CA2051119A1 - Use of fluid insoluble oxidizing agents to eliminate interfering substances in oxidation-reduction measuring systems - Google Patents

Abstract

Systems and methods are disclosed for removing redox-active substances from aqueous, partially aqueous or non-aqueous fluids. The invention involves contacting the fluid to be treated with a redox-active agent that is insoluble in the fluid, thereby oxidizing interfering redox-active substances. Electron transfer agents can also be employed. The residual redox oxidizing agent is removed from the treated fluid on the basis of its insolubility, so that no residual redox activity remains. The invention is useful for removal of interfering redox-active substances from liquid samples when analytes in the sample are to be measured using reduction-oxidation chemistry and the redox-active interfering substances removed by the disclosed method interfere in the reduction-oxidation analysis. Furthermore, this invention is especially useful to pretreat blood, serum, plasma or other bodily fluids prior to analysis or other use of these fluids, where the presence of reduction-oxidation active substances constitutes an interference in that analysis or use.

Description

WO 90~12113 - 1 - ` PCr/US90/01887 2~1119 :. . '' :

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O~2A~ON-~U~ Q ~U~G SY~S
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.
The invention g nerally relates to more accurate '~
redox measuring devices Analytical devices baQed on redox '' reactions can provide inaccurate measurements due to the pres-nce of redox-active contaminants This invQntion relates generally to a system for removing,;eliminating or rendering non-interfering redox- -active~cont~minants contained in fluids by the use of redox-active~in~oluble compounds (hereinafter, RA~C), methods for perfor~ing'th removal and the resulting fluid free of the ' ~' mterfering redox-contaminants The lnvention also rQlates to diagnostic kits which contain the redox-active insoluble compound us~d in accordanc- with the invention I~proved assays are obtained -as a result o~ th- invention , , Th- invention relates to disposable, machine-in,d pendent d vices and to machine or apparatus-dependent ''`-~"
devices, not disposable after each u8e. `i~
The fluid free of the interfering redox-active contaminant~ is then suitable for a variety of uses including a redox reaction based~analysis of analytes in the fluid using redox measuring systems The analysis is more accurate ~ ,- ' ., ~,. ... . .... .

Wo90~12113 PcT/US9oio1~7 2~5~ 2 than ~f performed without the use o~ the redox-active insolublQ compound This system depends on oxidation-reduction (redox) chemical reactions for thQ removal Or the unwanted or contaminating redox-active componentQ The behavior of the redox-active insoluble compound5 and the interfering redox-active contaminants which are sought to be rendered non-interfering (or inert) in the analytical reaction for the analyte (a~ well a~ various analysis technique~) can be understcod by rererence to the general principle~ o~
oxidation-reduction reactions For the purpo~es o~ racilitating the understanding ot the rollowing discussion, descr~ption and examples set rorth in this disclosurQ, th9 following derinition~ are given which are intended to supplement or complement the terms of art generally accepted Insoluble - a material is insoluble ir a fluid that passe~ over (or contacts it) picks up an amount ot said material that is wholly insignificant compared to the concentration ot the analyte(s) ot interest in said fluid As a general matter, insoluble material will not dissolve to an extent in oxcess o~ ~0 ppm, regardle~s of analyte concentration.
R dox roaction - a reAction in which electron~ are lib rated by on- sub~tance whil- simultaneously being con-u~ d by anoth r substancQ Th- substance that losQs the electrons is the rsducing agent, or reductant, and is itself oxidiz-d Th- ubstance that gains oloctrons 18 th oxidizing agent, or oxidant, and i8 it~lr roduc-d. In redox reactions, neith-r oxidation nor reduction c_n occur alone It can also b d-~ined in terms o~ chang in oxidation number - the oxidizing ag nt undorgoes an algebrAic decreasQ in oxidation number, and tho reducing ag-nt undergoes an alg~braic increa-e In redox measurement systems, redox reactions ar~ typically catalyzed ~eactions catalyzed by protoin~ includo onzymatio reaction~; reactions can also be .: ~
--" ' . . ' ' :.. " . : . . ' ' . "~ ' :' .' -' .': .. : " ' .' ~. : . ' ' -' ~ W090~12113 _ 3 _ 2 O a l l l g PcT,usgO,O,~, -non-protein catalyzed, ~-~i is the case ln the basic reducing sugar assay Redox measurement system - a redox reaction, or a set of redox reactions that act on qiome redox-active analyte i- ;
to give a product of reaction; the product then being converted to some signal that can be interpreted and used to detect the absence or pre3ence and determinQ the concentration of the analyte Examples of thls measurement include colorimetric measurement, as in the case where the product of the reaction is a color change in a dye molecule, and electronic measurement, as in the case where the redox reit~ction is linked to a semiconductor on a biosensor Redox-active - compounds which can undergo oxidation or reduction in redox reactions under the conditions present in the fluid, i o , reducing and oxidizing agent~
Analyte - a redox-active ~ubstance who~e absence or presence or concentration is measured by ~ redox measurement system Fluid - any liquid substance, either aqueous, non-aqueous or any mixture of the two, that can be contacted with an insoluble redox-active material The fluid may be subsequently analyzed in a redox measurement system, but does not have to bo so analyzed ~;
Interfering - a substance, especially a redox-active ~ubstanc-, that interferes with a redox measurement syst-m, usually by itself being oxidized or reduced, thereby gener~tlng an inaccurate result from the redox measurement syst-~ Thu a redox ba~ed analy-i~ of a fluid with an interf-ring substance would be 1-8- accurate than if the analy~i- wer- perform d without an interfering substance This term in the invention i6 interchangeable with ~conta~inating~ For fluids to be u~ed in ways other than analysis, "lnterfering" ~eans causing error or non-ideal performance in the use of the product fluid r t~

rwO 90~12113 ~ PCT/US90/0188i i 2~ ~ 4 ~ ~

The "interfering" substance will generally cause a lower reading of the concentration of the analyte, unless rendered~"non-interfering" in accordance with the invention Render non-interfering - refer~ to the effect the insoluble redox-active compound has on the redox based analysis of an analyte containing fluid, which is to make such analysis more accurate This is achieved by the inter~ction of the insoluble redox-active agent with redox-active contaminants in the fluid As a result the contaminant is oxidized if the RAIC i~ an oxidizing agent or the contaminant reduced if the RAIC is a reducing agent This interaction causes the contaminant or contaminants to be inert, inactive or to not interact with the compounds responsible ~or the redox based analytical determination The presQnce of the RAIC in the system causes the analytical reaction to be as accurate as if there were no contaminant present Although applicants do not intend to bo bound by any theory on the effect of the redox-active insoluble compound, lt is thought that the RAIC reacts with respect to the interfering contaminant by way Or a redox reaction, which precedQs the principal redox reaction which involves the analytical determination of the analyte It would appear that th ro is a net positivo electromotive potential between th~ RAIC and redox-active contaminant and a pathway exists for electron tran~fer betw en the~ while no effective pathway exists for l-ctron transfer between the RA~C and analyte of int r st Thus the RAIC reacts selectively with the conta~n~nt~ whil- not affecting the analyte When oxidized, tho r-ducing int-rf-ring compound i~ not int-rfering in the following r-dox reaction Oxldation - r-duction (redox) reactions are very co~mon and important chemical r-actions Many different typ-s of coupounds can undergo redox reactions As is well known, a ~olecul- i~ oxidized when it undergoe~ an algebraic incr~ in oxidation number, and a molecule is reduced when it und rgoe~ an alqebraic decrease in oxidation number .

W090~12113 PCT/US~/01887 ~ ~ _ 5 _ 20~ 9 .

Oxidation or reduction cannot occur wlthout the other also occurring. Thus a substance cannot be reduced without another substance being oxidized. When a substance is reduced, it i8 responsible for the oxidation of another substance, 80 the substancQ that is reduc~d is called the oxidizing agent. Conversely, the substance that is oxidized is called the reducing agent, because it is responsible for the reduction of another substance.
For redox reactions, the reader is referred to the literature, for instance House, Modern-~ynthetic Reactio~s (W.A. Benjamin, Inc., Menlo Park, CA, 1972) discusses at length various types of redox reactions. In chapter 2, House describes metal halide reactions: chapter 3 describe~
dissolving metal reductions; chapter 4 describes reductions with hydrazine: chapter 5 describes oxidation with chromium and manganese compounds: chapter 6 describes oxidation with peracids and other peroxides; and chapter 7 describes other methods of oxidation. Mortimer, Chemi~y: a Co~çeptual A~ h (D. Yan Nostrand Co., New York, 1979) gives an ;
example of a typical redox reaction which occur~ in acid -~
solution, wherein Mno4 1 is oxidized to Mn2 while As406 is reduced to H3AsO4:
24 H+ + 18 H20 + A~406 + 8 MnO4~ - - - > 20 H AgO + 8 Mn+2 Also see Manahan, Env~ro~mRn~al Chemi~stry tWillard Grant Press, Boston, 1984) which give~ an example of a redox --reaction in alkaline condition~, in which toxic, soluble chromiu~(V~ removed from waste water by reducing it to in~oluble chromium(III):

4 Cr2S3 + 3 S04 + 40 OH + 30 N~ .
The~e references are incorporated herein by reference.
The occurrence or non-occurrence of a redox reaction betwe-n any two chemical compounds will depend upon two factor~: (1) whether there i~ a driving force favoring the reaction, i.e., does the energy of the potenti~l reactants exce-d the energy of the potential product~ of a : ' -...

.. .. . . . .

W090~12113 -- PCT~US~/01~7 205~9 ~
rQdox interaction, and (2~ whether there is a pathway for the electrons to~transfer from reducing agent to oxidizin~ agent under the conditions prevailing in the ~ystem As is well known in the art, ir there exists a potential energy difference between reactant~ and products such that energy could be given up by the reaction rendering the products at a lower ~inal energy than the reactants, and i~ a means exists ror electrons to transfer from reducing agent to oxidizing agent under the conditions prevalent in the system, then the redox reaction will occur The second condition is important It is well known that most organic compounds o~ intermediate or high reduction statQ are essentially stable in the presence o~
atmo_pheric oxygen, yet they could give up energy i~ a redox reaction occurred between them and oxygen An aqueous ~olution containing ethyl alcohol and glucose, ~or instance, could oxidize to carbon dioxide and water by reaction with oxygen, as is well Xnown by those sXilled in the art But an energy barrier exists for the trans~er of electrons from thess molecules to ~xygen such that these potential redox reactions do not occur or occur at insignificant rates under conditions o~ neutral pH and temperatures Or 0-40 C Redox reaction between the3e materials and oxygen can be racilitated by a rise in ambient temperature that overcomes the energy barrier, or by the addltion Or catalyQts to pro~id a pathway for electrons between interacting redox compound~
In gen-ral organic compounds undergo redox reaction~, although, in comparison to inorganic compounds;
organlc molecul-~ more ~requently reqUir- a catalyst to ~acllitate redox reactions Enzyoe~ ar- especially important catalysts o~ organic redox reactions under condltions re~tricted to the neutral pH range and temperatures from 0 to about 40 C Enzymes catalyzing redox reactions ~all into clas--~ includlng, but not limited to, the dehydrogenases, th- oxidas-s~ the oxygenases and other oxidoreductases, as is well known in the art and described in ~oc~ trv~-~nd ed , ~ WO90/1211~ ~ PCT/US~/01887 ~ 7 20~1119 :-A.~. LQhninger, Worth Publishers, Inc. N.Y. (1975) pp. 477-508.
Other than enzyme catalysts, there are other known catalysts th~t facilitate redox rQactions of organic molecule~ with each othor, or with inorganic or organometallic redox partner These catalysts can generally be termed elQctron transfer agent(~) (etas) since they rQversibly transfer electrons among thQ redox-active compounds boing alternatively oxidized and reduced in a redox reaction. In the~e reactions, the etas are not con_umed but recycle betw on the oxidized and reduced forms, facilitating tho reaction. TherQ are a great nu~ber of chemical substancQs that can play this catalytic role in redox chemistry, including but not limited to simple ~etal ions such a8 iron in it~ (II-III) valenc-~, copper (I-II), Cr (II-III), among many other metal ions; and many organic compounds including, but not limited to phenazine metho~ulfate (PMS), 2,6-dichlorophenolindophenol (DCIP), 1,4-benzoquinone and many o~ itQ substituted forms, phenazine-, phenoxazine- and phenothiazine-based dyes including but not limlted to methylene blu , azure blue A, basic blue 3, neutral red, among a very large number of other redox-active dyes, as is well known to those skilled in the art.
It i~ to be noted that reduced che~ical co~pounds can be selectivoly oxidized in the presence of each other by addltion of r-dox catalysts that selectively interact with one or the oth~r roduced compound, for whatever reason. For ex~mple, at about pH 9 the addition of iron t~II) ion to the aqueous solution Or alcohol and glucosQ mentioned above will facilitato oxidation of the glucose, with concomitant reduction Or oxygen, but will not similarly facilitate oxidation Or the ethanol. Alternatively, addition of enzyme catalyst~ such as glucose oxidaso will cause oxidation of glucose wlthout oxidation of`ethanol, and addition of alcohol oxidase will CaU~Q oxidation of the alcohol without oxidizing the glucose.

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' Wo90~12113 PCT/US90/01887 ~ 9 - 8 - ~

Selective oxidation of reduced analytes in mixtures Or compounds by~the addition o~ a redox cataly~t that selectively interacts with an analyte o~ interest is a common and powerful means to determine the presence and to determine the coneentration of analytes, as is well known to those skilled in the art In general, the eleetrons provided by ehange in oxidation state of the analyte is eonverted to a measurable ~ignal by a number of means, ~uch a~ coupling the oxidation to a colorimetrie sy~tem a~ wa~ done in the test w ed to meaQure glucose, diselo~ed in U S Patent 4,391,906 Alternatively, a signal can be generated by coupling the oxidation to an eleetronic system, as is done with enzyme electrodes.
~ problem ari~es in using redox chemistry to detect and determine analytes when there are redox-active substance~
present in the sample along with the analyte ~he signals obtained in eolorimetric and electronic systems typically result from the total of eleetrons available from redox-active substances in the system Non analyte redox-active materials may be present in the sample that will give up their electrons to the colorimetric or electronie signal producing ~ystem~ and thereby provide a falsely high signal co~pared to th eone-ntration of analyte on eomnon means to eorrect for sueh redox-active interf-ring nat-rials is to measure the total redox signal of th yst a in two samples, one eontaining an analyte -s-l-ctive r-dox eatalyst and one without eataly~t, d t r~lning th eontent o~ analyt- by difr-r-ne- Obviously, thi~ approaeh r-quir - at least twie- as mueh er~ort, sample and analytie~l material~ to det-rmin- th- analyt-coneentration and is also inherently less aeeurate than a dir et analysis Another common means to correct for the presenee of the int-r~ering eontaminants is to destroy the~ prior to initiation o~ th analysi~ by addition o~ an exc-ss of an oxidizing ag nt ~-.

,.- - ,. ., , .. ,.. ..... , .,,.. ~ . . .. .. .. . .

WO90~12113 PCT/US90/01~7 9 - 2 ~

Rendoring interfering reducing sub6tances non-interfering by addition ot oxidants is fraught with difficulty First, the oxidant added mu~t not oxidize the analyte of interest Thus, it must be of an energy level able to spontaneously react with the interfering reducing substanc-s and it must have a pathway for electrons from interfering materials to itselr, but not from the analyte of interest More importantly, additlon of an oxidant typically leaves a residuQ ot oxidant in the sample This residu~
itselr constltutes an interfering contaminant because it is capable Or acc-pting Qleetrons from the oxidation Or the analyte in the analysis st-p, resulting in an incorrect, low signal relativ to the analyte Typically, as shown in the prior art, the oxidant added to remove interfering reducing sub6tances mu~t itselr be removed, without any ~igni~icant residu , by a back titration with a known reducing agent Alternatively, the rluid treat-d by tho oxidizing agent may be lert with the r-sidual agent in place, but the fluid is not subsequently w ed in a manner involving redox reactions This way ot correeting for the interfering materials is not satisfaetory The generalized dQscription of redox analysis of speeific analytes in complex tluids, and interference with this analy~ls by eonta~inating redueing (or oxidizing) substane-s, i~ fr quently eneountered For example, the colorl~Rtrle a~ay Or many biologically important analytes is mediatea by th eontAet with analyte-speeiric dehydrogenases ~nd th r ~ultant oxldation/reduetion ot nieotinamide adenine nuel-otid (NAD) or it~ phosphate analogue NADP In reduced form, NAD(P)H e~n bQ measured by reaetion with tetrazolium salt~ to yi-ld an equivalent a~ount of highly color~d formazan dye For instanee, NADH plw iodophenyl nitrophenyl ph nyltetrazollu~ (INT) in the pre~enee o~ a diaphorase will yield NAD and INT-~ormazan, which is dark red The amount of red color produeed by this redox reaetion is proportional to :
the eoneentration o~ NAD(P)H; however, if an interfering redueing agent were present in this sample, then a more .

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WO90~12~3 _ PCT/US90/01887 ~9 - lo ~

intense signal wlll be generated, thereby, causing an overestimation of the concentration of NADH This technology is utilized by U S Patent Numbers 3,867,259 4,024-,021;
4,247,833; and 4,556,834, all of which are subject to interference by reducing substance~ as de~cribed Another redox measurement technology that detsrmines metabolite or chemical levels in a ~luld utilizes enzyme oxidases Numerous examples of thi~ technology exist in the patsnt literature, for example ~ee U S Patent 4,391,906 U S Patent 3,164,534 and U S Patent 4,544,249 which deal with methods for eliminating bilirubin interfsrence, and U S Patsnt 4,186,251 All of thsss patents involve the very well known a88ay system in which the compound to b- measured i8 contacted with an oxida~e in the pr sence Or oxygen, resulting in hydrogsn peroxide production Th- resulting hydrogen peroxide is then utilized to oxidize a chromogen to produce an indicatlve dye It is -clear that the presence of redox-active components in these systems will lead to incorrect results, as is well Xnown in the art Indeed, the package insert accompanying the Boehringer Mannhei~ Reflotron Cholesterol diagnostic states "Th following substances, when pr~ent in high or pathologic concentrations, may produce depressed cholesterol value~
cysteino, ascorbic acid, methyldopa, gentisic acid, dipyrone, ampyrone, ho~og ntisic acid, or glutathionen All of these list d compound are interfering reducing agent~, a~ will be readily apparent to thosQ o~ aver~ge ~Xill in the art Young t al hav publish d a compr-h-nsiv list of redox-active co~pound~ th~t ~ay be pres-nt in blood that can lead to inaccuraci-~ in r-dox-ba-ed measurem nts Or blood analytes ~Clin Ch~ ~L, No 5, 1975) The list of these interfering cont~oin~nta i~ incorporated herein by refer-nce The interrer nce o~ all th reducing agents listed above referred to in Young et al , may b~ eliminated by the use of the pre--nt inv ntion Still another technology that makes use of redox reactions is th- enzyme electrode Por in~tance, the W090/12113 PCT/US~J01887 ~3 - 11 - ,.. ' 2051119 -' .
oxidation of gluco6e will produce hydroqen peroxide, the rate of whose production can be measured by a current at the surface of the electrOdQ. One such apparatus is disclosed in U.S. Patent No. 4,340,448, whlch discusses the potentiometric determination of hydrogen peroxid . Som other examples of this technology are U.S. Patent No. 4,547,280, which describes a maltose sensor, and U.S. 2atQnt No. 4,~56,074, which describes substrate-specific galactose oxidase ~ -electrodes. Since these electrodes are dependent on redox reactions, interfering substanc-s that are redox-active may generate incorrect readings.
Newer measurement ~ystems that use redox chemistry can com~ in the form of biosensors. This is a generic term --that i~ not specific to any measure~ent type, but one embodiment of bio6ensors involve~ attaching a redox-active biological material to a semiconductor. The behavior o~ the semiconductor and, therefore, the signal generated by the semiconductor, is dependent upon the redox state of the biological material. In this field, as in the more traditional bio-measurement field~ disc w sed above, it is important that the redox stats of the particular measurement system, in this case, the redox-active biological material, be a~Sected only by the ~nalyte. Therefore, if an interfering r-dox-_ctive substance is present in the fluid to be measured, an erronoous sign_l will be generated.
Pending patent application serial number 075,817, discloses a method for decreasing the color produced by colorl~ tric r-dox-measuring 6ystems. The dlsclosure discussQs the oxid_tion o~ NAD~P)H and the simultaneous reductlon o~ a chromogen, the reaction being catalyzed by diaphoras-. In that system, the concentration o~ NAD(P)H is such that too much color would be produced by the chromogen, so that the concentration o~ NAD(P)~ cannot be determined.
Therefore, an alternate electron acceptor is introduced that comp tes ~or electrons with the chromogen. Th~ alternate electron acceptor is by definition an oxidizing agent.
There~ore, problems may arise when a contamina~ing reducing -W09~ 3 5~3 - 12 - PCT/US~/OIU7 agent is present; the contaminating agent can reduce the alternate electron acceptor, thereby giving a ~al~e reading aRI~E DE~cR~E5lQN OF ~
Typical of the conventional technology which adds oxidizing or redox-active agent~ to biological rluids in which the addition of such agent~ are made ~or variou roa~ons are the ~ollowing i~ctued patents U S Patent No 4,342,740 describe~ th~t use of a soluble oxidizing agent which i5 added to ~aeilitate the labeling o~ red blood cell~ wlth Technetium - 99 M The re ulting fluid eontain~ re~idual oxidizing agent and the rluid is not sub~equently Analyzed by redox analysis U S Patent No 4,180,592 deseribes a process ~or deeolorizing blood by the addition o~ the soluble oxidizing -agont hydrogen peroxide and the removal o~ exee-s peroxide The end use Or the decolorized blood produet is not analyzed by redox method~
U S Patent No 4,298,688 deseribes a test strip to mea~ure glueo~e in body fluids The strip contain-a an oxidizing agent to d-stroy interferents The re~idual oxidizing ag nt and glueosQ are separate~d by ehromotography Th~ patent do-s not t-aeh nor sugg st the U8- Or insoluble oxidizing agents and their removal based on insolubility U S Patent No 4,255,38S deseribe~ a ~ethod, r-agont and t-~t kit rOr the deter~ination Or glycosylated he~oglobin wlth u8e ot the soluble oxidizing agent, t-rrieyanide The treated rluid eontainC~ residual oxidiz~ng ag nt and i- not analyz-d by redox r aetion~
~ S Pat-nt No 3,894,844 d-serib-~ a m thod for det-r~ining th ~oount Or triglyeerides, eholesterol and pho-pholipid present in blood by using oxidation reactions The thre- anaiytes Or interest are separated in a procedure which rooov ~ interf-ring redueing substanees as well U S Patent No 4,256,833 deseribe th preparation Or a p-roxida~--eoupled IgG antibody by foraation Or a Schlf~'- b~se. The ree~ulting produet is preeipitated and reeov r d fro~ th reduetion reaetion mixture er~eetively , . .

W090/12113 PCT/US~/01~7 - ~ - 13 - 20~1 119 removing it from the resldu~l roducing agent beforo subsequent use Insoluble reducing agent~ ~ro not taught nor ~uggested U S Patent No 4,645,660 doscribe~ th- addition of a soluble reducing agent to a radioactive diagnostic agent wherein iron ions compet- with the dQsired radioactive elements for formation of desired chelate compounds, and a reducing agent is added during formation of the~e chelates to reduce the iron to a form th~t does not effQctiv-ly chelate In this patent the resulting fluid contains the rQsidual reducing agents and the fluid is not subsequ-ntly an~lyzed by r-dox ~ethod~
U S Patent No 4,720,385 doscrib g the st-rilization of therapeutically or immunologically activ~ ~-protein ~olutions by contacting th -e solution~ with a metal chelat- complex such ~ copper ph nanthroline and a reducing agent, such a8 ascorbic aeid or a thiol The resulting solution contains the soluble rQsidual reducing agent and is -~
not subseguently analyzed by redox methods Patents deal$ng with the addition of oxidizing agent~ to solutions containing various contaminants, for the purpose of r-moving the conta~inants includ- U S Patent No 4,572,797, d scribing the addition of soluble oxidizing agents to aqueou~ solutions containing trace metals In this patent, the oxidizod product of the tr~ce contamin~nt is insoluble in th liquid i~ then sQparable from the fluid as a precipitata Th rc~idual oxldizing agent remain~ in ~olutlon and th resulting fluid 18 not analyzed by redox method~
Similarly, U S Patent No 4,431,847 d~scribes the addition oS an oxidizing agent to fluids containing halog nated phenolic compounds, to cause the polymerization and insolubilization oS th 8e compounds The resulting polymeric Sorm of the contaninants are easily renoved as precipitates The resulting fluid contain~ the residual oxidizing agent and i~ not subseguently analyzed by redox methods :

W090/~2113 ~9 _ PCT/US~/01~7 14 - ~

U S Patent No 4,249,939 describes the removal of copper from spent solutions o~ complexing agents containing cuprous aluminum tetrahalide~ In this patent, the copper is oxidized to the water soluble eupric form by contacting the fluid with an oxidizing agent organic materials in the solution are removed by extraction with an organic solvent, and the copper is subsequently recovered by contacting the aqueouQ pha~e with a metal of higher electromotive potential than copper, plating the copper out of the solution In this last step, the reducing agent, the metal of higher potentiai, i~ an in~oluble agont serving as a specitic means o~
recovering tho copper from solution Th$s review of the prior art shows that tho technology for pretreating fluids such as biological rluids for subsequQnt rodox-based analysis does not utilizo insolubl6 oxidizing agents which insoluble oxidizing agents aro sub~equontly removed by means based on their insolubility so as to produce a tluid suitable tor redox-based analysis;
there is no diselosuro either of etas used in conjunction with sueh ~y~tems This inv~ntion relates to a system including kits, rOr the $nact$vation or the render$ng non-interSering of intertering redox-~etiv sub~tances in a tluid by the use of r-dox-aet$v eo~pounds, a mothod ~or using the redox-active insolubl- eo~pound to render inert the interfering su~-tane-~ in th tluid and th purlti-d fluid freo of the int rt-rinq eo~pound Th -y to~ ot th inventlon prov$des tor highly improv d ~n~lytleal re~ult~ such a~ would be obta$ned in the ab~enc- ot th- eonta~nant under eomparable eondit$ons Th- inv ntlon rolates to disposable, non-maehine or in~trum nt~tion-ind p-ndent dispo~able deviees, and to maehin~ or in tru~ ntation dopendent devices, whieh are not eusto~rily dise~rd d a-tter eaeh U8-. -- Th invention diselosed overeomes the di~tieulties present-d by redueing agents contaminating a fluid The , . :. - . . . : . - . ............... --: : . - . .. : ....... ... . : - , . . .. - - -, ,- ~ ~ . . . . ~ i .. . . . .

W090/12113 PCT/US~/OlU~
~. - 15 _ 20~;1119 ... .

invention involves a process for providing an amount of an oxidizing agent in exces~ of that required to render the contaminants non-interfering, of energy level enabling the spontaneous reAction with the contaminant reducing agent The fluid also provides optionally an electron transfer agent to catalyze passage of electrons rrOm the reducing agent to the added oxidizing agent The oxidizing agent has the distinctive property to be insoluble in the ~luid, so that it can be simply removed from the treated fluid by rapid, uncomplicated m~ans ~uch as filtratlon, decantatlon, sedimentation or centrlfugatlon or other mQthods known in the -~
art The fluid treated in this manner i8 then prepared or ready by subseguent analy~is or other use free from interference by eith-r th~ cont~minating reducing substAnce~
or residual oxidizing agent This invention is particularly useful in the --pretreatment Or certaia biological ~luids including, but not limited to, blood serum or plasma, cerebrospinal fluid, semen, saliva, tears, sinovial fluid, lymph or urine since these contain not only many important analytes, but also contain unknown and variable amounts of contaminating reducing ag~nts The invention ls also usQful for veterinary applications in t~e analysis o~ the blological fluids o~
animals The invention may also be applied to complex fluids containing contaminating oxidizing agents, wherein these contaminant- would be destroyed by the addition o~ electron tran-f-r agent and an insoluble reducing agent, that~would be removed on th ba~is Or it~ insolubility prior to use or analysi- o~ the rluid Thus the invention is applicable to a system and method Or rendering non-interfering either oxidizing cont~in~nt~ or reducing contaminants by an insolubl~ oxidizing agent or r~ducing agent, respectively The fluld to bQ treated can be aqueous partially aqueou or a non-aqueous (or organic) fluid which contains an analyte to be assayed as well as redox-active contaminants The invention is broadly applicable to fluids which are , .

1 ' ' , . . , ' . , ~ . ., ,' ' . ' , ' ' . . 1~ ` , . . . ' ' c~,Q~J~9 - 16 - ~

single or multi-phase; multi-phase fluids may be eonsidered emul~ion~ or suspensions The ~ystem of the invention, provldes for the interfering-substanee containing fluid to be eontacted with the redox-aetive agent whieh is insoluble in the fluid The interfering substance can be a reducing or an oxidizing compound The insoluble redox-active compound undergoe~ a redox reaction with the interfering substanc~ In~oluble redox agent remaining in th~ fluid is then selectively remov d Srom the Sluld Th~ re~ulting ~luid free of the inter~ering substance ean then be analyzed for a particular analyte by oxidation-reduetion analy~i~ method-Q without the lnterfering redox contamlnant that would causQ inaccurate results Thus, the analy~i~ perform~d on the purlSied fluid is mor~ reliable and accurate Si~llarly any other use made of the fluid can be without the dlstortlng or interfering effect that the redox-aetive conta-~nants would eause In additlon to th- us~ in m~chine-lndependent d-vie-J, the invention is applieable Sor use in m~chine-dependent diagnostic ~yst-ms and methods whereby more aceurate analysis of a fluid's analyte or analytes is possible G n-rally the maehine-independent d~vices are disearded a~ter eaeh use, the maehin--dependent are not - , A particularly important embodiment Or the invention i- klt~ Th kits inelude a ~ystem for bringing th analyt- eontaining fluid into eontaet with the RAIC so ' ' , that tho RAIC r nder~ non-interf-ring substantially all the, r-doY-aetiv eontauinants in th- fluid Th- kit provides a ~ ns for th -leetive pass~ge of th- tr-ated fluid into a sep~rat- eo~p~rtu nt or vess-l without any r-sidual RAIC
Th kit provid-- eompounds ror an oxidation-reduetion based analysis to t~k- plae- in tho eomp~rtment This analysis is p rfor~ d on the fluid whieh has had any redox-aetive eont~-1nants a~ w ll as residual RAIC re~oved Thu~ an aeeurat- r-dox-bas-d analy~is Sor a partieular analyte in the ~luid e~n th n b- performQd --: .:
~-, -Wo90/~2~3 PCT~US~/01887 - ~ - 17 -The invention alQo provides a method by oxidation for the removal, inactivation or the rendering non-interfering of interfering, redox-active contaminants which are contained in fluids. The invention also encompa~sesi -fluids such aQ biological ~luids free of contaminants that would interfere in a redox reaction and render a redox-based analysi~ inaccurate. The invention encompassies biological fluids which are suitable for redox analy~is of the fluid~s redox-active analytes.
The sy~tem (including kits) and method of the invention also optionally contemplates the use of active electron transfer ~gents to facilitate the oxidation-reduction reaction between the insoluble redox-active agent and the redox-active interfering contaminant. The use of the electron transfer agent or agents in con~unction with the insoluble redox-active purifying compounds more effectively subject the intcrfering compound to a redox reaction.
Any residual redox-Active insoluble agent which remains after the fluid has been treated, is renoved by any means ConveniQnt to one s~illed in the _rt using the insolubility property of the compound. Typical means include ~ -but are not limit-d to filtration, sedimentation, -~
centrifugation or decantation. The fluid that remains i~
free of any residual redox-active agent as well as the redox~
active contaminant ~iaterlal.
In on- embodiment of the invention, the fluid to be purified is pretreated with the insoluble redox-active agent.
The r-dox-activ agent reacts with the redox-active conta~inants contained in the fluld. Aftor the fluid is recovered with th- redox cont~nants inactivated, removed or rendered non-interfering and any residual insoluble redox agQnt re~ov d, the fluid i8 assayRd for the particular analyte sought to be determined in a redox measuring system.
Thes- redox based measuring systems for analyzing a fluid include but are not limited to those described earlier in this application. ~ -.-. ... : , .. , ., , , , -., . ,.. ; ~.. . ;~. .. ... . . ,. - .

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W090/12113 - PcT/Us9o/o1 ~3~ 18 - ~

In another embodiment of the invention, the fluid to be purified and then analyzed passes into two reaction chambers The first chamber (or area) contains the insoluble redox agent and'may or may not contain an electron transfer agent The fluid containing the contaminants pa~ses through the first chamber where the redox contaminants are rendered non-interfering and then into a second chamber without carry over Or the insoluble redox agent, where the fluid is assayed for particular analytes These analytes are detected and coneentration determined by a redox mea~uring system for analyzing a fluid including but not limited to those d-scribed earlier in this application This embodiment permits the ~luid or analyte to be treated and measured in one clos-d system, in one proeedure and without having to reeover the purifled rluid from on~ sy~tem and transferring it to another syste~
The insoluble redox-aetivQ agent-~ selectively react with the redox-aetive eontaminant when the analyte and the insoluble redox-aetive agent are presQnt in the same fluid; ' ' th~y do not affeet the ~luid's analyte which i~ subsequently to b- analyz-d Thus, the insoluble redox agent can be eon~idered Qub~tantially inert with re~pQet to the fluid~s analyte to b- analyzed ' G-nerally, the amount Or insoluble redox-active ag nt used i8 that amount suf~ieient to eliminatQ the adverse e~r-et o~ any interfering redox-aetive eontaminant in the fluid to be analyzed Any YC-~8 in~olubl- r-dox-aetiv- ag nt i~ th-n r-uov d a~ d ~eribed ls-wh-re in thi~ applieation Co~only the amount Or insoluble redox-active agent u -d to treat on ml o~ bodily rluid ranges ~rom about 1 to about 100 ~g, prererably rrou about 5 to about 20mg Pr-r-rably th insoluble redox-aetive agent is plaeed in a physical ~tate th~t is mo~t e~eetive to render the fluid~s ~' eont~inants non-int-rrering It is desirable to inerease as mueh a~ po~aible its reaetive sur~aee areas For instance, ; - ,. ' , W090~12113 PCT/US~/01~7 - 19 - 2 ~ 9 such a physic~l state include~ that o~ a finely-ground material Some of the more preferred insoluble redox-active agents are lead (IV) dioxide (PbO2), cuprous 3ulfide (Cu2S), ceric dioxide (CeO2), silver sulfide (Ag2S), sllver ferrocyanide (Ag4 Fe(CN)6), silver oxide (Ag20), manganese -dioxide (MnO2), barium permanganate (BaMnO4), cuprous carbonate, potasfiium dichromate (X2Cr207), potassium permanganate (RMnO4), cuprous ferrocyanide, lead permanganate and the peroxyacetic acid derivative of carboxymethyl cellulo-e The particular insolubl~ redox-~ctive agent select-d will depend on the fluid and analyte to be analyzed The redox-active agents in the invention belong to cla~ of compounds that can be identified as follows Variou~ electron transfer agent~ specifi~d -~
hereln~ ter can be used to facilitate the reaction o~ RAIC
and contamtnants However, the use Or electron transfer agents is not necessary rOr this invont$on and is an optional ingredient Thes- electron transfor agents are generally used in an amount in the range of about O l-lOmM The preferred rango of ~mount of electron transfer agent is about 0 5mM-2mM These electron transfer agents do not interfere with ~ -the subsequ-nt redox analysis of tho rluid It should be noted that this invention has special application wh ro there i8 a need for an accurate analysis of a biological fluid In particular blood, plasma or serum is ort n analyz-d rOr particular analyt-~ of interest such as chole~t-rol, blood urea nitrog n, ketones, glucwe, lactate or triglyceride~ Interfering contaminants with which this invontion deal~ includ , without limitation the ~ollowing class-s Or compounds redox active acids, thiols and bilirubin In blosd these may includo organic acid~ like ascorbic acid, as well as other substancss such as uric acid, genti~ic acid, bilirubin, glutathione, cysteine, and thiol-containing peptides and proteins such a~ albumins The Wo90~12113 PCT/US90/01887 ~ 20 -speeifle nature of many contaminants that intQrfsre-in the redox-based analysls of a fluid are g-nsrally unknown Various proteins thQmsolves may bs a~soeiated with contaminants which may be substituQnts ther-on In thos-eircum~taness, these are oxidiz-d The as~aying or analyzing Or other bodily fluid for glucosQ, or monosaeeharides is also of intersst for patiQnts with dlabete~ IntQrtsring eontaminants in ~uch bodily fluids inelude uric aeid, a~corbic acid and thiol-containing proteins and peptides Noverth~ s thi~ invention ha~ broad r application than the an_lysis of biologieal fluid~ Thu , it is appar-nt trom th~
de~cription provided that it is ~n ob~ect o~ the invention to - -render non-interfering tho-e r-dox-activ- conta~inant~
eontained in th fluid to b- _nalyzed that int-rfer- with the d t-ction and d termin~tlon ot th conc-ntration Or an analyt- that ls to b analyz-d ualng nzymatic or oxidation-r duetion ehQ~istry Sinil_rly lt is an ob~-et Or the invention to render non-interfsring thos- r-dox-aetiv eontaminants in a tluid that intorf-re in th w of th tluid and to provide a fluid fr- of int-rt-ring rsdox-activ eonta~inant~ so that ~aid tluid will not b~ affeeted by th- intQrfering --sub-t_nc~
Thw it i~ _n ob~-et of th inv ntion to provide a sy~t ~ and ~ thod of s-l-etiv!ly reaeting with the eon ~-tnant and whieh do-s not d -troy, alt r or in_etivate th analyt and whieh do-~ not _tt-et th analyte to b analyz-d or ~ -ay d by a r-dox-ba--d r aetlon It i- an ob~-et of th lnv ntlon to provid a y t ~ and ~ thod rOr r-~ovlng or lnaetlvating rro~ a tluid r-dox-aetlv sub-tane-a without l-aving a n w r~dox-_etive eonta~inant or a r idu Or in-olubl- r dox-aetiv _g-nt aft r th fluid h~s b -n treat d for th r noval of the int rt-ring eont_~inant~
It l- also n ob~-et Or th inv ntlon to provide a ~y t-n and ~ thod whieh r move~, inaetivat ~, or r nd rs non-int rr-ring tho-- int r~ering eont~ninants eontain d in-a - . - ..
.:

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~ 90/12113 - 21 - PCT/US90/01887 2 ~ 9 fluid, when said fluid is to b- analyzed by oxidation-reduction means a~ woll a~ a me~n- for a~-aying the analyte within one closed ~ystem without the noed for removal and transfer of the fluid aft-r the fluid i~ purifiQd to another separate syst-m It is al~o an ob~ect of the invention to provide disposable diagnostic test kit~ or apparatus-independent kits for accurately det~rmining the presQnce or concentration of an analyte in a fluid by pr-treating the fluid with the insolubl~ redox-active compound and analyzing the pretreated fluid by redox-b~sed mQthods all within one kit It is also an obj-ct of the inventlon to pretr-~t biological fluid~ to remove contaminant ~o the pretreated fluid can be analyz-d for particular analyt-s or used for oth-r purpos-s fre- from interfering or distorting redox-active agents The invention is not limit-d to the objects listed above and th-s- ob~ects as well as oth r u8e8 o~ the invention will become app rent to those skilled in the art ~-and by the description which follows Th invention provid - a systQm of tre~tment or method perfor~ d on a fluid which can be agueou~, non-aqueou~, or a mixture The ~luid generally contains a redox-activ analyt or analytes which fluid contains a redox-activ int rf-ring contaminant Th SQ contaminant~ are interf-ring in the sense defined arll-r in that th-y interf r- in the accurate analy-is of a redox-active analyte in th ~luld which is sought to b- nalyzed by a redox m asurement syst m The contaminants are also interfering in the sense that th y cause error or non-ideal performance in th~ us- of the fluid T~e inv ntion provides a ~yste~ and method whereby the fluid iB tr-ated wit~ an in~oluble redox-activQ agent It-is important to note that regardl-ss of whether the fluid is aqueous, non-aqueous or parti~lly aqueou~, the redox-active agent used in this invention should be insoluble in ~ ..

t . .... . .. . ... . . ..... . . ..
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... : . - - . -. . - . . .. .
:- . . . . .. . . . - .. : .

WO ~12113 -- PCT/US~/0188 - ~ 3 22 -the fluid The amount of insoluble redox-active agent introduced will depend on the ~luid itselr and the redox-active component to be inactivated by the redox reaction The amount o~ insoluble redox-activQ agent used i~ that amount which i~ sufficient to render inter~ering redox-active contaminant~ non-interfering In general an amount of insoluble redox-activQ agent is added which is in excess of that needed to insure the complete oxidation-reduction reaction o~ the insolublo redox-active agent wlth the redox-active contaminant~ -Th~ temperature at which thQ reaction is carried out is generally ambient temperature such as in the range of about lO C to 50 C
The roaction botween th~ in~oluble redox-active ag-nt or ag-nts or tho redox-activ contaminant or contaminant~ c~n b~ facilitated by the introduction of one or mor- electron transfer agents to th ~luid After the rluid has beon treated so that all or substantially all the interfering redox-active contaminants hav- undergono a redox reaction or have beon rendered non-interfering, any remaining insoluble redox-active agent is ~`
renoved This removal is aeeomplish d by m ans based on ~he insolubility of th- r-dox-aetiv agent Th- r ~ulting fluid is free of redox-active eonta~inant~ a- w ll as residual redox-aetivo agent This fluid i- id al for aeeurate analysis for redox-aetive analyte by r-dox-r-aetion ~ asure~ nt sy~t~mJ Th- invention provld ~ a fluld which i- fre- in gen-ral of any re~idual non--p~ei~le r-dueing or oxldizing ag nt whieh would eonstitut- an interferenee for the subseguent use or the fluid - Thu~ th kit, systen and ~ thod di~closed herein gener~lly involves a set of two sQquential redox reactions Th fir t r aetion involve~ th- RAIC and interfering redox-activ cont~ inunt Th seeond reaetion i8 generally a NAD-NADH dep nd nt r-dox reaetion or a non-NAD-NADH oxido-r duet~ - (-nzy~atie) redox reaetion eoupled to color W090~12113 PCT/US90101887 changing reactions which involves the determinatlon Or the concentr~tion or presence of an analyte A large number of color changing reactions such a~ the peroxidase-chromogen reactions for hydrogen peroxid- can be coupl-d to these r~dox reactions The kit include~ both thos- compounds necessary for rendering the contaminants non-interfering, and those ~-compoundQ for A redox-b~d analysis The kit typically include~ a physical support or housing for two layers of filter~ betw-en w~ich is contained the ~AIC The fluld to be treated i~ generally passed through the fir~t filter which may contain an lectron transfer ag nt The fluid dissolves th- electron tran~fer agQnt, contact~ th- RAIC and paJse~
through the second rilter with the RAIC retain-d This invention i~ useful for r-moving interfering reducing substance~ from ~ liquid medluc This invention is particularly u eful ir the fluid to bQ treatQd i8 a sample that i8 to be analyzed by redox chemistry, or a sample where residual non-specific reducing or oxidizing agent constitutes an interference for the fiubsQquent use Or the fluid The invention i8 e~pecially U8QfUl i~ th ~luid i~ a bodily fluid such a~ blood sQru~ or plas~a, amniotic fluid, cerebrospinal fluid, sinovi~l fluid, urine, saliva, tears, semQn and the like, and the Jub~equent use i~ the analyJis of the fluid for the concentr~tion of a component of the fluid by enzymatic and other r doY reaction AJ di-cu~od above, the basic principl-s ot redox chemistry ~nd it~ use in measur-ment systems ar- w-ll known When ~n an~lyt- is r-dox-active, it c~n b- u~ed ln redox rQ~ctions to det-ct its pr-~ence and determin- its concentr~tion Det-rmination Or an analyte by redox rQ~ctions, how ver, opens the possibility of interference by any non-~n~lyt redox-active cont~min~nt in the sample Ther fore, it i- w eful to have a means by which interfering redox-active substance~ can be eliminated without affecting the analyte~ that will subsequently be as~ayed by a redox - ' .
:

., , ~ . . ,, . . .- ..... . ... . .. , . . . : . i , .: , . .... .

WO90/12113 _ PCT/US90~01~7 ~ 24 - ~

reactlon, and without leaving a new redox-actlve contaminant in the fluid, such as residual oxidizing agent To accomplish the removal of interSering reducing agents, the use of insoluble oxidizing agents was contemplated In this invention, in~oluble meana the inability to dissolve to any appreciable extent in the fluid of interest As a general characteristic, appreciable solubility refer~ to the residual concentration, if any, of the insoluble agent relative to the analyte o~ interest, and in typical situations, the residual will be les~ than l/looo of the analyte concentration, and in any casQ, this residual concentration will typically be less than about 10 ppm in the product fluid The oxidlzing agent used in this invention is insoluble in the fluid to be treated or analyzed Obviously, the solubility o~ an oxidizing agent, as for any chemical composition, will depend upon the nature of the fluid Manganese dioxids, for example, is insoluble in water but soluble in llN HCl Potassium dichromate is soluble in water but insoluble in alcohols Solubilities of organie and inorganic oxidizing (and reducing) agents in a wide variety of solvents are well known to those skilled in t~o art, and are extensively compiled, for instance, in the Tables o~ Physieal Constant o~ Inorganie and of Organic Compounds, ~ound in the Handbook Or Chemistry and Physics, CRC Publications, Clevoland, OH Also for the purpose of this invontion, oxidizing agent~ will moan any chemical eo~po-ition having an eleetromotive potential ~uch th~t it would r aet with anothor ehemieal eomposition, ealled the r-dueing ag nt, having di~orent ol-etromotiv potential, wh ro olsetrons ar- transferred spontaneously from the redueing ag nt to th- oxidizing agsnt if a means or pathway ~or th el-etron trans~or was available ~n aeeordanee with the inv~ntion an oxidizing eompound may bo used with an eleetro tiv potontial sueh that it ean react with an int-r~-rinq rodueing eontaminant Numerous treaties deseribing the very wide seope of redox-aetivo ehemieal eompositions have been published and ~ ;

W090/~2113 PCT/US90/01~7 ~ - 25 ~ 2 0 ~

are available in chemical references, as i~ well known to those of avQrage skill ~n the art. The extsnsive compilations Or the standard reduetion potentials of inorganic and organie compounds may be consult-d to readily determine the relative redueing or oxidizing strength of any particular inorganic or organic compound. The compounds characterized by a highly posltive standard reduction potential are preferred redueing agent~. ~ho actual oecurrence of a redox re~etion depends upon the combined potentials Or the oxldizing and reducing agents undor a particular set of conditions, including the avallAbillty of a pathway or ~eans rOr the el~ctrons to transfer between the two agQnts. In particular, the standard reduction potentlals rerer to redox-activQ species at 1 Molar concentration in solution.
Thus rrom among the published compendia, it would not involve undue experimentation to select compounds having both a relatively potent oxidizing (or reducing) activity, and insolubility in any particular solvent. The means to make such seleetions are quite well Xnown to anyone of average ~kill in the art.
While the seleetion of insoluble oxidizing (and redueing) agent~ eAn b- 80 made it is not obvious that insoluble oxidlzing tor reducing) agents would aetually effeetively reaet with other redox partners. This may be beeauso the insignirieant solution eoncentration o~ insoluble agent~ would ~bolish the inherent redox eapability or that the roaetion would be prevented by tho neeessity tor eleetron trans~er aeros~ a physieal phase barrier, or for other specific reason-.
In faet, attempts to destroy the eontAminating redueing activity of human plasma by addition of various insolu~le oxidizing agents were only partly suecessful.
When, for example, plasma was eontacted with erystals of manganese (rV) dioxide, there oeeurs some diminution of the plasma eapaeity to reduee indieators like ferrieyanide ion, but surrieient redueinq aetivity remains to eonsume several .~, . . . . . - - - . . .......................... .

.. . .. . .

WO90/12113 PCT/US~/~1887 2~ 9 - 26 - - -tenths of mN o~ thi~ agent; and, for ex~mple, to reduce the tetrazolium prodye MTT to give a colored formazan This reducing capacity of plasma did not change even after hours of agitated contact of the plas~a with the insoluble oxidizing agents An analysis of the contaminating reducing activity remaining after MnO2 treatment demonstrated that all of the remaining acti~lty wa-~ located in the trichloracetic acid (TCA) precipitated fraction The reducing contaminants in the TCA soluble ~raction were destroyed by manganese dioxide treatment It is well known that TCA will only precipitate macromolecul~r polymers, e g , proteins Therefore, apparently manganese dioxide is incapable of reacting with protein associated reducing cont~nants, either because it is not of su~ficient oxidizing strength, or because no pathway exists for transfer of electrons between the polym ric contaminant and the in~olubl- mAngan-se dloxide It was therefore surprising, wh-n treatment of the plasmA-MnO2 suspension by very ~mAll amounts of a wide variety o~ electron transfer agents, overcame this poor r-action between the contaminating reducing qubstances and th insoluble ox$dizing agent Plasma treated with 1 mM
electron transfer agent and contacted for a few seconds with -~
solid, insoluble oxiiizing agent, completely lost the ability to r duc- ferricyanid-, or to reduc- tetrazolium salts to the for~az_n Mor over, pretreatment Or plasma in thi_ manner did not arrQct the concentration Or any Or the common co~pon nts Or blood, serum Or plas~a usually assayed by redox ch-~i-try, except those components th_t _ro highly redox-_ctiv _nd dir-ctly able to reduc- oxidizing agents like f-rricyAnlde Thu~ uric acid, glutathione and ascorbic acid, for x_nple w re destroy d, but cholesterol, triglycerides, A~no _cids (excopt cysteine), and ~o ~orth wer- unarfected There are occurrences where complete destruction of all r ducing contaminant_ in plasma would occur in the ab~nco o~ an electron transfer agent These occurrences are adv_nt_geou~ in Qituations where, for example, the electron ~
,. .

WO ~/12113 PCTtUS~/01887 . ~ .
2 0 ~ 9 transrer agent would interfere wlth subseguent analy~is. we discovered that lead (IV! dioxide insoluble oxidation agent is capable of destruction of all the contaminating reducing activity in plasma. An electron transferring substance is not needed for the action of PbO2, and both the TCA soluble and insoluble contaminants are deQtroyed. It is surprising that PbO2 should be an acceptabls agent. The standard oxidat~on potential of lead is 1.455 voltQ, considerably greater than the 1.229 volt reduction potential of oxygen.
Thus, lead (IV) dioxide has the energetic capacity to split water, sugge~ting that it might not be stable in an aqueous solution. Stability is observed, however, probably due to th- extreme insolubility Or the lead oxidizing agent.
Thus in accordance with the invention the removal of contaminating reducing (or oxidizing) ~gent W~8 accomplishsd by the pretreatment Or a fluid with an insoluble oxidizing (or reducing) agent, QspQcially when the fluid was also treated with a soluble electron transfer agent to provide more effective pathways for electrons to the insoluble agent. The resultant fluid is free of both oxidizing and reducing activity and will be especially suitable for uses where such re~idual activity constitutes an interference (but where the presence of the electron transfer agent does not constitute such interference). Of special utility would be fluids to be subsequently analyzed by methods involving the unmasking of specific reducing (or oxidizing) activity by ~pecific redox catalysts such as redox-activ enzyne~. or course, the invention is not intended to be lim~ted to analytical uses Or fluids pretreated in accordance with the invention, and indeed, the general spirit and teaching of this invention makes obvious a multitude Or other wes to those of average skill in the art.
In accordance with the invention, the systQm comprises the treatment of some fluid, that fluid either hydrophilic (aqueow solutions), hydrophobic (lipid-like), or any mixture of the two. An insoluble oxidizing agent is introduced into the fluid so that the agent will contact the : . . - . .

' WO90/12113 _ PCT/US90/01887 ~,,r~ - 28 -interfering contaminants. Some of the more preferred insoluble redox-active agents are: cuprous sulfide (Cu2S), ceric diox$dQ (Ceo2), silver sulfid~ (Ag2S), silver ferrocyanide (Ag4 Fe~CN)6), silver oxide (Ag20), manganes~
dioxide (MnO2), barium permanganate (BaMnO4), potassium dichromate (~2cr2o7), potas~ium permanganate (KMnO4), cuprous fQrrocyanide, lead permanganate and the peroxyacetic acid derivative of carboxymethyl cellulos~. The use of a particular insoluble redox-active agent will depend on the rluid and analyte to be analyzed.
Generally the amount of insoluble oxidizing agent to be used i5 that amount sufficient to render non-interfering any interfering reducing agents in the fluid to be analyzed, but is usually an amount in excQss o~ that amount needed to in~ure the complete oxidation of all contaminants. This amount is a funct$on of thQ surface area the insolubl~ redox-activQ agent provides rOr effective contact with the interfering contaminant in the fluid. Since the redox-active agent is insolublQ it can only react with the interfering contaminant at its surface area in contact - -with the interfering contaminant. Thus a smaller guantity of an insoluble rQdox-activQ agent with a largs surface area ~-such as a fin~ly ground powder would render contaminants non-interfering ~ust as effectively as a larger guantity of the insolubl~ r~dox-active agent such as on~ lumped mass.
It i8 also be notQd that the liquid medium itself and the contaminant to be rQndered non-interfering also att-ct th ~ount of insoluble redox-active agent to be used.
The r~ction or the insolubl~ oxidlzing (or reducing) agent may be furthor ~aeilitatQd by inclusion of small amounts, usu_lly in th~ rang- of about 0.1-2 mM o~ any of a variety of electron tr_nsf-r agents, not limited to but including the group con~isting o~ phenazin~ methosul~ate, 2,6-dichlorophenolindophenol, substituted 1,4-benzoquinones including l,4-bonzoguinone, ~ono-, di-, tri- and tetra- -substituted 1,4-benzoquinones where the substituents may include, but ars not limited to, simple alkyl groups WO90~12il3 PCT/US90/01~7 ` - 29 -2 Q ~
containing 1 to 5 carbon~, halogen~, alkoxyl radicals with 1 to 4 carbons, among other posslble substitutions Substituted 1,4-benzoguinones of particular utility in thi~ regard are 2,5- and 2,6-dimethyl-1,4-benzoquinone, 2,5- and 2,6-dichloro-1,4-benzoquinone, 2-isopropyl-5-methyl-3,6-dibromo-1,4-benzoquinone, 2-methoxy-5-methyl-1,4-benzoquinon-, and 2,3-dimethoxy-5-methyl-1,4-benzoquinone Substitut-d and unsUbstitutQd 1,4-naphthoquinones, and 1,2-benzoquinono~ ar- also generally userul as electron transfer agents in this invention A large collection of redox-active dyes generally members of the phenazine, phenoxazine, phenothiazin~ and ind~in f~milies of dyes also function well as electron trans~er agents, although they aro usually not pre~erred becau-e they impart a color to the final treated ~luld An ext-nsive compilation o~ dyes in these families, all Or whlch function to some degree a8 electron transfer agents in thi- invention, is givon in ~H J Conn~s Biological Stains 9th Ed,~ R D Lillie, Ed, Williams and Wilkins, Baltim~re, MD (1977) Treatment methods in accordance with the invention can be any method which will accomplish the objectives described her-in In one method analyte is passed over a bed the insolublo oxidizing (or reducing) agent The bed may contain an amount Or oxidizing agent so that all cont~inating r~ducing agents are oxidized a8 the analyte pas~es over th b d The surface area of the bed is prer-rably ~ufriciently large 80 that all analyte comes in contact wlth oxidizing agent as the analyte flow6 through the bed T~e b d of oxidizing agent may be supported on a porous filter matrix that rQtains the oxidizing agent while allowing the fluld to pass through rapidly In another embodim nt of the invention, th insolubl- oxidizing agent is sandwiched between two membrane- Th analyte is introduced through the top of one membrane, rlow8 through the layer Or oxidizing agent, then passes through the second membrane The membrane allows the rapid passage of ~luid, but retain the oxidizing agent so WO ~/121~3 - - PCT/US90/01 ~.J~'~ 30~

that $t is ~eparated from the fluid The kit represents a specific physical disposable embodiments of the system In a further embodiment oS the invention, the oxidizing agent i~ bound to a substrate The substrate i~
insoluble, and can consist of, but is not limited to membranes, polymers, and columns of some supporting material Binding the oxidizing agent to an insoluble support has the effect of rendering the oxidizing agent insoluble Analyte is then passed through the oxidizing agent which is attached to the support substrate In this e~bodiment, filtration is-not reguired since the analyte will not carry any o~ the bound oxidizing agent with it ~
In a further embodim~nt of the invention, insoluble ~ -oxidizing agent and analyte can be mixed together in so~e container, thon mixed for an amount of tim~ sufSlcient to allow all analyte to be contact-d by the oxidizing agent The oxidizing agent c n then be removed from the analyte by filtration, decantation, aedimentation or centrifugation In each of tho embodiments mentioned above, to the ~luid ther- may be added about 0 1-2 mM of an electron transfer agent prior to contacting the insoluble oxidizing agent In embodiments involving the ~andwiching of oxidizing agent betwe-n me~brane or filter ~upports, or involving oxidizing agent attached to an insoluble substrate, the electron transrer agent may b- introduced to the Sluid dried on a upport lay r that constitutos the first layer of the sandwich, ~uch that th fluid cont~ct- this lay-r ~ust prior to contacting th oxidizing agent In such ~n embodiment, so~ of th~ el-ctron transfer ~g nt leach-s into the fluid in pa~ag~ to th oxidizing agent The embodiment involving a support layer with electron tran~Ser agent dried on, followed by a sandwlch of oxidizing agent may, in certain - configuration-, permit the ~luid to be actively driven through th~ structure holding the ~andwich layers This e~bodinent is particularly efficie~t and rapid for --pretreating the fluid -WO ~/12113 PCT/US90/01887 ~ - 31 - 2~3 ~ 1 1 9 In th- practice of thi- inv-ntion, th ~ount of insoluble oxidizing agent us-d i- set by the din n-ion- of the support sy~te~ Th- support 8yste~ i- d-sign-d in such a way to offer nough surfac- ar-~ of in-olubl- oxidizing agent so that all of the analyt- flowing through th- ~yste~ will be contacted by th oxidizing ag nt Optionally, if the surface ar-~ of th- oxidizing ag-nt is not larg , as in the case wh r- oxidizing ag nt and an~lyte ar- ~ixed tog~th r in a contain-r, th n the length of contacting tima has to be sufriciently long to allow th oxidation of all contaminating r-ducing ag nt~
In th preferr~d embodim-nt of th- invention, there ~ -are two reaction chamb rs into which th analyt- is ~ade to pa~ A chamb r in this case consi~ts of any nclo--d ar-d-signed to contain th fluid b ing an~lyzed, and can b- open at one or both nd and be of ~ny de~ir d shape and size he ~ir~t chanb r contain~ th insolubl- oxidizing agent, and is~de~1gned for th analyt- to b- introduced into one end of th ch~ber, th n pas8 through to the other end readily, wher it nt rs th ~econd ch~ber This second cha~ber contains th r dox ~na~uring sy~t n, and can include but is -not~1i ited to~on Or th~- r dox _ a~uring sy-te~s d scrib~d in~the~ ckground and Prior Art Section of this p~tent appli~c~tion~ The ~ir~t ch~nber or pretreat~ent cha~ber, cont~n- ~ ~in oluble oxidizing ag nt 80 that ~ll con ~ nat ~ ~r docing substanc-s are rapidly reduced as the fluid~pa~ tbrough ~I~ th- oxidizlng ag nt u-~d consists o~ loo~ in olubl- p~rticl~s, th r- i- provid~d ~ filt-r b tw n th two ch~b r ~f th oxldlzing ~g-nt i~ bound to ~ an~in olubl- upport, such filt-r i- unn-ce-~ry. It i9 an dv~nt~g o~thi- inv ntion that t~ analyt- doe~ not have to r~t~b ~treat-d ~-parately by on- procedur wholly apart fro~th r dox~n-a~uri~ng ~yste~, then introduc-d into th ur$ng~y-t~ p~rate ~t-p - the analyte is tre~ted th n~pa~-e- dir ctly to th~ ~ ~suring ~yste~ ~t i~ also an dN~nt g ~th~t tr-at3ent is rapid, and do-s not~interfere : ~ ~ . . ..

WO90~12113 J ~- PCT/US90/01887 ~ 32 -with obtaining accurate results from the redox mea~uring system The rilter~, f ilter matrices, and membranes which have been discus~ed in connection wlth embodiments of the invention are insoluble, hydrophilic, synthetic or natural polymers Examples o~ this invention are presented below It is not intended for these example~ to limit the invention Furthermore, it i~ not intended that this invention be limited only to the pretreatment o~ rluid that will subsequently be analyzed This invention is applicable in all cases where fluids contain redox-active components that can b- eliminated by the U8e of redox-active insolublQ
compounds. ~, The rollowing exa~ple~ illustr~te the ~ystem and method of the invention and said examplQ are orfered by way Or illu~tration and not by w~y o~ limitation ~ '- ' ,:
A golution of 100 mg/dL glucose was prepared, and dosed with 1 mM dithiothreitol (DTT) This solution was then measured with a gluco~e enzyme elQctrode An erroneous result wa~ obtained ba~d on th- known amount Or glucose put in thQ solution Two ml Or th solution was run through a bed of insoluble oxidizing agent This b d wa- created by laying down 200 mg Or cuprous sulfide (Cu2S) evenly over a 1 cm di~ot~r piec- Or rllter paper, seated in a cylinder over a rec-iving v ~--1 Th- gluco-- solutton wa- then measured ag~n u~ing the ~am enzym el-ctrode, and the re~ult was correct, bao-d on the concentratlon o~ gluco6e placed in the solution Th r-rore, DTT was acting as a contaminating reducing ag nt Examole 2 A ~olution Or sarcosine and the chromogen dimethyl thiazolyl dlphenylt trazoliu~bromid- (MTT) was mad- up When ~rco~in d~hydrogena~e is added, sarcosine is oxidized and MTT i9 reduc~d, yielding formaldehyde, glycine and colored ,-, . ....... . . . ,.. .. . .. ,, .; . .. . -...... ... ,.. ,,. . ~.,. .. ~ .- . ..

: . . . , :- ~ , ,~ ', . .-. ,. -.. :, - . ' . . ' , ' : -WO ~/12113 PCT/US~/01887 ~ - 33 ~

MTT-~ormazan Sarcosine concentration cAn b- m a-ured colorimetrically, since MTT-formazan will absorb at 580 nm A re~ction wa~ run, with conditions as follows 50 uM ~arcosine l IU/mL sarcosine dehydrogena~-- 0 5 mM MTT
20 mM potas~ium pho~phate; pH 7 5 The rQaction was run to endpoint, then absorbance W_8 read on a ~pectrophotometer at 580 nm, and tound to be The re~ction was repeatQd, but 0 5 mM ~odium ascorbate was introduced This time, color production w~s 80 intens~ that the absorbance wa~ c~f scale Ascorbate is an -interf~ring reducing subst~nce, and will readlly r~duce MTT
A l ml solution ot sarcosine containing ascorbate was placed in a sealed l 2 ml microcentrifuge tube with 50 mg of cerium oxide (CeO2), then inverted gently for 5 minutes This tre_t-d fluid was then run in the above colorimetric redox measuring system again, _nd the ab~orb_nce obtained was 0 49, very clo~e to the expected value Therefore, ascorbate was oxidized but sarcosine was not ~ .~ .
Th t-st ln example 2 was repeated, but Ceo2 was replaced wlth ~ilv r sulfid- (Ag2S) Results were similar to those obtain d in ~YamplQ 2 Before treatment with Ag2S, color productlon wa~ o~f scale due to the pr~sence o~
_~ascorbate, but a~t r tr-atment, the expected ~mount of color production occurr-d 0th r ln-oluble silver (I) co~pounds, such _8 sllver ferrocy~nide (Ag4 Fe(CN)6) ~nd silver oxide (Ag20), were al80 ~ound to perform similarly ~ .
In a r action th~t cont_in~
100 mM Tris acetate pH 9 5 0.5 ~ ', 2 mM NAD

.

Wo90~12113 _ PCT~US90/01887 ~9 2 mM ferricyanide 5 IU/mL diaphorase - ~ ;
2 IU/mL lactie aeid dehydrogenase a dark purple color is generated ir greater than l mM lactic aeid is added, but the reaetion will remain light yellow w~en les~ than l mM lactie acid is added This i~ because .` ferriCyanidQ i9 preferentially redueed by NADH before the formazan, and l mM is the thre~hold concentration This set of condition~ wa~ run with 0 2 mM uric acid Concentration~ of only 0 9 mM or greater Or lactic acid were required to get purple color Urie acid i~ss a contaminating reducing agent; in the example, it reduced ferrieyanide, thereby lowering the threshold re~ponse The urie aeid-eontaining analyte fluid was run through a filt~r apparatw that w~ overcoated with rinely ground MnO2, then run again in this eolori~etrie redox measuring system This time, the threQhold eolor response was ~ound to be at the expeeted value Therefore, urie aeid Wa8 oxidiz~ssd by the MnO2, but laetle aeld wa~ not Exa~Lg_~ -The condition~ in example 4 were repeated, but the MnO2 wa~ replaced with in~soluble per~anganate salts, sueh as b~riu~ per~anganate (BaMnO4) Before treatment with these ~;
oxidizing agents, the thre~sssshold response wa~ lowered - color produetion oeeurred with lower concentrations oS laetie acid th~n ~xp et-d ba-ed on the ferrieyanide eoneentration After tr-at~ nt, th threshold respons- wa~ baek up to th- xpeeted vol B~nO4 w~ eff-etive in oxidizing th- urie ~eld, but dld not ar~-et th laetle aeid Exam~lg_ In a p~rtleular reducing ~sugar ass~y, nonossaeeharide- are det r~s,ined by oxidation ln an al~aline ~olution by pota~iu~ f-rrieyanide, and the reduetion of f~sssrricyanido is ~ _sured by t~e deerease in absorbanee at 420 nm Thi~ i~ a redox m-~s~surement system that does not employ a prot-in eatalys~t '~ .. -.
- ,. ' ~ ~ - ' , . .

W090/12tl3 PCT/US~/01887 ~ 35 _ 20~119 When a 50 mg/dL solution of D-mannooe was spiked with uric acid, then run in the reducing sugar as~ay wlth ferricyanide, the decreas- in A420 was greater than expectQd An insoluble oxidizing filter was formed by m~king a slurry Or MnO2 and celluloso fi~ers, then drying down to form the f$1ter The mannose/uric acid solution was passed through this filt-r at neutral pH The solution was made basic by the addition Or sodium hydroxide, since manno~e is not reducing unle~s the pH i8 high, then run in the reducing sugar assay again, this time with the exp cted results Therefore, uric acid was oxidized but manno~e was not Exam~l* 7 Some oxidizing agents are soluble in aqueous solutions, but they ar~s not soluble in an organic solvent, that is, non-aqueous fluid This is true for potassiu3 dichromate (X2Cr2o7), which is ~oluble in w t-r, but not in ethanol WhQn a solution of formAldehyde in ethanol is diluted into a solution containing sodium carbonate and silver nitrate, a black color forms, indicating the presence and oxidation Or the formaldehyde The ~ame result will be ~ --obtained if mercaptoethanol is diluted into the carbonate/
silver nitrat~ solution Therofore, if formaldehyde and mercaptoethanol are ln solution together, thls redox mea~ure~ent syate~ cannot distingulsh between the two If fo~maldehyde i- th compound being detected, mercaptoethanol can b- consld red a cont~minatlng reduclng compound 1 mL solutlon of 5 mM formaldehyde was mAde in absolute thanol with 1 mM mercaptoethanol To remove the merc~pto-thanol, thls solutlon was run through a 1 cm diameter pl-c- of filter paper on top of whlch had b--n spr ad 100 ~g ~2Cr2o~, and collected lnto a t-st tube An aqueous solution contalning 0 28 M ~odtum carbonate and 12 mM
silver nitrate was added to an allquot of the flltrate, whereupon a black color formed, indlcating the presence of formaldehyde The filtrate was further analyzed by the additlon of c~rbonate-buffered Ellman's reagent Th$s would ,- , . , i ., , i,,-WO ~/121~ 9 - - 36 - PCT/US90/Ot887 cause a bright yellow color to be generated if any mercaptoethanol had passed through the dichromate rilter. No yellow color occurred, indicating that the mercaptoethanol had been oxidized by the dlchromate ilter. When potassium pormanganate replaced the dichromate, essentially the same results were obtained.
ExamDle 8 Human plasma was analyzed for its lactate concentration by incubating an aliguot of ~reshly prepared plasma in a reaction mixture containing: -3 mM NAD
8 U/ml lactate dehydrogenase 40 ~M HEPES buf~er, pH 8.5 50 % pla~ma ~-5 U/ml Clostridial diaphorase 2.00 mM potassium rerricyanide Lactate was oxidized by the lactate dehydrogenase with concomitant reduction o~ NAD to NADH. In the presence of diaphorase and ferricyanide, the NADH was reoxidized with concomitant reduction of 2 mols/mol of ferricyanide.
Ferricyanid~ reduction was directly determined by ab~orbance at 420 nm in a spectrophotometer. For a particular plasma sample, th~ apparont total lactate content was l.9 mM. When this pla-ma wa- incubated without the lactate dehydrogenase ;~ -und r th conditions above, 0.50 mM ferricyanide was reduced dir-ctly, with an additional 1.40 mM ferricyanide being reduc-d wh~n the lactate dehydrogenase was add-d. Thus, the pla-~a contaln d an inter~ering reducing activity eguivalent to O.S mM lactat- in the n~tiv plas~a.
Wh-n this pla~ma was pr-tr-ated by amendment with 1 mM 2,5-dichloro-l,4-benzoguinone and ~ddition o~ lOO mg/ml C-02, followed by filtration of th in~oluble oxidizing ag~nt, tho a-~y now show d no m a~ureable reduction of the torricy~nid- in the absence of the lactate dehydrogenase and a valu corr-sponding to 1.4 mM lactate in the plasma.

WO90/12113 PCT/US90/01~n ~ _ 37 _ 29~ 9 - -Measurement of pla~ma cholQsterol in a machine-independent, disposable devic- 50 ul Or human plasma were placed in a pla~tic tub- containing a sandwieh of filt-r papers Suitable filter paper- were prepar-d from c-llulo~e, nitroeellulose, nylon, fiberglass or polycarbonat- polymers The first filter paper contained l umol Or 2-isopropyl-5-methyl-3,6-dibromo-l,4-benzoqUinon- Ben-ath thi- paper a bed l mm thick of finely ground MnO2 wa~ contained, hQld in plac- a bed 1 ~m thick of finely ground MnO2 was contained, held in plac- by a -cond rilt-r papQr The nd Or this tube was constrict-d, m ehanically holding the s~ndwich in plac-, with an op ning going to a 20 ul capillary containing a driod film that, when rehydrated by entrance of aqueous fluid, would giv- a ~olution Or O l M potassium phosphate, p~ 7 4, 2% sodium cholate, 200 U/~l porcin- paner-atie eholest-rol esterase, 20 U/ml ehol-~t-rol oxida~e, 1 mM thiazolylblue tetrazoliu~ (MTT), 3% tran--l,2-eyelooetanediol, and 12 4 mM
potassium ferrieyanid A firmly fitting plunger is u~ed to drive the plasma through the sandwich and into the eapillary In th- eapillary, the plasma hydrates the reagents, eausing hydrolysi~ of eholesterol esters by th cholest rol est-r hydrolase, and oxidation of the eholesterol by the oxida-- In th presenee of th quinone eleetron transfer ag~nt and th exelw ion of oxygen by th walls of the eapillary, the electrons mad- available by th oxidation r due- th ferrieyanid Whon all th- ferrieyanid- i~
redue-d~ leetron- th n and only th n r~due- th tetrazolium dy giving a ~harp eolor ehange by formation of the highly eolored f omazan dy The eoneentration of eholesterol in a partieular plas~a wa- 230 mg/dl, qual to 5 94 ~M This did not exe--d th equivalent ferrieyanide eoncentration (12 4/2 - 6 20 rH) ~o thi~ plas~a eaused reduetion of ll 9 ~
ferrieyanid , leaving 0 5 mM ferrieyanide intaet and eausing no reduction of MTT and no formation of the darkly eolored dye WO ~/12113 ~~ PCT/USgO/01887 8 - ~

When the insoluble oxidizing agent was removed from the sandwich, and the test repeated in an otherwi~e identical devico, a dark color formed, incorrectly indicating that the cholesterol content excesded 240 mg/dl, the concentration at which the ferricyanide is just consumed and color changes begin. This ~alsely high reading was due to unknown reducing - -substances in the plasma that lowered the ferricyanide -concentration, allowing cholesterol oxidation to reduce the tetrazolium. In the system containing both electron transfer agent and insoluble oxidizing agent, these unknown reducing substances were advantageously removed, permitting the che~ical color switch to correctly and precisely indicate the presence o~ les8 than 240 mg/dl cholesterol. In other similarly constructod devices, the manganese dioxide was replaced with the insoluble oxidizing agents cuprous ferrocyanide, or le~d per~anganate, with e~sentially equivalent results.
Eyam~le 10 Measurement o~ hydrogen peroxide. A solution of 0.05 M Tris chloride, pH 7.7 contained 2.4 mM hydrogen peroxid~ and 0.4 mM ascorbic acid. The hydrogen peroxide content o~ th- solution was measured by dllution o~ 1 volume into 19 volu~en o~ a solution containing 20 Ujml horseradish peroxidas-, 0.1 N potassium phosphatQ pH 7.3, 0.8 mM 4-a~ino_ntipyrin _nd 20 mN 4-hydroxybenzoic acid. The solutlon wa- allowed to react for 10 min, and the absorbance w_s r ad At 500 n~ in a spectrophotometer. Fro~ a standard cur~ produced with standard solution~ o~ hydrogen peroxide, th eolor o~ th test solution indieated that the peroxide eone-ntr_tlon o~ the original solution w_s 2.0 mM, 0.4 mM
l-~s than tho true coneentration. Passage o~ the test solution over a b d of the insoluble oxidizing agent barium p r~ang_n_tQ prior to as~ay as above, resulted in a color eorresponding wlth the true coneentration. Thu , the ascorbic _cid contaminating the solution inter~ered with the peroxide-dependent oxidation of the prodye eomponents, giving a falsely low eolor intensity, and the insoluble oxldizing ' ''; .
;
~ ..

WO 90/12113 ~ ~ ~/US90/01887 r ` ~ 3 9 agent quickly and completely removed thl- interferlng reducing agent without affecting the peroxld- concentratiOn When similar measurements were carried out, but the hydrogen peroxide ~olution was cont~nated wlth cystein-, or uric acid, or ~-dopa, similar result~ w-re obtain-d, that i8, without the pretreatment the essentially true concentration of peroxide was not indicated by the color reaction, but the e~s4ntially true value was obtain-d if the fluid was pretreated with the insoluble oxidizing agent Exampl~ 11 Measurement of glucose in bodily fluids A 20-ul aliguot o~ human blood was diluted to 1 ml in a reaction mixtur- containing 15 U/ml D-glucos- oxida-e from AJpergillu~
niger and a hydrogen peroxide measuring mixture consisting of 1 mM of 0 1 M sodium phosphate, pH 7 0 20 mM 3,3~,5,5~-tetramethylbenzidine and 25 U/ml horseradish peroxidase The color at 660 nm was determined in a spectrophotometer after 10 min reaction at 37 C and th glucos~ concentration obtained with reference to a standard curve, the glucose content was found to be 4 85 ~H When the same blood sample was assayed by measurement of oxygen con~umption in an oxygen electrode, correcting for any oxygen consumQd before addition of th- enzyuo, the glucose content was found to be 5 25 mM
When this blood sample was filtered tkrough a bed of glass fibers to remov red blood cells, and then passed through a sandwich containing 5 umols 2,6-dimethyl-1,4-benzoquinon nd a 1 mu doep b~d of MnO2, the resultant pl~a m a~ur-d 5 23 mM gluco-- in the glucose oxidase, - peroxidase/benzidine color reaction describod above Thus, the interfering reducing substances present in the blood were removed from the pla3~a by the insoluble oxidizing agent and electron transf-r agent prior to analysi~, permittlng an essentially correct m~asurement of the glucose content When this analysis wa8 repeat-d, but using a sample of neonatal cerebrospinal fluid, the gluco~e content measured without pretreatment by insoluble oxidizing agent was 1 82 mM
glucose After pretreat~ent the glucose content was found to .. . - . ~ , , . . . ,, -. - ~ . ~ : - : . . . .. . .

WO90/12tl3 - - PCT/US90/01887 ~5~9 - 40 - ~ ~

bo 2 45 mM and in the oxygen electrode, the content was determined to be 2 45 mM As in well known in the art, -cerebrospinal fluid is subject to greater relatlve error in glucose determination than i9 blood due to the typically low r glucose concentrations and the typlcally higher content of reducing substances like ascorbic acid Exam~le 12 Mea~urement o~ chole-terol in plasma A sample of hu~an plasma was diluted 20-~old into a measurement reaction containing 0 1 M Tris chloride pH 7 5, 20 U/ml pancreatic cholesterol e~tera~e, 20 mM sodium cholate, 0 2% Triton X-100, 5 U/ml Nocardia cholesterol oxidaae, 0 4 mM 4-a~inoantipyrin-, 10 mM phenol and 25 U/ml hors-radish peroxida~e Thc mixture wa~ incubated ~or 20 min at 3~ C and tho color d-termined at 500 nm in thQ usw al manner By comparison to a standard curve prep red wlth authentic chol-nterol standards di~solved in a mixture o~ 5% 2-propanol and 1% Bri~ 35, the color ot the te~t ~ixture was related to cholesterol concentration The chole~terol content was found to b 4 87 mM (equivalent to 188 mg/dl) However when this pla~a wa- r aeted with cholest-rol oxida~e in an oxygen -~
eloctrod , in th pre ence Or the reagent~ speci~ied above but without th antipyrine, ph nol and p roxidaae, oxygen consumption corr ~pond d to a chol-~t-rol content o~ 5 23 mM
(202 mg/dl) lt the pla-m~ was pretreated by amendment with 0 5 r~ ot 2,S-d~ehloro-1-4-b nzoguinon and pas~ag through a b d ot fin-ly ground e-rlu- dloxld , and then a~ay d aJ b-~or- in th colorl~otrle r-aetion mixture, th m a~ur-d chol--t-rol cont-nt wa~ tound to b 5 20 mM, in exeell-nt agr~-ment with th valu d t-r ined by th l-etrod- Th w the pretreatment wlth in~olubl- oxidlzing ag nt and eleetron transfer agent r-mov~d unidentltl-d redueing ~ubstanee~ in the pla~ma that w r r--pon ibl- ~or th fals-ly low measurement ~ .:
Int rt-ring r-dueing ~ubstanee~ w r- rendered non-int rt-ring by an organic oxidizing agent attached to an :

i: . - :` : : - :- : ` .:: : . : : . . -::~ .:- . . . ::.:. : . - ~ - :.: ::. :: . .,: . - : . . , , W090~12tl3 PCT/US90/01887 ~- - 41 - 2Q ~ 1119 insolubl- m_trix The peroxyacetic acid deriv_tlv of carboxymethyl cQllulose (CMC) was prepared by incubating 100 g CMC with 100 ml 90% hydrogen peroxide for 6 h at room temperature, and the derivatized product washed xt-nsivQly wlth water to remove the hydrogen peroxide The w t ;~ peroxyacetic derivative wa~ mixed with an egual weight of cellulo~e carrier and the mixture dried to 50% relative humidity at 20 C Twenty mg Or thi- matrix w_~ plac-d in the interior of a glass tube _nd us-d for the pretreatm~ent of 100 ul Or the plasma described in example 12, by forcing the plasma through the tube with a plunger The cholest-rol cont-nt o~ th- plasm. following this treatm nt w_~ m _sured by th- e6tera~e/oxidase/peroxida~- color sy~tem deQcribed in exampl- 12 to b 5 24 mM, in excell-nt _gr--~ nt with th-corr-ct value Thu~, thls in~olubl- organic oxidizing _gent also destroyed the contamin_ting reducing substances in the pla~m_, without affecting the analyte concentration E~ ~."., Coating Glass Fiber Filter Material with Lead (IV) Dioxide A suspen~ion of 5 grams of lead (IV) dioxide in 100 ml of a 2% ~ethylcellulosQ (15 cp) aqueous solution was spread over 600 sguare centimet-r of glass fiber filter materi_l having a mean pore siz- of 3 0 microns (S&S 31 glass fiber~ available from Schleich r ~ Schuell) The coated filter was w~ h d fre- of methylcsllulo6e with water and dried at lOO C Prior to use, sm_ll circles of the material w r- punch d or cut out of this material Trat-d filter mat-rial could be mada in advance and stored for l_ter usage ... =~, Pretreatment of Plasma by $e_d (rV) Dioxide Prior to Cholesterol Measurement A small circle of gl_~s ~iber filter material pr co~ted with l-ad (IV) dioxide, ~-discus~-d abov , w_~ inserted into the bottcm o~ a 1 ml disposabl- plastic syringe A sample of pl_sma, of approximately 0 5 ml was added to the syringe, and the plasma pushed through the filter at such a rate so that at least 1 sec of ~ilter time occurred The cholesterol content of the - . ~- i, .. - .. ; - . . .. . .. . .. . ... . . . . .. .

WO90/12113 --- PcT/US9o/o1~7 ~ A~9 - 42 -treated plasma was subsequently measured in a Ciba CorningImpact 400E clinical analyzer, using the manu~acturer~s cholesterol reagènt and run accoring to the manu~acturer~s specification~.

;-.. ;

Claims (47)

IN THE CLAIMS:

1. A diagnostic instrumentation-independent kit disposable after use which is adapted to accurately determine by means of an oxidation-reduction reaction, the presence (or absence and concentration of an organic analyte in a biological fluid which contains redox-active contaminants which are likely to interfere with the analytical redox determination of the analyte, which kit comprises a fluid insoluble oxidizing compound and separate therefrom, the reactant for a redox reaction which measure the analyte in the organic fluid wherein the contaminants are devoid of adverse effect on the redox reaction, which redox reaction determines more accurately the concentration of the analyte than would be determined in the absence of the insoluble oxidizing compound.

2. The kit of claim 1 wherein the insoluble oxidizing compound is positioned in a first area and the reactants for a redox reaction are positioned in a second area.

3. The kit of claim 2 wherein the first and second areas are separated by an insoluble hydrophilic natural or synthetic polymer, which is impermeable to the insoluble oxidizing compound.

4. The kit of claim 2 wherein the contaminants in the fluid have been reacted with the insoluble oxidizing compound in the first area thereby causing the contaminants in the fluid to b oxidized and non-interfering in the redox reaction in the second area.

5. The kit of claim 3 wherein the insoluble oxidizing compound is selected from the group consisting of lead (IV) dioxide, manganese dioxide, cuprous ferrocyanide, lead permanganate, cuprous carbonate, cuprous sulfide, ceric dioxide, silver sulflde, silver ferrocyanide, silver oxide, barium permanganate, potassium dichromate, potassium permanganate or a peroxyacetic acid derivative of carboxymethyl cellulose.

6. The kit of claim 2 wherein the insoluble oxidizing compound is insoluble in the fluid which contains the analyte.

7. The kit of claim 6 wherein the insoluble oxidizing compound is water insoluble.

8. The kit of claim 6 wherein the insoluble oxidizing compound is organic solvent insoluble.

9. The kit of claim 6 wherein the insoluble oxidizing compound is an oxidizing compound which changes the contaminants to the oxidized state.

10. The kit of claim 6 wherein the insoluble oxidizing compound is supported by a porous polymeric matrix which is permeable to the fluid containing the analyte.

11. The kit of claim 2 which also contains an electron transfer agent positioned in the first area wherein the insoluble oxidizing compound is positioned, which electron transfer agent promotes the redox reaction of the insoluble oxidizing compound with the contaminants.

12. The kit of claim 2 wherein the biological fluid is selected from the group consisting of blood, plasma, serum, amniotic fluid, cerebrospinal fluid, sinovial fluid, saliva, urine, semen and tears.

13. The kit of claim 12 wherein the contaminant substance of the biological fluid is selected from the group consisting of cysteine, ascorbic acid, mercaptoethanol, uric acid, dithiothroitol, methyldopa, gentisic acid, dipyrone, anpyrone, homogentisic acid, glutathione, thiol-containing peptides and proteins, and thiols.

14. A system for purifying a biological fluid of contaminant redox-active reducing substances which are likely to adversely interfere with an analytical redox determination of an analyte in the biological fluid.
which system comprises the biological fluid containing the contaminant reducing substances and an insoluble oxidizing compound in the fluid which renders by redox reaction the contaminant substances non-interfering in the analytical redox determination of the analyte in the fluid, whereby after reaction of the insoluble oxidizing compound with the contaminant substance, an improved analytical redox determination of increased accuracy of the analyte in the biological fluid can be carried out without adverse effect due to the contaminants.

15. The system of claim 14 which includes means for removing from the fluid the insoluble oxidizing compound after the interfering contaminants have bee rendered non-interfering.

16. The system of claim 15 wherein the removing means is a means adapted to remove the insoluble oxidizing compound using the insolubility property of said insoluble oxidizing compound.

17. The system of claim 15 wherein the removing means is an insoluble support substrate.

18. A system for purifying a biological fluid of contaminant redox-active reducing substances which are likely to adversely interfere with an analytical redox determination of an analyte in the biological fluid.
which system comprises a first area which contains the biological fluid and an insoluble oxidizing compound in the fluid which is capable of rendering by redox reaction the contaminant substances non-interfering with the analytical redox determination of the analyte in the fluid, a second area which comprises the reactant for a redox-reaction of the analyte in the biological fluid to determine the presence (or absence) or concentration of the analyte in the biological fluid, and means for separating the first and second area, which means is permeable to the analyte in the fluid but not to the insoluble oxidizing compound.

19. The system of claim 18 which includes also in the first area an electron transfer agent which promotes the redox reaction of the insoluble oxidizing compound with the contaminants.

20. The system of claim 18 wherein the biological fluid in the first area also contains the contaminant reducing substances.

21. The system of claim 18 wherein the contaminants in the fluid have been reacted with the insoluble oxidizing compound in the first area thereby causing the contaminants in the fluid to be oxidized and non-interfering in the redox reaction in the second area.

22. The system of claim 18 wherein the separating means also holds the insoluble oxidizing compound.

23. The system of claim 14 wherein the insoluble oxidizing compound is selected from the group consisting of manganese dioxide, cuprous ferrocyanide, lead permanganate, potassium permanganate, cuprous sulfide, ceric dioxide, silver sulfide, silver ferrocyanido, silver oxide, barium permanganate, potassium dichromate, cuprous carbonate or a peroxyacetic acid derivative of carboxymethyl cellulose.

24. The system of claim 18 wherein the insoluble oxidizing compound is selected from the group consisting of lead (IV) dioxide, manganese dioxide, cuprous ferrocyanide, lead permanganate, potassium permanganate, cuprous sulfide, ceric dioxide, silver sulfide, silver ferrocyanide, silver oxide, barium permanganate, potassium dichromate, cuprous carbonate, or a peroxyacetic acid derivative of carboxymethyl cellulose.

25. The system of claim 14 wherein the biological fluid is selected from the group consisting of blood, plasma, serum, amniotic fluid, corebrospinal fluid, sinovial fluid, saliva, semen, urine and tears.

26. The system of claim 18 wherein the biological fluid is selected from the group consisting of blood, plasma, serum, amniotic fluid, cerebrospinal fluid, sinovial fluid, saliva, semen, urine and tears.

27. The system of claim 14 wherein the contaminant substances are selected from the group consisting of cysteine, ascorbic acid, mercaptoethanol, uric acid, dithiothraitol, methyldopa, gentisic acid, dipyrone, ampyrone, homogentisic acid, glutathione, thiol-containing peptides and proteins and thiols.

28. The system of claim 18 wherein the contaminant substances are selected from the group consisting of cysteine, ascorbic acid, mercaptoethanol, uric acid, dithiothreitol, methyldopa, gentisic acid, dipyrone, ampyrone, homogentisic acid, glutathione, thiol-containing peptides and proteins and thiols.

29. A method for the treatment of an organic fluid and for measuring with improved accuracy the presence (or absence) or concentration of an organic analyte in the organic fluid which contains redox-active contaminants which are likely to adversely interfere with an analytical redox determination of the analyte by means of an oxidation-reduction reaction which comprises:
adding to the fluid which contains said contaminants an insoluble oxidizing compound, reacting said contaminants and the insoluble oxidizing compound, thereby causing the contaminants not to adversely interfere, thereby causing the contaminants not to adversely interfere in the oxidation-reduction reaction, removing the insoluble oxidizing compound from the fluid, mixing with the fluid which is free of the insoluble oxidizing compound, reactants for an oxidation-reaction of the analyte, and measuring the presence (or absence) or concentration of the analyte in the fluid, which measurement of the analyte is more accurate because the reduced contaminants do not adversely affect said measurement.

30. The method of claim 29 wherein the fluid which contains said contaminants and the insoluble oxidizing compound are located in one area and the reactants for the oxidation-reduction reaction for determination of the analyte in the fluid are located in a second area, the first and second areas being separated by separating means permeable to the passage of the analyte in the fluid but not to the insoluble oxidizing compound.

31. The method of claim 30 which comprises passing the fluid from the first area to the second area through the separating means thereby also causing the passage of the contaminants which are now devoid of adverse effect on the redox determination into the second area.

32. The method of claim 31 wherein the fluid in the first area also includes an electron transfer agent which promotes the redox reaction of the insoluble oxidizing compound with the contaminants.

33. The method of claim 31 which comprises also removing the insoluble oxidizing compound after contacting the contaminants by means which uses the insolubility property of said compound in the fluid.

34. The method of claim 33 wherein the removing is performed by precipitation, decantation, sedimentation, filtration or centrifugation.

35. The method of claim 31 wherein the contaminants in the fluid have been reacted with the insoluble oxidizing compound in the first area thereby causing the contaminants in the fluid to be oxidized and non-interfering in the redox reaction in the second area.

36. The method of claim 30 wherein the fluid is selected from the group consisting of blood, plasma, serum, amniotic fluid, cerebrospinal fluid, sinovial fluid, saliva, urine, semen and tears.

37. The method of claim 30 wherein the insoluble oxidizing compound is selected from the group consisting of lead (IV) dioxide, manganese dioxide, cuprous ferrocyanide, lead permanganate, potassium premanganate, cuprous sulfide, ceric dioxide, silver sulfide, silver ferrocyanide, silver oxide, barium permanganate, potassium dichromate, cuprous carbonate, or a peroxacetic acid derivative of carboxymethyl cellulose.

38. The method of claim 30 wherein the contaminant substances of the biological fluid are selected from the group consisting of cysteine, ascorbic acid, mercaptoethanol, uric acid, dithiothreitol, methyldopa, gentisic acid, dipyrone, ampyrone, homogentisic acid, glutathione, thiol-containing peptides and proteins and thiols.

39. A biological fluid which contains an organic analyte the presence (or absence) of concentration of which is to be measured in an organic fluid with increased accuracy which fluid comprises reactants for a redox reaction to determine the presence (or absence) or concentration of the analyte, and redox-active interfering contaminants that have been rendered non-interfering by redox reaction with an insoluble oxidizing compound, which insoluble oxidizing compound has been removed from the fluid.

40. The biological fluid of claim 39 which is selected from the group consisting of blood, plasma, serum, amniotic fluid, cerebrospinal fluid, sinovial fluid, saliva, urine, semen and tears.

41. The biological fluid of claim 39 in which the interfering contaminant is selected from the group consisting of cysteine, ascorbic acid, mercaptoethanol, uric acid, dithiothreitol, methyldopa, gentisic acid, dipyrone, ampyrone, homogentisic acid, glutathione, thiol-containing peptides and proteins and thiols.

42. The biological fluid of claim 39 in which the insoluble oxidizing compound is selected from the group consisting of lead (IV) dioxide manganese dioxide, cuprous ferrocyanide, lead permanganate, cuprous sulfide, ceric dioxide, silver sulfide, potassium permanganate, silver ferrocyanide, lead permanganate, cuprous sulfide, ceric dioxide, silver sulfide, potassium permanganate, silver ferrocyanide, silver oxide, barium permanganate, potassium dichromate, cuprous carbonate, or a peroxyacetic acid derivative of carboxymethyl cellulose.

43. The kit of claim 1 wherein the fluid insoluble oxidizing compound is lead (IV) dioxide.

44. The system of claim 14 wherein the insoluble oxidizing compound is lead (IV) dioxide.

45. The system of claim 18 wherein the insoluble oxidizing compound is lead (IV) dioxide.

46. The method of claim 29 wherein the insoluble oxidizing compound is lead (IV) dioxide.

47. The fluid of claim 39 wherein the insoluble oxidizing compound is lead (IV) dioxide.