Note: Descriptions are shown in the official language in which they were submitted.
21071~
P~T~JS 9 2 10 23 9 3
IP~A/US 2 7 0 CT l992
-1-
i
CONTROLLE~REL~ ASE SOLID PHASE AS~AY Dl~:VICE
INC~ E~CAPSULAT13D R~AGl3NT~ D~I~I~G
C~UICAL SUBSTANCl3S
BACKGROUND OF INVENTION
1. Field of Invention. This invention relates to a solid phase
method, and deviee for praeticing this method for the detection of
endogenous and esogenous ehemical substanees in body fluida
2. Baekeround of Invention. A wide variety of analytical
10 methods are known for the detection of ehemical substances (both
endogenous and e~cogenous) in body fluids. Among the endogenous
substances for which rapid and sensitive assays are frequently requ~red
are sugar in urine, serum cholesterob and liver enzyme in blood. The
detection of e~ogenous ehemieal substanees in body fluids is often
15 necessary as well. Among sueh e~cogenous substanees frequently
a~sayed are eentral nervous system stimulants (ç~, cocaine and
amphetamines), depressanb (~L. barbiturates), psyehopharmacological
drug3 (ç~, cannabis) and nareotics (~, opiate alkaloids). Many of the
known analytieal methods are complicated, costly and require a trained
20 analyst or clinician. Gas-liquid-chromatography (GLC) coupled with
flame ionization or thermionic detection ~ystems is perhaps the most
widely used instrumentation method for drug abuse testing. The
sensitivity and selectivity of GLC make it the standard to which new
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methods of analysis or screening are frequently compared. Thin layer
chromatography (TLC) is also often applied to drug abuse testing.
Separation of constituents of body fluids on a thin layer of silica gel or
aluminum oxide using an optimized solvent mixture carrier can be
achieved with the aid of a suitable sample preparation procedur~. The
separated constituents can be visualized with an appropriate staining
reagent and quantified by reI1eclance densitometry. Immunologicai
assays such as radioimmunoassay (RIA~ and enzyme multiplied
immunoassay technique (EMIT) have also been used for drug Pbuse
testing. These latter techniques combine the use OI an~ibodies -.ha~ are
highly specific to individual drugs with either radiometric or
spectrophotometric measurements for quantitation. Gas
chro;natography coupled with mass spectrometry (GC/MS) is perhaps
the most sophisticated analytical technique available at this time for
drug abuse testing, but, is also the most costly and complex of the
testing methods mentioned thus far. GCtMS, however, offers nearly
complete characterization of the constituents in body fluids. Drug
identification and detection is based not only on the chromatographic
retention indices of constituent drugs, but also on their unique mass
fragmentation pattern.
During the past forty years, the use of chemical test paper and
solid solution chemistry has been widely accepted by medical
practioners and patients in areas other than drug-testing, for example
in the detection of glucose and ketones in urine and blood. The known ~ .
25 paper tests, however, although quantitative and cost effective, do not
lend themselves readily to testing substances of abuse because of their
lack of specificity. Tests of this nature (~, color reactions) depend on
the participation of analyte functional groups in the chemical reaction.
Hence, the detection of common, and, in many cases, ubiquitous,
functional groups provides information only about a characteristic
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region of the ana]yte with consequent loss of specificity. In a clinical
setting, the apolic2tion of such paper tests to drug-testing would lead to
a high frequency of false positives due to the similarities in chemical
functional groups of the analyte of interest with those of constituents
5 that are endogenous to biological function or drug therapy.
Sul~stanc~ abuse has reached epidemic proportions, affecting all
leveis of socie~-. The most commonly abused substances today are CNS
stimulan,s W~, cocaine and amphetamines), depressants (~,
barbiturates), psychooharmacological drugs (~, cannabis) and
iO narco~ics ~, opia~ alkaloids).
Some abused drugs are metabolized extensively by the liver. The
metabolis;n of cocaine proceeds by hydrolysis to yield benzoylecgonine,
the principal metabolite appearing in the urine. Though only between
1 to 12% of the total ingested dose is excreted in the first 24 hours as
15 unchanged drug, the amount of unchanged cocaine appearing in the
urine is dependent on urinary pH. The excretion of amphetamines iæ
also influenced by urinary pH. Between 30 and 40% of amphetamine
itself is e2~creted unchanged in the urine within 48 hours. After
ingestion of large doses, however, unchanged amphetamine may be
20 detected in urine for up to seven days. Metabolites of amphetamine,
including p-hydroxyamphetamine and conjugated benzylmethylketone,
also appear in urine in relatively small amounts.
Of the opiate alkaloids, morphine is the most widely used
narcotic analgesic. Only a minor fraction (~, 7%) of the original
25 morphine dose is excreted in the urine as unchanged drug.
Appro~imately 45% i8 excreted as morphine-3-glucuronide.
Many of the endogenous and exogenous substances discussed
herein give distinct colors when brought into contact with appropriate
chemical reagents. Upon the application of a test substance to a filter
30 paper impregnated with reagent solution, colored reaction products
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become fixed on the surface of the paper and produce distinct colored
rings. In this way, the products of chemical reactions may b~ separated
from the resction sphere and fixed in the surface of the test paper. The
simplicity of such tests allows for a fast and effective identi~1cation
procedure for drugs of abuse.
Generally, such colorimetric techniques are referred to as solid
solution chemistry and dif~er considerabiy from conventional solulion
colorimetric methods which use dilute solutions of color-producing
reactants. Interaction of the reactant with a functional group of the
drug or metabolite in solution yields a color .eaction identifiable by
visual observation or spectroscopic absorbance. If, however, a molecule :
of drug or metabolite interacts with a reservoir or solid matrix of
color-producing reactant at infinite concentration, that drug or
metabolite may exhibit very different properties with regard to
reactivity and specificity as compared to those obtained using
conventional dilute solution chemistry. For example, Friedenberg et al.
(Clin. ~ox., 18(~), 619-633 (1981)) has observed that the detection
sensitivity for alpha-amino acid residues of amino moieties with ~ -
ninhydrin (1,3,5-triketohydrindene, a common diagnostic reagent) using
solution chemistry techniques is typically on the order of one part per
thousand, whereas solid solution chemistry will yield color reactions in
the sensitivity range of one part per million to one part per ten million.
Since sensitivity is essential in a practical drug screening device,
solid solution chemical reactions (j~, reactions of drugs or their
metabolites with highly reactive color-forming species at infmite ::
concentration) have tremendous potential value for drug testing.
However, reproducibility and sensitivity in many currently used solid ~ .
phase assays are often hampered by the instability of the reagent . . .
employed, as well as by the inability to carry out sequential reactions
necessary to conduct many of such assays.
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SUMMARY OF INVENTION
This in~ention provides a cost-effective, yet sensitive solid state
method and screening device used in this method, to detect both
endogenous and exogenous chemical su~stances in body fluids. More
6 particularly, this invention provides solid phase assays for the detection
of endogenous qnd exogenous chemical substances in body fluids, and
means for conducting such assays, in which at least one reagent is
present in Lle ,oiid phase in encapsulated form. Encapsulation of one
or more rea~ents provides greater stability for reagents which tend to
lû decompc. ~ eby providing a longer shelf-life for the assay means
when in the form of a kit. The encapsulation of certain reagents also
permits delayed release of a reagent or reagents into the reaction
sphere, thus permitting sequential or timed release of one or more
reagents required in one or several assay steps.
The assay means of this invention can comprise a solid phase
substrate, such as a chemical test paper, strip or spot, a glass or plastic
tube, stick or bead, or the like, to which has been adhered at least one
encapsulated target chemical immobilizing reagent, or a potentiating
reagent or a visibly detectable marker. Such assay means can be used
to assay chemical substances, including most commonly abused
stimulants and narcotics in body fluids, such as urine, saliva, serum,
perspiration and the like. The solid phase test system of the present
invention is easily utilized and does not require sophisticated laboratory
equipment or techniques.
In one embodiment, the present invention comprises a
drug-screening test paper for the detection of abused drugs, such as, but
not limited to, amphetamines, aporphines, catecholamines and various
morphine alkaloids (~, morphine and codeine) in urine and other
body fluids. In a preferred embodiment, the device of the present
30 invention employs a metal complex in conjunction with controlled,
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delayed release of a potentiating agent to produce a colorimetric
reaction with drug-positive urine. Hyperflow kinetic encapsulation of
one or more of the reagents adhered to a device prepared in accordance
with this invention provides a delayed release mechanism for
5 introducing any of the encapsulated reagents, such as a potentiating
agent, at the appropriate time during the colorimetric chromatographic
reaction.
This inven"ion may be utilized to monitor any chemical
substance in body fluids or in any other aqueous ~luid. Endogenous
substances such 2S hormones, lipoprotein-associated choles"erols (~,
HDL- and LD~cholesterol), or sugars may be monitored utilizing the
concept of the present invention. In addition to detection of abused
substances in body fluids, the present invention may also be utilized to
monitor levels of prescribed or administered drugs.
15 DETAILED DESCRIPTION OF INVENTION
The present invention provides solid phase assays, and means for
conducting such assays, in which at least one reagent is present in
encapsulated form. The encapsulation of one or more reagents provides
greater stability of reagents which tend to decompose, thereby,
20 providing a longer shelf-life for the assay means when in the form of a
kit. The encapsulation of certain reagents also provides a means to
delay the release of certain reagents, allowing a sequential addition of
the encapsulated reagent into the reaction sphere. The assay means
comprises a solid phase substrate, such as a chemical test paper, strip
25 or spot, a glass or plastic tube, stick or bead, or the like and one or
more reagents selected from the group consisting of a target chemical
immobilizing reagent, a potentiating reagent and a detectable marker,
at least one of these reagents being encapsulated in a water swellable,
pH- or heat-dependent or maleable polymer. The detectable marker is,
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preferably, visually detectable. However, markers may also be selected
from the ~ou? ~hich are detectable by other means such as iluorescent
markers, radioactive markers, chemiluminescent markers when the
appropriate 2pparatus for detecting such markers is available to the
6 assay user.
One Ot skill in the art can select the type of encapsulating
matcrial (e lcapsri!ating r~2tL~) 3.CCO~ ding . o the iype of release
mechanls~n ~esired tO reiease the encapsulated substance or reactant.
Encapsulated materials can be released by mechanical rupture, thermal
release er -~rmPatier ChoicD of a som.bination of encapsulating
materials can provide ~ven further control oî the release of the
encapsulated materials. The encapsulating material can be selected, for
instance, from a wide variety of natural and synthetic polymers, as well
as natural gums, waxes and resins. Aqueous materials may be
encapsulated in a variety of materials. For instance, in one
embodiment, aqueous solutions may be encapsulated in a wax or
polyvinyl alcohol shell and released by mechanical rupture. In another
embodiment, chemical reactants may be encapsulated in a water
swellable polymer such as gelatin or polyacrylate. Fatty acids may also
be utiLized as an encapsulating material. These useful encapsulating
materials are pH dependent and will release the encapsulated contents
by dissolution at pH below 7Ø Encapsulating materials such as saran
are also useful when an application of heat is appropriate for the
release of the encapsulated reactant. Other encapsulating materials
u8eful in the present invention are known to those of skill in the art
and their choice and use will be apparent to one of skill in the art.
- The device of this invention comprises, in its broadest aspect, (1)
a known solid substrate, (2) at least one known "reporter substance
forming' reagent (~, one that interacts with an analyte to give a
colored substance or some other indication of the analyte's presence)
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for detecting the presence of an analyte. The reporter reagent is
adhered to the solid substrate and is often one of the encapsulated
reagents. In all cases, encapsulation protects the reagent(s) from
atmospheric attac~, i.e., provides storage stability. Therefore, it is
evident that the assay device of this invention may be applied to the
assay of any chemical wherein the stability of the reaction reagents will
be increa, ~' by er.capsulâ'ion.
T". solid ~hase assay of this invention also provides
encapsulation of reagents which, although stable to atmospheric
condilionc~ a. q 'o be rel~ased in a delayed manner into the reaction
sphere. ln one aspect, this invention contemplates the use of
microcapsules of different capsule thickness. Thus, by varying the
composition and/or the thickness of the encapsulating material, one can : -
control the time of release of the encapsulated material into the
1~ reaction sphere. For instance, if microcapsules of the same reagent are
made using the sa~ne capsule-forming substance (polymer) but having
different dissolution rates, for instance, by varying the polymer . . .
thickness, a single reagent can be released in stages over time ("timed
release").
If microcapsules of more than one reagent are made using the
same capsule-forming substance for each, but a different wall thickness
for each, or using different capsule-forming substances, having different
dissolution rates, for each, then several reagents can be released
sequentially. Hence, one reagent can, for e~ample, react first with one
analyte and a second reagent can then react with another analyte, or
one reagent can react first with an analyte and a second reagent can
then react with the reaction product of the first reagent and the
analyte.
Microcapsules can be made utilizing a variety of encapsulating
materials. Microcapsules may also contain more than one encapsulated
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2 ~ JS ~ 2 / 0 23 9 3
IPEAIUS 2 7 0 CT 1992
reagent within the ~ame microcapsule.
The enc~p~ulatin~ materials may be used singly or in
combination. The choice of encapsulating material(~) will be dependent
upon a number of ~actors.
In one aspect, the present invention comprises:
A 30!id su~strata, a binding ~u~starc~ to bind the target
substance to "na assay theat~r anci a report~r substance for detecting
andlor indicatin~ tha p;~senc~ OI said ~araet substance. In one
embodiment. the assay of the present invention may also comprise an
agent or aO-n~3 .ur potantia.ing o~ enllancillG ~he indicator or reporter
substanc~ signal. In a preferred embodiment, tne ~ignal is a visual
signal and the signal is a color produced or generated by the
substance(s) resulting from the reagent's reaction with the analyte.
Depending on the choice of reagents, one or more of the reagents are
encapsulated. In one embodiment, the reporter substance is not
encapsulated and the potentiating substance is encapsulated 80 that the
reporter substance reacts with the analyte and the potentiating
substance is then released to potentiate the indicia obtained from the
reagent-analyte reaction.
In yet another embodiment both the reporting ~ub~tance and the
potentiating substance may be adhered to the substrate.
The foregoing aspects may be more fully understood by reference
to the e~amples wbich follow. Thus, in E~ample 1, the substrate is
filter paper, the reporter molecule is e~emplified by potassium
2~ ferricyanide and the potentiating agent is encapsulated ninhydrin, withthe ninhydrin being later released to potentiate the color of the
hexacyanoferrate-drug comple~ formed by the earlier potassium
ferricyanate-drug (analyte) reaction.
E~ample 2 illustrates the use of two encapsulated potentiating
agents, ninhydrin and trichloroacetic acid. Thus, in E~ample 2, the
- SUBSrlTUrE SHEET
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substrate is filter paper, the reporter agent is potassium ferricyanide
and the potentiatin~ agent is the primary- (trichloroacetic acid) and
seconda~ (ninhydTin) encapsulated potentiating agents, whereby the
primary agent is re!eased at a greater rate than the secondary reagent
to allow for denaturation of endogenous proteins followed by color
potentiation of ne~ac Janoferrate-drug complex.
In ~A -mple '', al ~LISA assay~ .he ~ub;"iate is the filter paper,
the reporter--.o~^-cu~ ~ an enzyme sub3tr2te, and the potentiating
agent is t'ne antigen-enzyme hapten.
13 A. ~oiid ~ubs~r2tes
Because interferences from drugs and metabolites of similar `
chemical structure or endogenous material present in a biological test
material (~, urine) can hinder the sensitivity and specificity of the
test, it is sometimes necessary that the interfering substances be
16 separated from the reaction theater prior to reaction of the analyte of
interest. The target chemical may interact with or be bound to a
binding reagent on the solid substrate and then react with analyte
detecting or visualizing reagent. In the alternative, the target chemical
may be separated from interfering body fluid constituents or
metabolites by a chromatographic or other separation step(s) on the
solid substrate prior to reaction with the detection reagent or prior to
contact with the solid substrate. For e~ample, color reactions with
chromatographic separations on an inert absorbent substrate capitalize
on the interaction of color-producing reactant with drug or metabolite,
but with the additional migration or segregation of drugs and
metabolites within the contacted area or areas of test substance on the
chemical test paper. In the presence of a highly potent color-producing
reactant (~g, ninhydrin) or other indicator reactant, interaction of
drug or metabolite with reactant caused identifiable indicia of the
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WO 92/17769 PCr/U592/02393
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reaction product such as colored rings or bands to be formed as the
molecules, me$abolites and endo~enous rnaterial migrate at different
rates away from the application site OI the test urine. Thus, this
approach allo~Ned one to differenti2te the drug or metabolite derivative
of interest from similarly structured derivatives or endogenous material
derivatives. Among the solid substretes suitable for use in the device of
the present invent.on are cel1ulo~c, nr;c.n, and propyleIle.
B. Tar~et Chemicais.
A1II1OS~ 2n~ Ild'`ge-'C'~ S cr _i~gc.i~ u;, chcmic.-l ~ubs~ance may be
detected in body i1uids by tne method of the present invention. For
instance, it is often necessary to monitor the levels of endogenous
chemicals such as microbacteria, estrogen and androgen in body fluids.
Most often the steroid hormones (estrogens and androgens), such as
estradiol and testosterone, are monitored by radioimmunoassay (RIA).
However, these methods often require prior extraction from the
biological matrix and the shelf life of the available kits is limited by the
radiochemical half-life of the iodinated ('25I) antigen.
The levels of exogenous chemicals such as antibiotics,
chemotherapeutic agents and the like must often be monitored.
Administration of the levels of many such drugs is often based on an
average half-life rather than the known excretion or metabolism of the
drugs. Utilization of the present invention allows an efficient, stable
and cost effective method to monitor the levels of therapeutic drugs.
It is also desirable to monitor for the presence of exogenous
substances such as central nervous system stimulants (~, cocaine and
amphetamines), depressants (~, barbiturates), psychopharmacological
drugs (~, cannabis) and narcotics (~, opiate alkaloids) in body
fluids.
Thus, any target chemical substance in any fluid medium may be
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assayed utilizing the assay of the present invention. While especially
useful for the d~tection of chemicals in body fluids, the assay of the
present invention may be utilized to detect chemical substances in any
fluid medium. The assa~ of the present invention may be utilized to
detect environmental pollutants, pesticides and the like.
C . . l ~ ~ 2 ~ ~ n t
~ .vic~ ^f i. icating r~agent will be dependent on the type of
chemicai subs~ance to be targeted. In the preferred embodiment, a
color inai r~ oa30nt is ..ti! ~d. ~ n~ color indicating reag2nts are
;O organic coior inaica~ing reagents, the specificity of which depends on
the participation and availability of a specific functional group in the
chemical reaction. Detection of the group gives information only about
the presence of that specific chemical functionality, but not its origin.
Thus, the detected functional group may originate from the compound
of interest, i_, an abused drug or metabolite thereof, or from an
interfering compound containing the same functional group. Thus,
there is a loss of reliability as to the actual reactant detected, with
consequent loss of sensitivity to the amounts of reactant actually . ...
present. In one aspect, the present invention provides a solution to this
problem by incorporating color indicating metallic complexes whose
specificity for the drug or metabolite of interest relies on the
participation of more than one functional group, their relative
orientation in space, and the size, nature and oxidation state of the
central metal atom. In many cases, the metallic complex can chelate to
more than one molecule of the drug or metabolite of interest, thereby
enhancing the intensity of the colored products f~xed in the surface of
the paper. Metal complexes, for instance, K3[Fe(CN)~,], K3[Co(CN)~],
Na2[Fe(CN)sNO], Na[Fe(EDTA)] and Na2[Cu(EDTA)], can be . ~.
potentiated by agents such as ninhydrin, 5-sulfosalicylic acid, 2, 4- .
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Dinitrophenylhydrazine and phthaiic dicarboxaldehyde, respectively.
Other analvte indicators useful in the present invention include~
but are not limited to, colbalt(II) thiocyanate, Dragendorfi's reagent
(OBiNO3, KI and acetic acid), lead acetate, mercuric chloride,
5 carbazone, and iodoplatinate.
D. Potemiatin Agent
In addition to increasing sensitivi~J Dy solid soiution chemistTy,
the present invention provides the use of a potentiating agent. The
addition of trace quan.ities vf a pv"en~ia~ g a;,-enL, sub~e~tl nt to "he
10 indicating reaction, greatly enhances the de~ection or visualization of
reaction product such as colored rings or bands on the substrata.
However, the provision of the potentiating agent at the proper stage of
the reaction provides reliability, specificity and sensitivity. Unless
introduced in sequence, the use of the potentiating agent is nonselective
15 since it can react with all reactive components, including those
unrelated to t~e drug or metabolite of interest.
E. Controlled Release i~Iechanism
According to the present invention, microencapsulation of the
potentiating agent or other indicator or color-producing reactants
20 provides their sequential introduction into the reaction theater. For
instance, the nascent drug-positive urine spot is propagated by the
absorbing medium and visualized (detected) via sensitized release of a
potentiating reagent or color-producing reactant at the appropriate time
during the course of the colorimetric reaction. The delayed release
25 mechanism is produced by the microencapsulation of the potentiating
agent or color-producing reactant in a water swellable, pH-dependent or
malleable polymer matrix. The microencapsulated formulation is
adhered onto the surface of the solid phase support or chemical test
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paper.
The controlled release mechanism provided by encapsulation of
some or all of the reactants and incorporation in chemical test papers
provides the sensitiv~ ~nd reliable de~ce and method for detecting
chemicals, metabolites or substances such as abused drugs and/or their
metabolites in boày -1uids. The present invention may also be utilized
for nume.ous o~'ne~ âpplicâl,iûns i~ di~ostic ana medical screening
procedurDs. r~he p 2ge~t i~ Yen~ion may also be utilized to provide
chemicai test papers impregnated with encapsulated reagents for
selective re~ eSs~cif^ immlu-v-l~âgen~s and eIlzymes, both of
which are time-dependent reactions involving more than one reagent.
Equally important, microencapsulation is useful in preserving the
potency of the agent(s), thereby prolonging the shelf-life of the
diagnostic or medical test kit or chemical test paper.
Preferably, the potentiating agent or color-producing reactant iæ .
encYpsulated in polymer microcapsules of approsimately 100
micrometer in diameter. Encapsulation procedures useful in practicing
the present invention are known to those skilled in the art of hyperflow
kinetic encapsulation.
- Briefly, in the manufacture of one embodiment of small capsule
spheres (microspheres) useful in practicing the present invention, a
slurry or emulsion contair~ing the potentiating agent or color-producing
reactant and a suitable polymer are pumped to the center of a rotating
disk. An even distribution of the feed material, along with control of
the rotational velocity of the disk, provides control over the size
distribution of the microspheres. Optimum dynamical conditions allow -~;
disengagement of individual microspheres at the periphery of the disk.
The disk is positioned at a distance above the collection surface to allow
sufficient time for the microspheres to d~r, cool and solidify before
impact.
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Wo92/17769 2 ~ ~7:~ 3 Pcr/US92/02393
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However, other encapsulating procedures known to those in the
art may be used to provide the microspheres for the controlled,
sustained or sequential release mechanism of reactant or reactants
essential to the colorimetric reaction. For instance, the encapsulating
procedure taught and disclosed in U.S. Patent No. 3,389,194
(Somerville), incorporated herein by reference, may be used to
encapsulate reactants used in the present invention. This method
` produces filler material (reactant) that is contained within a seamless
film material (3hell). A centrifugal extrusion de~ice '.3 used to
manufacturè the microcapsules. The device consisls of an
encapsulation head with two or more nozzles and concentric feed tubes ;
which enter the head through a seal arrangement. The device is
attached to a rotating shaft such that the direction of rotation is ~
around its vertical alcis. Shell and fill materials are pumped separateb ~ ~ -
15 through a feed tube into the head and to the nozzles which consist of
concentric orifices. As the head rotates, shell material flows through
the outer orifice of the nozz!e and fill material flows through the inner
orifice of the nozzle, thereby creating a rod of filler material
surrounded by a sheath of shell material. This e~truded rod of material
eventually breaks into individual capsules~which are collected by
appropriate means.
Many different types of polymers may be used in preparing the
microencapsulated reactants. Microcapsules or microspheres of varying
dia~neter or capsule-wall thickness (j~, microcapsule) may be prepared
using polymers such as, but not limited to, cellulose acetate trimellitate
(CAT), cellulose acetate phthalate (CAP), polyac~ylate-polymethacrylate
copolymers (Eudragit RL~100 and RS-100 and others) and
polyisobutylene. ln the preferred microencapsulation methods of the
present invention, the mechanical parameters useful in varying the
manufacture of microcapsules or microspheres, and which such
r t I . ' . ,' . ' . ` ' ' j~ .' '.,, ` . . ~ ' . " ', . ' ', ; " . , ' ' ", ., , ' '' ' : . ' ' ' .' '
2 / 0 23 9 3
lPEA/US 2 7 0 CT 1992 _,
parameters consequently vary the release characteristics of the
encapsulated reactant, are the feed rate~ of the fill and matri~
materials, the rotational speed of the disc or extrusion head, and the
diameter of the disc or e~trusion orifices.
The release rate of reagent through the polymer wall (~,
microcapsule) or matrix (~, microsphere) is controlled by Yarying the
initial ratio of reagent to wall or mat;L materi~. A d~cr~asa i~ "hia :
ratio causes a decrease in the release rate of tha r2agant. (~3anita, ~t
al., J. Microencapsulation, 2(3), 207-222 (1986)). HoweYer, the
diffusion rate of the reagent is dependent upon it,3 sclubilitJ 2I.~d
molecular size. Therefore, two different reagenta micrcencap~ulated oy
the same method and having approximately the same reagent to wall or
matris ratio may have considerably different release rates if the
solubility or molecular size of either reagent is also different.
Microcap~ules or microsphere~ may be adhered to any solid
substrate by air drying, as described in the preferred form of the
present invention, or by the use of polymer binters such as cold-water
soluble polyvinylalcohob (PVA), water soluble cellulose derivatives (~,
methylcellulose) and starch derivatives.
In one embodiment, the present invention describes a method
ant device for detecting the presence of morphine alkaloids, aporphine,
catechol, amphetamine or metabolites thereof in urine or other body
fluid~. A solid substrate, such as, for in~tance, filter paper was treated
with a pH-adjusted solution of a transition metal comples such as, for
2~S i~tance, potassium ferricyanide. After the impregnated papers have
dried, a microencapsulated potentiating agent, such as, for instance,
ninhydrin, was coated on the test paper. To.assay for the presence of
one of the target drug~ of abuse, a drop (approximately 100 uL) of
urine or another appropriate body fluid (~, saliva) was applied to the
paper strips. However, timed-dipping may also be used. Drug or
SU8STITUTE SHER
.. - ... , , . ., . ,, , . . . i -' -
, .. , , .. ... - . . , - .. . . . . .. ... . . , .. .. .; , ..
; . .. i ,-. . . ,..... ., -, . . , .. . ,. , , " . .- . , .,~ . .
. . ' ~ . ' ; . , ; . . i .
- t~ 9 2; 0 23 9 3
IPEA/US 2 7 Q CT 1992
-17-
metabolite constituents resembling morphine alkaloids, aporphine,
- catechol or amphetamine species migrated at different rates away from
the application site of the absorbent substrate and immediately
interacted with he~scyanoferrate ion to yield highly specific
5 he~acyanoferrate-drug complexes. These complexes become
immobilized within the fibers of the absorbent paper substrate. The
controlled, delayed release of potentiating agent was acti~atsd whan the
polymer matriY of each individual microsphere comes in contact ~nth
the specimen. The polymers used for microencapsulation are capable of
10 swelling and releasing active ingredients by diffusion. Thu~, the release
of the potentiating agent from the microcapsule is delayed for the
period of time needed for the swelling of the microcapsule shell and the
diffusion of agent through the shell. This delay period provides that
sufficient time is allowet for migration of the trug, comples formation
15 of he~w~anoferrate~drug, ant immobilization of specific
he~a~anoferrate-drug comple~es (i,~L, he~a~ranoferrate-alkaloid,
-aporphine, -amphetamine, and -catechol compleses). After a period of
time the color reaction of immobilized drug compleses became
intensified by the induction of potentiating agent to give a blue color
20 for drug positive specimen and no color change for drug free urine
specimen~.
The potentiating agent may be any agent that: 1) removes from
the reaction theater any con~tituent endogenous to the sample
~pecimen that my interfere with the esact determination of the target
26 analyte; 2) manife~S~, through the action of chemical derivatization,
complesation or enzymatic catalysis, the presence of a target analyte
that is immobilized or fL~ed on a chemical substrate; or 3) intensifies a
colorimetric reaction that has occurred on a chemical substrate.
Preferably, the potentiating agent is selected from the group consisting
30 of ninhydrin, trichloroacetic acid, dansyl chloride, fluorescamine,
. ,:
SUBSTITUTE SHER
wo 92/17769 Pcr/usg2/02?"~
'"'
- 2107 l ~3
-18-
--
2,4-dinitrophenylhydrazine, phthalic dicarboxaldehyde, and enzyme -(~, alkaline phosphatase) ;
The following examples are offered by way of illustration and are
not intended to limit the invention in any mann~r. In all e~amples, all
percentages are by weight if for solids and by volume if for liquids, and
all temperatures are in degrees Celsius unless otheF~se n~t~id.
~:~MPL~ 1
Chemical test papers were prepared by saLur~ g o ~ ~ô ~m
strips of WhatmanTM No. 3 filter paper with a 6% (w/v) solution of
potassium ferricyanide (K3[Fe(CO6]) p.~par~id i;l û.1 /I ~r~c3~)hât~
buffer, pH 9.3. After allowing the test papers to air dry,
microencapsulated potentiating agent, ninhydrin, was dispersed in a
carrier and spray-coated onto the strips. Microspheres were applied to
filter paper by di~persing them in a volatile carrier such as hesane in
'~ 15 appro~imately 1% (w/v) concentration and immediately spray coating
the misture using a spray gun. Microspheres were kept dispersed
during the coating process by constant agitation of the mixture in the
- solvent reservoir. - -
J Because the microsphere matrix is slightly soluble in hexane (~,the di~solution time is 6 hours) the outer surface of the matrix ~oftens
and subsequently increases adhesion of the microspheres to the paper.
Leakage of the encapsulated material was prevented by spray coating
this misture in a period of time that was less than the dissolution time
of the matris and by volatilizing the carrier solvent immediately after
26 adhesion.
Substantial quantities of the potentiating agent, ninhydrin, were
prepared in the form of microspheres (20 - 100 micrometers in
diameter) using the spinning disk apparatus previously described for
the preferred embodiment. Approximately 30 grams of milled
ninhydrin v~as suspended in a slurry consisting of 10% (w/~)
L
~ ' ' '
`:' ' ' . '
wo 92/17769 ~ 3 Dcr/US92/02393
-19-
methacrylate copolymer Eudragit RL-100 in chloroform. The slurry
was fed to the center of a disk rotating at 4300 rpm. The resulting
microspheres of ninhydrin were collected on paper and subsequently
spray coated onto the pre-saturated paper strips.
Urine specimens were applied by means of a small bore pipette
to previously treated test papers by permitting 100 microliters of the
specimen ..o spread from the center point on the paper in a manner
that is analogous to the capillary action of developing paper
chromatograms. Once the urine specimen was applied to the paper
~0 strips, drl~g or metabolite constituents resembling morphine alkaloids,
aporphine, catechol or amphetamine species migrated at different rates
away from the application site of the absorbent substrate and
immediately interacted with hexacyanoferrate ion to yield highly
specific hexacyanoferrate-drug comple~es which became immobilized
15 within the fibers of the absorbent paper substrate. Coincidentally with
the migration of sample specimen, the controlled, delayed release of
potentiating agent was activated when the polymer matrix of each
individual microsphere came in contact with the specimen. The
polymers used are capable of swelling and releasing active ingredients
20 by diffusion. This allowed for induction of the potentiating agent after
- immobilizing specific hexacyanoferrate-drug complexes (j~,
hexacyanoferrate-alkaloid, -aporphine, -amphetamine, and-catechol
complexes). After a period of time the color reaction of immobilized
drug complexes became intensified by the induction of potentiating
25 agent to give a blue color for drug positive specimen and no color
change for drug-free urine specimens.
Qualitative comparison study of positive donor urine samples
tested by EMIT and GC/MS versus a novel test paper prepared in
accordance with this invention is shown on Table 1.
: :
wo 92/~7769 ; Pcr/~'s92/02393
21Q711.i~
-20-
Table 1.
TABLE 1. COMPARATIVE QUANTITATIVE TESTING OF
DONOR URINE SAMPLES
POSITIVE DONOR URINE
TESTED BY EMIT AND TEST PAPE~
GCtMS DRUG IDENTIFICATION
AMPHETAMINE -:
2 "
3 "
" :
8 ~ .
9 ~ .
11
12
13 PENTOBARBITAL
14 "
BUTALBITAL
16 '~ -
17
18 '~
19 "
PENTOBARBITAL
21 "
22 . '~
23 ~
24 "
MORPHINE
26 "
27 "
28 "
29 DIHYDROCODEINE
~ -
31 ~ :
32 "
33 MYDROCODONE
34 " ~.
~ :
36
. . ~ ..... - :, i,~"- .,, . ,. ~", ,"; . ~.".,,: ~, .: ." ; ,,,
WO 92/17769 PCI'/US92/02393
2 10 ~
-21-
a) SAMPLE 1 THRU 12 TESTED POSITIVE FOR
AMPHETAMINES IN RANGES OF 0.6 to 6.0
MICROGRAMS/ML OF URINE.
b) SAMPLES 13 THRU 24 TESTED POSITIVE FOR
BARBITUDES IN RANGES OF 1.9 to 19.0 MICROGRAMS/ML.
c) SAl~PLES 25 THRU 36 TESTED POSITIVE FOR OPLATES IN
RANGES OF 0.9 to 8.2 MICROGRAMS/ML.
This study and subsequent specificity studies indicate a correlation of
90 perc~nt ~ositi~e results when compared to the GC/MS method. The
10 other ten percent were positives; however, the specific chemical
compound was not comparative. This reaction could have been caused
by multiple metabolites in the sample or other drugs taken by the
donor which have similar but unknown side chains.
The results of a study using 31 spiked samples of drug free urine
15 reagent in pre-test paper prepared in accordance with this invention at
three different levels of concentration i8 shovrn on Table 2.
,
Table 2.
FIGURE 2 RESPONSE TO TEST PAPERS TO DRUG-SPIKED
URINE (USING TWO DIFFERENT MICROENCAPSULATED
20 POTENTIATING AGENTS)
.
RESPONSE
10 ug/mL 5 ug/mL 1 ug/mL
Sub~tance (-) (+) (-) (+) (-) (+)
Acetaminophen a x x x
b x x x
Amobarbital à x x x
b x x x
Apomolphine a x x x
b x x x
.
~ ~ '
wo 92/17769 Pcr/us92tO2~t
2 ~ ~ 7 ~ 3 -22- ~
Table 2 (con't)
RESPONSE
10 uglmL 5 ug/mL 1 ug/mL
Substance (-) (+) (-) (+) (-) (+) ;
Atropine a x x x
b x x x
Barbital a x x x
b x x x
Benzoyl ecgonine a x x x
b x x x
Cocaine a x x x
b x x x
Codeine a x x x
b x x x
15Dextromethorphan a x x x
b x x x
Dihydromorphone a x x x
b x x x
d-Methamphetamine a x x x
b x x x
Ephedrine a x x x
b x x x
Hydromorphone a x x x -
b x x x ~
25Hydro~camphetamine a x x x . .
b x x x : - .
Methadone a x x x
b x x x
. 6 monoacetylmorphine a x x x
b x x x
WO 92/17769 PCI/US92/02393
21~71~3
-23-
:
Table 2 (con't)
RESPONSE
10 ug/mL 6 ug/mL 1 ug,/mL
Substance (-) (+) (-) (+) ( )
6 Morphine a x x x
b x x x
Morphine
glucuronide a x x
b x x x
Nalorphine a x x x
b x x x
Naloxone a x x x
b x x x
Sodium
pentobarbital a x x x
b x x x
N-norcodeine a x x x
b x x x
Oxycodone a x x x
b x x x
Oxgmorphone a x x x
b x x x
Phenobarbital a x x x
b x x x
26 Phenylephrine a x x x
b x x x
Phenylpropanolamine a x x x
b x x x
Pseudomorphine a x x x
b x x x
Salicylic acid a x x x
b x x x
., ' ' . ;
,~' ' ' , : :, , ' ' . , , ' , . . ":
WO 92/17769 PCl/US92/02?~3
2~ 07113
-24-
Table 2 (ccn't)
RESPONSE
10 ug/mL 5 ug/mL 1 ug/mL
Substance (-) (+) (-) (+) (-) (+)
Secobarbital a x x x
b x x x
Thebaine a x x x
b x x x
Drug Free a x x x
b x x x
Two controlled release potentiating agents were used. It is noted
that only three potential abused drugs (secobarbital, sodium
pentoBarbital and methadone) were not positively detectable at the
Ievel of 10 micrograms/mL. l"nis indicates that adtitional standards
varying the pH of the isolation of the test paper and modification of the ~ ~ -reagents may be necessary to deterrnine these materials. However, the
¦ ~ ~ other abused drugs which showed a positive response demonstrated
that this diagnostic technique is specific, quantitative and effective.
EXAMPLE 2
Test papers described in Example 1 were further developed by
spray-coating a primary and secondary microencapsulated potentiating
agent onto the absorbent chemical substrate. Microspheres of
ninhydrin and trichloroacetic acid were prepared using the method and
apparatus of the previous.e~cample. However, ninhydrin was
encapsulated in a polymer matrix consisting of the methac~late
. copolymer Eudragit RS-IOO, which is less permeable than the RL-IOO
polgmer matrix of trichloroacetic acid. The present embodiment,
~ therefore, consisted of an absorbent substrate saturated with 5% (w/v)
r~ solution of potassium ferricyanide (K~[Fe(CN)~l~ prepared in 0.1 M, pH
.~ .
~ . .
wo 92/17769 Pcr/uss2/o2393
~la~7l 1 ~
-2~-
9.3 phosphate buffer onto which is spray-coated microspheres of
ninhydrin (RS-100) and microspheres of trichloroacetic acid (RL-100). ~ -
Urine specimens were applied to the surface of the test papers as
described in the previous example. Urine constituents resembling
5 morphine alkaloids, aporphines, amphetamine or catechol species
migrated at different rates from the application site of the specimen
and forrl2id h ~.acyanofi~rrate-dru~ comple~es which were again ~ ~ -
immobili~id ,~lithin +he fibers of the paper substrate. Controlled,
delayed reiease of both potentiating agents (~, ninhydrin and ~ .
trichloroac^it c ac-d) was activ2ted when the polymer matri~ of each
indi~iduai ~nicrosphere came in contact with the specimen. However,
trichloroacetic acid was released at a greater rate than ninhydrin ~. .
because the polymer matri~ of trichloroacetic acid microspheres is more
permeable than that of ninhydrin microspheres. After a period of time
the color reaction of immobilized he~acyanoferrate-drug complexes : :
became intensified by the induction of trichloroacetic acid for the -
denaturation of endogenous proteinaceous material present in the
sample specimen that can interfere with the simultaneous induction of
ninhydrin for color intensification.
- 20 EXAMPLE 3
This example illustrates the use of the invention as an
enzyme-linked immunoassay technique to determine drugs of abuse
with a high degree of specificity. The present e~ample consists of the
following components: a) a solid substrate as in Examples 1 and 2, ~ . . .
as except that the said substrate is saturated with an enzyme substrate
(ç~, 5-bromo-4-chloro-3-indolyl phosphate); b) a controlled, delayed
release mechanism imparted by water swellable microspheres or
microcapsules; c) a hapten comprising of antigen (~, morphine, ''
benzoylecgonine, tetrahydrocannabinol, barbiturate, amphetamine) ~:
30 colyugated (covalently linked) with enzyme (~, alkaline phosphatase);
:. . : ..
' ' ... .
.. . ..
''"~, ' ' ''
WO 92/17769 PCI/US92/023".~
210~l 1, 3
-26-
and d) an antigen specific mono- or polyclonal antibody. The hapten of
the present example is analogous to the potentiating reagent of the
previous examples in its action upon the substrate to produce a color
change for a target drug positive ~pecimen.
Microspheres or microcapsules containing hapten bound to
antigen specific antibody are manufactured in accordance with the
method of hyp2rf~.0w ~inetic ancapsulation or any other encapsulation
method ~nown to those in the art. The hapten-antibody coupling pair
may be prepared by incubating the two components prior to ~ -
microencapsulatioIl. Subsequent to manufacture of protected,
immooiiized and immunochemically active hapten- antibody coupling
pair, the microencapsulated product was applied to the surface of the
solid phase substrate which was previously saturated with enzyme
substrate as described in previous e~atnples.
In the method of the present invention, a narrow strip (6 x 0.5
ctn) of the coated paper substrate is contacted with a urine specitnen
containing an antigen of one of the trugs of abuse. The
hapten-antibody coupling pair is sensitized upon contact of the polymer
matri~ of each microsphere with the specimen. The immunochemical `
and enzymatic activity of the hapten-antibody coupling pair is delayed
for a period of time determined by the characteristics of the polymer
matrix selected for the encapsulating microYphere. This delay allows
-~~ =, sufficient time for the antigen to migrate and become separated from
endogenous constituents. As the microspheres become fully activated
25 (fll~lly 8welled), constituents interact with the hapten-antibody coupling
pair in the pores and confines of each individual microsphere. Within
this space, the antigen, in the case of drug-positive specimen, competes
with hapten for antigen-specific antibody. Consequently, antigen
becomes bound competitively to antibody binding sites in the
30 microspheres and unbound, enzymatically active hapten is released
,.~
wo 92/17769 Pcrtus92/o2393
21071~3
-27-
from the microspheres. A distinctive color is then produced when the
released hapten comes in contact with the enzyme substrate which is ~ .present in the absorbent substrate. When no target antigen or drug is
present, no color change is observed since no antigen is available to
compete with the hapten.
EXAMPLE 4
This e ~am.~le illust- ates the use of the in~ention as a
multi-en~ -catal~zad assay for serum triglycerides. The technique
capitalizes on tne sequential or delayed release of multiple enzymes
containen ~n ~.ic-ospher~s or ~.icrocapsules of ~arying polymer-matrix
compositions t~ producP a dye on a solid substrate that can be detected
and quantitated visually for various concentrations of serum
triglycerides. The release of multiple enzymes and reagents is achieved
by exploiting different porosities and ratios of reagent or enzyme to
polymer matrLx material of the polyacrylate- polymethacrylate - -
copolymers t ype as described elsewhere. The present example con~ists
of the follov~ng components: a) a solid substrate as in previous ~-
examples, except that the said substrate is saturated with adenosine
triphosphate (ATP), surfactant (~, æodium dodecylsulfate) and a leuco
dye (ç~, 2-(3,5-dimethoxy-4-hydroxyphenyl)-4,5-bis -(4-dimethylaminophenyl)imidazole); b) controlled, delayed release : -
mechanisms imparted by water Awellable microspheres or
microcapsules; c) lipase enzyme; d) glycerol kinase enzyme; e)
glycerophosphate oxidase enzyme; and f) peroxidase enzyme. ~~
Microspheres or microcapsules of individual enzymes, lipase
(LP), glycerol phosphatase (GP), ~-glycerophosphate oxidase (GP0), . :. :
and peroxidase (P0), are manufactured in accordance with the methods
of the previous esamples. However, the porosity of the enzyme-polymer
matris and ratio of enzyme to polymer matrix composition is chosen s
such that the rate of release of each enzyme follows the order: LP >
`~ -"'''~'' `" "' '' `' "" ' '' '''''" ' ''' ' ' ,'"'' ' , '
wo 92/17769 PcrtuS92/02343
2~7 ~ 13
-28-
GP > GP0 > P0. Preferably, the rate of release of enzyme from its
corresponding polymer matrix is formulated such that induction of each
enzyme into the reaction theater occurs sequentially. The protected
and enzymatically active enzymes are applied to the surface of the solid
5 phase substrate which has been previously saturated with ATP,
surfactant and leuco dye as described in previous examples.
Thus, in the method of the present invention, a narrow strip of
coated paper substrate is contacted with a serum specimen containing
endogenous lipoproteins. The release of each enzyme is sensitized upon
10 contact of the corresponding microsphere- or microcapsule polymer
matrix with th~ serum specimen. During this sensitization period, the
serum specimen migrates through the absorbent substrate to allow for
separation of lipoproteins into their triglyceride and protein :~ :
constitutive components. As LP is released from the corresponding
15 microspheres or microcapsules at a rate greater than GP, GP0 and P0,
the separated trig~ycerites are hydrolyzed catalytically by LP in the
presence of water (from serum) to glycerol and fatty acids. After a
period of time, induction of GP into the reaction theater occurs at a
rate greater than GP0 and P0. In the presence of ATP (saturated on
20 the substrate), glycerol.formed from the previous step is phosphorylated
catalytically by GP to L-a-glycerophosphate ant adenosine diphosphate
~ADP). Subsequent to the release of GP, induction of GP0 occurs at a
rate that is greater than P0. The L.-a-glycerophosphate that is formed
from the previous step is osidized catalytically in the presence of air by
26 GP0 to dihydro~lyacetone phosphate and hydrogen peroxide. In the
final step of the colorimetric reaction, P0 is released from the
corresponding microspheres or microcapsules. A distinctive color is :
produced, with intensity proportional to the concentration of
triglycerides originally present in serum specimen, when the leuco dye
30 that is saturated in the absorbent substrate is oxidized catalytically by
.
, . . .
wo 92/17769 PCr/US92/02393
21~ 7113
-29-
PO to form a dye that can be detected by visual observation.
One skilled in the art will readily appreciate that the present
invzntion is well adapted to carry out the objects and obtain the ends ~; . .and advantages mentioned, as well as those inherent therein. The
5 components, methods, procedures and techniques described herein are
presently representative of the preferred embodiments, are intended to
be e~amplarJ, and ara not intandad as limitations on the scope of the ~ -
prssent in~2ntion. Chan~,ras tharein and other uses will occur to those
skilled in the art which are encompassed within the spirit of the
invention and are defined by the scope of the appended claims. .
~:
What is claimed is~
" :' :' -
:~:
...,~ .... ...
~ .... . ... . .. . . . .. . ... .. .. .. . . . . .. . . .. . . ..... . . . . . .... .. . . . .. . . ...
. . .. . . . .
