bar. At the end of the reaction interval. the hydrazine modified hydrogel is washed with 0.1 N NaCl aqueous solution until the supernatant tests negative for the presence of free hydrazine, as indicated by a failure of the development of a pale violet color in the reaction of the supernatant with trinitrobenzenesulfonate. The hydrazine modified hydrogel, obtained as described above, is reacted with stoichiometric quantities of cold (-4C.) l N HN0 for about 5 minutes and then the acyl azide modified hydrogel washed with 0.1 M Bicene buffer until the hydrogel pH is raised to between 7 and 8.
The acyl azide modified hydrogel is then transferred to a beaker containing 5 milliliters 0.2 M Bicene buffer, pH 8.0 and 0.20 grams D-serine deaminase (same activity as in Example I), the buffer-enzyme solution being pre-cooled to about to 4C. The resulting mixture is maintained at about this same temperature while continually stirred for 5 hours. The resulting hydrogelenzyme conjugate is separated from the unreacted enzyme by centrifugation, and then washed with 200 milliliters 0.01 N glycine buffer.
A D-serine specific electrode is prepared utilizing the insolubilized D-serine deaminase obtained above according to the technique described in Example IV. The D-serine specific electrode thus prepared is referenced to a constant potential electrode and potentiometric assay of an aqueous fluid sample conducted as described in Example 1.
EXAMPLE VI Two grams of a sample of powdered porous 96% silica glass (950A i 50A pore size, l6m. /gm. surface area) is washed in 0.2 N HNO at 80C. with continuous sonication for at least three hours. The glass sample is washed further by decantation with distilled water and dried by heating to 625C. overnight (-12 hours) in an oxidizing environment.
The following day the glass sample is cooled, placed in a round bottom flask with 100 milliliters ofa 10% solution of y-aminopropyltriethyloxysilane in toluene. The mixture is refluxed overnight (-12 hours), cooled, and the particulates separated from the reaction mass by filtration. The filter cake is then washed with acetone to remove traces of solvent and unreacted silane, air dried and then stored, if desired, or used immediately thereafter. The product of this reaction (hereinafter referred to as aminoalkylsilane modified silica), is calculated as having 0.171 meq. ofsilane residues/gram of glass sample as determined by total nitrogen.
One gram of aminoalkylsilane modified silica is added to a flask containing 3.5 milliliters distilled water and 0.10 grams D-serine deaminase (same activity as in Example I). This mixture is then combined with a second solution comprising 0.5 milliliter N, N'-dicylohexylcarbodiimide (DCCl) in 0.5 milliliters tetrahydrofuran (THF). The resultant solution is then stirred overnight (-12 hours) at room temperature (-22C.), the reaction product separated from solution by filtration. and washed exhaustively with alternating solutions of NaHCO 0.001 M HCl and distilled water. The
insolubilized D-serine deaminase is stored in 0.1 M Bicene buffer, pH 7.0, at 0to 3C.
A D-serine specific electrode is prepared utilizing the insolubilized D-serine deaminase obtained above according to the technique described in Example IV. The D-serine specific electrode thus prepared is referenced to a constant potential electrode and potentiometric assay of an aqueous fluid sample conducted as described in Example 1.
What is claimed is:
I. A potentiometric method for selectively determining the concentration of alkaline phosphatase of an aqueous fluid sample, comprising;
a. diluting the aqueous fluid sample with a buffered aqueous solution having a pH in the range of about 8.5 to about 9;
b. providing a potentiometer electrically connected in series to a D-serine specific electrode and a reference electrode, wherein the D-serine specific electrode is provided with an ammonium ionsensitive membrane in contact with and substantially encapsulated by a coating of insolubilized D- serine deaminase, said coating containing a catalytically effective amount of D-serine deaminase and being permeable to both the aqueous fluid sample and products of the reaction of the aqueous fluid sample and the D-serine deaminase;
c. contacting the D-serine specific electrode and the reference electrode with the diluted sample;
d. recording the ionic background activity of the sample;
e. adding to the diluted sample about 5X10 to about 5X10" moles D-phosphoserine per liter of diluted sample;
f. and recording the incremental change in potential at regular intervals for a period ranging from about 5 to about 15 minutes.
2. The potentiometric method as defined in claim 1, wherein the D-serine specific electrode is referenced to a constant potential reference electrode.
3. The potentiometric method as defined in claim 2, wherein the diluted aqueous fluid sample is pre-treated with a strongly acidic cation-exchange resin.
4. The potentiometric method as defined in claim 1, wherein the diluted sample is periodically agitated after addition of D-phosphoserine during recordation of changes in enzyme electrode potential.
5. A potentiometric method for selectively determining the concentration of alkaline phosphatase in an aqueous fluid sample having a pH in the range of about 8.5 to 9, the method comprising the steps of:
a. providing a potentiometer electrically connected to an enzyme electrode and a variable potential reference electrode, in which the enzyme electrode is provided with an ammonium ion-sensitive membrane in contact with and substantially encapsulated by a coating of immobilized enzyme, said coating containing a catalytically effective amount of D-serine deaminase, said coating being permeable to both the aqueous fluid sample and products of the reaction of the aqueous fluid sample and the D-serine deaminase; and in which the variable potential reference electrode is provided with an uncoated ammonium ion-sensitive membrane of substantially the same ionic sensitivity and selectivity as the enzyme electrode; and
b. contacting the ammonium ion-sensitive membranes of both the enzyme electrode and the variable potential electrode with the sample of aqueous fluid and about 5 10 to 5X10 moles D- phosphospherine per liter of sample.
6. A method as defined in claim 5 in which the further step is provided of measuring the generated electrical potential on the potentiometer.
* i i i
|Cited Patent
|Filing date
|Publication date
|Jul 11, 1957
|Sep 12, 1961
|Warner Lambert Pharmacentical
|Diagnostic preparation and process for the determination of serum alkaline phosphatase
|May 9, 1963
|Feb 28, 1967
|Corning Glass Works
|Glass electrode apparatus and process for making same
|Nov 14, 1966
|Feb 4, 1969
|Smithkline Corp
|Laboratory reagent for assay of alkaline phosphatase
|Jan 27, 1964
|Nov 18, 1969
|Beckman Instruments Inc
|Electrochemical transducer
|Apr 18, 1967
|Nov 24, 1970
|Du Pont
|Enzyme electrode
|Jul 15, 1968
|Dec 26, 1972
|Measuring system
|Citing Patent
|Filing date
|Publication date
|Jun 26, 1975
|Aug 17, 1976
|General Electric Company
|Method and apparatus for detecting molecules in solutions
|Mar 12, 1975
|May 3, 1977
|The University Of Utah
|Selective chemical sensitive FET transducers
|May 29, 1979
|Aug 12, 1980
|Albert Huch
|Dish-electrode concentration meter with detachable transducer
|Dec 5, 1979
|Jun 9, 1981
|Eastman Kodak Company
|Buffer overcoat for CO2 ion-selective electrodes
|Dec 19, 1980
|Oct 19, 1982
|Radelkis Elektrokemiai Muszergyarto Szovetkezet
|Molecule selective enzyme electrode
|Dec 19, 1980
|Mar 1, 1983
|Radelkis Elektrokemiai Miszergyarto Szovetkezet
|Molecule selective sensor for industrial use and procedure for its preparation
|Aug 11, 1982
|Sep 27, 1983
|Blass Karl G
|Electrochemical determination of orthophosphoric monoester phosphohydrolase activity (EC 220.127.116.11 and EC 18.104.22.168: alkaline and acid phosphatases)
|Jul 7, 1982
|Dec 25, 1984
|Claudio Calzi
|Electrochemical cell provided with selective electrodes and at least one chemical reactor, for indirect measurement of clinical-chemical parameters
|Aug 12, 1987
|Mar 14, 1989
|Unitika, Ltd.
|Enzyme sensor for determining a concentration of glutamate
|Jun 9, 2004
|Jan 6, 2005
|Vilmos Keri
|Ion-exchange filtration of fermentation broth
|Feb 15, 1990
|Jun 21, 1995
|Beckman Instruments, Inc.
|Automatic chemistry analyzer
|Feb 15, 1990
|Jul 19, 1995
|Beckman Instruments Inc
|Title not available
|U.S. Classification
|205/778, 435/817, 204/420, 435/180, 435/176, 204/403.1, 435/178, 435/21
|International Classification
|Cooperative Classification
|European Classification
|Jul 5, 1988
Owner name: TECHNICON INSTRUMENTS CORPORATION
Free format text: MERGER;ASSIGNOR:REVGROUP PANTRY MIRROR CORP.;REEL/FRAME:004912/0740
Effective date: 19871231
Free format text: MERGER;ASSIGNOR:REVGROUP PANTRY MIRROR CORP.;REEL/FRAME:4912/740


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Glencoe Literature Texas Treasures: American Literature
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Texas Tech University womens gymnastics
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As a student athlete, you can play college sports competitively at an intercollegiate level, or just for fun via intramurals. Please keep in mind that few students will qualify for athletic scholarships, and that college coaches sward available sports scholarship money judiciously.
Approximately 126,000 student athletes go to college on either a partial or full athletic scholarship, but the vast majority of college athletic teams have open tryouts, and walk-ons may land a position on the team if they are talented enough. If you've been recruited or made it past a walk-on tryout, that's when the real challenge starts. You still have to make it through daily practices, and several rounds of cuts in order to make the team. San Antonio College football game tickets may indicate that it's an option to delay college entrance by a year. An extra year in high school or prep school can allow players to mature mentally, bring grades and SAT test scores up, put on weight and gain strength. Beyond consideration for Texas Tech University womens gymnastics, you may think about Loras College schedule of courses for complementary electives to your primary course of study.
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"post_date": "2017-04-10 06:45:09"
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"post_media_url": "\/media\/website_posts\/54\/",
"post_category_id": "1",
"post_title": "Is Your Website Generating New Business?",
"post_content_short": "It wasn't that long ago that most websites were simply electronic brochures. They simply listed the who, what and where of the business but times have changed.",
"post_icon": "Lead-Generation.jpg",
"post_content_type": "page",
"post_content_url": "http:\/\/www.nexusaccountants.com.au\/blog\/is-your-website-generating-new-business\/",
"post_date": "2017-02-22 03:13:07"
"post_id": "53",
"post_media_url": "\/media\/website_posts\/53\/",
"post_category_id": "1",
"post_title": "How to Get Your Business Off to a Flying Start",
"post_content_short": "Starting a business can be a maze of research, registrations and red tape. No amount of passion can guarantee your business success but the proverb, 'failing to plan is planning to fail' serves as both great advice and a warning for new entrepreneurs.",
"post_icon": "marketing-your-start-up-business.gif",
"post_content_type": "page",
"post_content_url": "http:\/\/www.nexusaccountants.com.au\/blog\/how-to-get-your-business-off-to-a-flying-start\/",
"post_date": "2017-02-10 01:52:01"
"post_id": "52",
"post_media_url": "\/media\/website_posts\/52\/",
"post_category_id": "1",
"post_title": "More Website Traffic with Search Engine Marketing (SEM)",
"post_content_short": "When Google displays its search engine results page they display three different types of results including organic, pay per click and local search results. Let's explore how they all work.",
"post_icon": "search-engine-marketing.jpg",
"post_content_type": "page",
"post_content_url": "http:\/\/www.nexusaccountants.com.au\/blog\/more-website-traffic-with-search-engine-marketing-sem\/",
"post_date": "2016-12-19 04:58:37"
"post_id": "51",
"post_media_url": "\/media\/website_posts\/51\/",
"post_category_id": "1",
"post_title": "How Does Your Website Work?",
"post_content_short": "Your website should effectively be your home base on the internet but how does it actually work and what is hosting and domain names?",
"post_icon": "website-icon-WWW-finger-click.png",
"post_content_type": "page",
"post_content_url": "http:\/\/www.nexusaccountants.com.au\/blog\/how-does-your-website-work\/",
"post_date": "2016-12-15 05:20:42"
"post_id": "50",
"post_media_url": "\/media\/website_posts\/50\/",
"post_category_id": "1",
"post_title": "Your Online Presence in the Digital Age",
"post_content_short": "In the digital age your online presence could make or break your business. While your website should be at the hub, your digital presence needs to extend beyond a website.",
"post_icon": "digital-tool-kit.jpg",
"post_content_type": "page",
"post_content_url": "http:\/\/www.nexusaccountants.com.au\/blog\/your-online-presence-in-the-digital-age\/",
"post_date": "2016-12-14 05:35:30"
"post_id": "49",
"post_media_url": "\/media\/website_posts\/49\/",
"post_category_id": "1",
"post_title": "Negative Gearing Traps Exposed",
"post_content_short": "Creating wealth through purchasing a negatively geared investment property is a well established practice in this country. We have identified some common areas where property investors often trip up including claiming interest on loans.",
"post_icon": "House-Dollar-Balance.jpg",
"post_content_type": "page",
"post_content_url": "http:\/\/www.nexusaccountants.com.au\/blog\/negative-gearing-traps-exposed\/",
"post_date": "2016-12-12 01:53:30"
"post_id": "48",
"post_media_url": "\/media\/website_posts\/48\/",
"post_category_id": "1",
"post_title": "Business Structures – The Family Trust",
"post_content_short": "In Australia there are a range of business structures available and in this blog post let's specifically look at the family trust option.",
"post_icon": "Business-Structures-Options.png",
"post_content_type": "page",
"post_content_url": "http:\/\/www.nexusaccountants.com.au\/blog\/business-structures-the-family-trust\/",
"post_date": "2016-11-18 03:44:02"
"post_id": "47",
"post_media_url": "\/media\/website_posts\/47\/",
"post_category_id": "1",
"post_title": "Thinking of Buying a Business?",
"post_content_short": "Starting a business can be a mine field and the risks are high which explains why so many budding entrepreneurs look to buy an existing business or franchise operation rather than start from 'scratch'.",
"post_icon": "Business-For-Sale.jpg",
"post_content_type": "page",
"post_content_url": "http:\/\/www.nexusaccountants.com.au\/blog\/thinking-of-buying-a-business\/",
"post_date": "2016-08-17 06:19:56"
"post_id": "46",
"post_media_url": "\/media\/website_posts\/46\/",
"post_category_id": "1",
"post_title": "Starting a Business – The Right Foundations",
"post_content_short": "As any entrepreneur will tell you, starting or buying a business involves research, risk, passion and planning. To be successful you need to make the right opening moves and ...",
"post_icon": "Business-Start-Up-Specialist.png",
"post_content_type": "page",
"post_content_url": "http:\/\/www.nexusaccountants.com.au\/blog\/starting-a-business-the-right-foundations\/",
"post_date": "2016-07-29 05:36:14"
"post_id": "45",
"post_media_url": "\/media\/website_posts\/45\/",
"post_category_id": "1",
"post_title": "Starting a Business - Do the Numbers Stack Up?",
"post_content_short": "Starting a business is exciting but stressful. Before you get too far down the track you need to make sure the numbers stack up with these four steps.",
"post_icon": "Do-the-business-start-up-numbers-stack-up.jpg",
"post_content_type": "page",
"post_content_url": "http:\/\/www.nexusaccountants.com.au\/blog\/starting-a-business-do-the-numbers-stack-up\/",
"post_date": "2016-04-27 08:02:29"
"post_id": "44",
"post_media_url": "\/media\/website_posts\/44\/",
"post_category_id": "1",
"post_title": "Before You Buy A Business",
"post_content_short": "When you're investigating buying a business it's important to do a detailed review of the business's operations, finances, reputation and industry as part of what is called the due diligence process.",
"post_icon": "Business-For-Sale-Yellow-Sign.jpg",
"post_content_type": "page",
"post_content_url": "http:\/\/www.nexusaccountants.com.au\/blog\/before-you-buy-a-business\/",
"post_date": "2016-04-05 06:33:33"
"post_id": "43",
"post_media_url": "\/media\/website_posts\/43\/",
"post_category_id": "1",
"post_title": "Helping You With Your Online Marketing",
"post_content_short": "As accountants, the number one thing our business clients want is advice to help them grow their business. In the digital age we believe the secret to growth is marketing and it could be the difference between boom and gloom for any business.",
"post_icon": "Marketing-Strategy.jpg",
"post_content_type": "page",
"post_content_url": "http:\/\/www.nexusaccountants.com.au\/blog\/helping-you-with-your-online-marketing\/",
"post_date": "2016-02-12 04:54:18"
"post_id": "42",
"post_media_url": "\/media\/website_posts\/42\/",
"post_category_id": "1",
"post_title": "Grow Your Business by Increasing Process Efficiency",
"post_content_short": "In this final post in the series we look at the fourth way to grow your business - increase the effectiveness of each process in your business.",
"post_icon": "4-Ways-Individual-Icons_No-4.png",
"post_content_type": "page",
"post_content_url": "http:\/\/www.nexusaccountants.com.au\/blog\/grow-your-business-by-increasing-process-efficiency\/",
"post_date": "2016-02-02 01:10:19"


text:
Business Free Directory.biz » Internet » File Sharing
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|Zootopia Movie Review & Picture Summary (2016) - http://zootopiafullmovie.com
I wouldn't recommend this film for stereotypical little girls who aren't used to seeing/enjoying violence and anger the manner stereotypical little boys are or for very young children. »» Details for Zootopia Movie Review & Picture Summary (2016)
|Services Entreprises - http://www.localrwa.com/member.php?action=profile&uid=67047
Ρroducts connectᥱd to HPE leverage 24x7 monitoring, pre-failure alerts, rapid diagnosis, automatic call logging and partѕ dispatch for hаrdware support and to help avoid outages. The American Farm Bureau Federation (AFBF), the nation's largeѕt group representing faгmers, saiⅾ сounty-stage USDA staffing cuts and decreased statistical providers might damage members. »» Details for Services Entreprises


text:
Managing Partner & CEO
Kevin Brown has been in the property management industry for 15 years. He began his career as a leasing agent in college. Kevin later made the move to association management when he was appointed the community manager of a large HOA comprised of 5 sub-Associations consisting of flats and townhouses in the Philadelphia suburbs. He then transitioned into high-rise management where he further developed his skill set for large, complex building operations. Kevin has worked in executive positions with the two largest national homeowner management companies both in Philadelphia and Florida giving him a unique perspective on how a local company can deliver superior services. Kevin is a people person who likes to stay in contact with his clients.


text: New Search 622930 Scott W. Eberle, MD (707) 778-6242 Primary Specialties Hospice/Palliative Medicine Primary Office Address: 416 Payran St. Petaluma, CA 94952 Phone: (707) 778-6242 Fax: (415) 600-7625 Recommend this Physician Request an Update for this Physician Get Directions The patient rating score is based on responses and comments from Press Ganey. Responses are measured on a 10 point scale, with 10 being the best score. These scores are then translated to a 5 point scale in order to display results in a 5 Star rating. Comments are also gathered from the same survey and displayed in their entirety with the exception of any language that may be considered libel, or may contain private health information, or is unrelated to the provider being reviewed. Learn more about Press Ganey star rating calculations


text:
Center for Mathematical Research
Center for Mathematical Research
Shamah-Levy T.,National Institute of Public Health |
Mundo-Rosas V.,National Institute of Public Health |
Mendez-Gomez-Humaran I.,Center for Mathematical Research
Journal of Nutrition | Year: 2014
Background: Anemia is a major cause of maternal mortality. Household food insecurity (HFI) may increase the risk of anemia among women of reproductive age although this hypothesis remains largely untested in representative samples from low- and middle-income countries. Objective: Our objective was to investigate the association of HFI with anemia in a nationally representative, cross-sectional sample of Mexican women of reproductive age (12-49 y old). Methods: We tested the association between HFI and anemia among 16,944 women of reproductive age using the multiple logistic regression among adolescent (12-20 y) and adult women (21-49 y). HFI was measured with the use of the Latin American and Caribbean Food Security Scale. Hemoglobin was measured with capillary hemoglobin with the use of HemoCue photometer (HemoCue, Inc.) and anemia was defined with the use of WHO standards. Results: The association of HFI and anemia was not significant (P > 0.05) for adolescent women (12-20 y), whereas in adult women (21-49 y), the adjusted odds of having anemia were 31-43% higher among those living in mild to severely food insecure households than adult women residing in food secure households (P < 0.05). Conclusions: HFI is associated with anemia among adult Mexican women. Programs that reduce HFI may also be effective at reducing the risk of anemia among Mexican women. © 2014 American Society for Nutrition.
Goring S.,University of Wisconsin - Madison |
Williams J.W.,University of Wisconsin - Madison |
Blois J.L.,University of Wisconsin - Madison |
Jackson S.T.,University of Wyoming |
And 5 more authors.
Quaternary Science Reviews | Year: 2012
Age-depth relationships in sedimentary archives such as lakes, wetlands and bogs are non-linear with irregular probability distributions associated with calibrated radiocarbon dates. Bayesian approaches are thus well-suited to understanding relationships between age and depth for use in paleoecological studies. Bayesian models for the accumulation of sediment and organic matter within basins combine dated material from one or more records with prior information about the behavior of deposition times (yr/cm) based on expert knowledge. Well-informed priors are essential to good modeling of the age-depth relationship, but are particularly important in cases where data may be sparse (e.g., few radiocarbon dates), or unclear (e.g., age-reversals, coincident dates, age offsets, outliers and dates within a radiocarbon plateau).Here we assessed Holocene deposition times using 204 age-depth models obtained from the Neotoma Paleoecology Database (www.neotomadb.org) for both lacustrine and palustrine environments across the northeastern United States. These age-depth models were augmented using biostratigraphic events identifiable within pollen records from the northeastern United States during the Holocene and late-Pleistocene.Deposition times are significantly related to depositional environment (palustrine and lacustrine), sediment age, and sediment depth. Spatial variables had non-significant relationships with deposition time when site effects were considered. The best-fit model was a generalized additive mixed model that relates deposition time to age, stratified by depositional environment with site as a random factor. The best-fit model accounts for 63.3% of the total deviance in deposition times. The strongly increasing accumulation rates of the last 500-1000 years indicate that gamma distributions describing lacustrine deposition times (α = 1.08, β = 18.28) and palustrine deposition times (α = 1.23, β = 22.32) for the entire Holocene may be insufficient for Bayesian approaches since there is strong variation in the gamma parameters both in the most recent sediments and throughout the Holocene. Time-averaged gamma distributions for lacustrine (α = 1.35, β = 19.64) and palustrine samples (α = 1.40, β = 20.72) show lower overall deposition times, but variability remains. The variation in gamma parameters through time may require the use of multiple gamma distributions during the Holocene to generate accurate age-depth models. We present estimates of gamma parameters for deposition times at 1000 yr intervals. The parameters generated in this study can be used directly within Bacon to act as Bayesian priors for sedimentary age models. © 2012 Elsevier Ltd.
Velasco Elizondo P.,Center for Mathematical Research |
Lau K.-K.,University of Manchester
Journal of Systems and Software | Year: 2010
Component-based development is based on the idea of building software systems by composing pre-existing components. Connectors are the 'glue' for composing components. Therefore, it is important to consider connectors as first-class entities and provide adequate descriptions of them to facilitate their understanding and promote their reuse. We have defined a catalogue of component connectors to support the process of 'development with reuse'. The categories and connector types in the catalogue were obtained through an analysis of the activities involved in this process as well as considering the syntax and semantics of a new component model. © 2010 Elsevier Inc. All rights reserved.
Vasquez-Gomez J.I.,National Institute of Astrophysics, Optics and Electronics |
Sucar L.E.,National Institute of Astrophysics, Optics and Electronics |
Murrieta-Cid R.,Center for Mathematical Research
IEEE International Conference on Intelligent Robots and Systems | Year: 2014
The task addressed in this paper is to plan iteratively a set views in order to reconstruct an object using a mobile manipulator robot with an 'eye-in-hand' sensor. The proposed method plans views directly in the configuration space avoiding the need of inverse kinematics. It is based on a fast evaluation and rejection of a set of candidate configurations. The main contributions are: a utility function to rank the views and an evaluation strategy implemented as a series of filters. Given that the candidate views are configurations, motion planning is solved using a rapidly-exploring random tree. The system is experimentally evaluated in simulation, contrasting it with previous work. We also present experiments with a real mobile manipulator robot, demonstrating the effectiveness of our method. © 2014 IEEE.
Dwivedi V.,Carnegie Mellon University |
Velasco-Elizondo P.,Center for Mathematical Research |
Maria Fernandes J.,University of Aveiro |
Garlan D.,Carnegie Mellon University |
Schmerl B.,Carnegie Mellon University
Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics) | Year: 2011
Computations are pervasive across many domains, where end users have to compose various heterogeneous computational entities to perform professional activities. Service-Oriented Architecture (SOA) is a widely used mechanism that can support such forms of compositions as it allows heterogeneous systems to be wrapped as services that can then be combined with each other. However, current SOA orchestration languages require writing scripts that are typically too low-level for end users to write, being targeted at professional programmers and business analysts. To address this problem, this paper proposes a composition approach based on an end user specification style called SCORE. SCORE is an architectural style that uses high-level constructs that can be tailored for different domains and automatically translated into executable constructs by tool support. We demonstrate the use of SCORE in two domains - dynamic network analysis and neuroscience, where users are intelligence analysts and neuroscientists respectively, who use the architectural style based vocabulary in SCORE as a basis of their domain-specific compositions that can be formally analyzed. © 2011 Springer-Verlag.
Dalmau O.,Center for Mathematical Research |
Rivera M.,Center for Mathematical Research |
Gonzalez A.,Ibero-American University of Mexico
Optics Communications | Year: 2016
We first present two closed formulas for computing the phase shift in interferograms with unknown phase step. These formulas obtain theoretically the exact phase step in fringe pattern without noise and only require the information in two pixels of the image. The previous formulas allows us to define a functional that yields an estimate of the phase step in interferograms corrupted by noise. In the experiment we use the standard Least Square formulation which also yields a closed formula, although the general formulation admits a robust potential. We provide two possible implementations of our approach, one in which the sites can be randomly selected and the other in which we can scan the whole image. The experiments show that the proposed algorithm presents the best results compared with state of the art algorithms. © 2016, Elsevier B.V. All rights reserved.
Estrada E.,University of Strathclyde |
Estrada E.,Center for Mathematical Research |
Sheerin M.,University of Strathclyde
Physica D: Nonlinear Phenomena | Year: 2016
A random rectangular graph (RRG) is a generalization of the random geometric graph (RGG) in which the nodes are embedded into a rectangle with side lengths a and b=1/a, instead of on a unit square [0,1]2. Two nodes are then connected if and only if they are separated at a Euclidean distance smaller than or equal to a certain threshold radius r. When a=1 the RRG is identical to the RGG. Here we apply the consensus dynamics model to the RRG. Our main result is a lower bound for the time of consensus, i.e., the time at which the network reaches a global consensus state. To prove this result we need first to find an upper bound for the algebraic connectivity of the RRG, i.e., the second smallest eigenvalue of the combinatorial Laplacian of the graph. This bound is based on a tight lower bound found for the graph diameter. Our results prove that as the rectangle in which the nodes are embedded becomes more elongated, the RRG becomes a 'large-world', i.e., the diameter grows to infinity, and a poorly-connected graph, i.e., the algebraic connectivity decays to zero. The main consequence of these findings is the proof that the time of consensus in RRGs grows to infinity as the rectangle becomes more elongated. In closing, consensus dynamics in RRGs strongly depend on the geometric characteristics of the embedding space, and reaching the consensus state becomes more difficult as the rectangle is more elongated. © 2015 Elsevier B.V.
Velasco-Elizondo P.,Center for Mathematical Research
CCE 2011 - 2011 8th International Conference on Electrical Engineering, Computing Science and Automatic Control, Program and Abstract Book | Year: 2011
Several works on using Component-based Development to construct Software Product Lines have been reported. However, shortcomings in both approaches have limited the benefits of combining them in practice for the development of software asset variants. This paper explores the feasibility of using composition operators to construct such variants from pre-existing software components. The variants are implemented as composite components, which provide variant behaviours that can be reused in a Software Product Line. Via a case study, we will show that the use of these operators enables a consistent and systematic approach to variant construction. Other benefits of our approach are design preservation, modularity and automation. © 2011 IEEE.
Velasco Elizondo P.,Center for Mathematical Research
Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics) | Year: 2010
Software Product Line (SPL) approaches enable the development of software product variants by reusing a set of software core assets. These assets could have variant features themselves that can be configured in different ways to provide different behaviours. Unfortunately, in many SPL approaches software core assets are constructed from scratch and in an ad hoc manner. © 2010 Springer-Verlag Berlin Heidelberg.
Vigueras J.F.,Center for Mathematical Research |
Rivera M.,Center for Mathematical Research
Pattern Recognition | Year: 2010
We introduce a two-step iterative segmentation and registration method to find coplanar surfaces among stereo images of a polyhedral environment. The novelties of this paper are: (i) to propose a user-defined initialization easing the image matching and segmentation, (ii) to incorporate color appearance and planar projection information into a Bayesian segmentation scheme, and (iii) to add consistency to the projective transformations related to the polyhedral structure of the scenes. The method utilizes an assisted Bayesian color segmentation scheme. The initial user-assisted segmentation is used to define search regions for planar homography image registration. The two reliable methods cooperate to obtain probabilities for coplanar regions with similar color information that are used to get a new segmentation by means of quadratic Markov measure fields (QMMF). We search for the best regions by iterating both steps: registration and segmentation. © 2009 Elsevier Ltd. All rights reserved.


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Le Moyne College geophysics
Think about what career is best suited for you, and where you want to study. Request more information now.
Think about what career is best suited for you, and where you want to study. Request more information now.
Think about what career is best suited for you, and where you want to study. Request more information now.
Get info about Le Moyne College geophysics, and registering for elective degree courses. Which college degrees give you the best chances of finding employment after graduation? There are a variety of engineering training programs that may appeal to you, such as mechanics, vehicle design, and alternative energy production. Given Le Moyne College geophysics, for example, you may gain an advantage in job recruiting on-campus. Check with the geothermal engineering department for course registration deadlines, and physics degree program requirements. Please use the form above to request admissions info for Le Moyne College
Mining techniques have dramatically transformed over many years, with technological advances improving efficiency and safety. In the discovery stage of mineral exploration, mining engineers work closely with geologists to identify minerals and assess reserve quantities. Mineral engineers may then drill core samples and conduct surface surveys, searching for specific compounds and ores. Unless the mineral exploration is done on public lands, the property owners may play a significant role in mining exploration.
Generally, mining is either done from the surface or underground. Surface mining requires the stripping of surface vegetation, dirt, and if necessary, layers of bedrock in order to reach buried ore deposits. Additionally, explosives may be called for in order to facilitate breakage. Hard minerals such as limestone, sand, gravel, and slate are generally quarried into a series of benches. Strip mining, on the other hand, is more appropriate for softer minerals such as clays and phosphate. In-situ mining can be done from the surface using dissolving agents, retrieving the ore via pumps. Finally, hydraulic jets may be utilized to wash away the overburden or to clear out the ore itself. Within the geothermal engineering department, you may consider Henry Ford Community College biochemistry major as an adjunct to your primary course of study.
Arkansas Department of Health
The Engineer, Professional Engineer is responsible for conducting engineering studies, developing design drawings and/or specifications, and performing related..
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Thomas & Betts Corporation
Test Engineer will design efficient Production Test Equipment,luding. The..
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The Manufacturing Process Engineer responsibilitieslude, but are not limited to, the tasks outlined below..
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Exxon Mobil
Lead Mechanical Engineer. Reviews engineering packages and gives technical guidance to assigned engineers. Acts as the main point of contact for the assigned..
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text:
Apartment Therapy in Red River Moms
In this month's issue of Red River Moms, Maxwell shares some of his best tips for giving kids rooms (from babies to big kids) a new look for the new year. Often as much about what you don't need as what you do, his ideas help create rooms that are comfortable, personal and cool.
Check out the online version for the full article (we're on Page 10): Red River Moms, January 2013
(Image: Red River Moms)


text:
a meme about writingNov. 22nd, 2010 08:58 am
From ashen_key:
I think it would be fun to talk about stories, but the usual memes are like, "What happens next?" "Tell me about Character A?" Which isn't so much talking about stories as it is writing more of a story. But you know how sometimes you read something and you're like, "I got ___ out of this story, I wonder if I have that right?" or "What on earth was ____ supposed to be?" and it's too awkward to ask the author? Now you could totally ask!If you are looking for things I wrote so you can ask questions about them, you might wanna hit up peahen or my WIP post.
I've heard people say that writing is hard because you have to make decisions, but we never really talk about the decisions we make with stories or why we make them. We talk about plot bunnies, but not about how we actually turn them into a story.
And it seems like a lot more fun to do that than to do work.
So, if you want, ask me questions! (Or use this to ask your flist to ask you questions).
What were you trying to do [here]? Why did you decide to ____? This is what I thought about XYZ, is that what you were going for? What made you write ____? Why did you decide to do this? And so on.