Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
1316212
CYCLING BIOCHIP SENSOR
Field of the Invention
This invention relates to sn apparatus for selectively adsorbing a
ligand from aqueous solution in which the adsorption apparatus may be used as anS element of a sensor. Specifically this invention relates to substratum having
biologically active molecules immobilized on the surface. The biologically
active molecules are capable of forming a complex with a ligand in aqueous
solution, the formation thereof causing a variation in 8 physical property of the
substratum which may be detected by electronic means. Subsequent to forming
10 the complex the biologically active molecules are reversibly denatured to
recover the ligand and regenerate the active adsorber.
Background of the Invention
For a variety of reasons, ;t is often desirable to selectively com-
plex small molecules from aqueous solution. First, selective complexing may
IS provide a method for isolation of unique ligands from solution. Biological~y
active molecules can selectively bind to ligands with extremely high
selectivity. For example, biologically active molecules such as proteins can
easily different;ate chemical isomers. This high selectivity can provide a meansfor isolation, purification, or sensing unique small molecule ligands, if the bio-
20 logically active molecule is ligated to a substratum and can be selectively in-
duced to release its bound ligand, and if tlle biologically active molecule does not
transform the small molecule ligand into another species. It is also often
desirable that release of the small molecule ligand from the biologically activemolecule be accomplished without the introduction of other substances such as
.
~ ,- " '
-2- 1316212
competitive inhibitors, ions or denaturing agents that would complicate the
isolation, purification or sensing procedures.
Second, selective complexing of small molecule ligands can pro-
vide a means for accurately detecting and quantitsting specific small molecule
5 ligands in aqueous solution. For example, it is often desirable to ~ualitatively
and quantitatively measure substances dissolved in an aqueous medium, without
the necessity of withdrawing an aliquot and measuring the included substances bystandard techniques. For example, rapid and continuous measurement of sub-
stances dissolved in body fluids such as serum is often critical to making a proper
10 diagnosis of a patient's condition. Present techniques often involve withdrawing
a sample of blood or urine and sending it to a clinical chemistry lab for
analysis. It would be a great advantage to be able to measure levels of dissolved
substances without withdrawing these fluids or waiting for analysis.
Similarly, continuously monitoring solute levels is often important
15 to the chemical and food processing industries in order to control levels of
important constituents, and to make sure that effluent discharge requirements
are met.
Accordingly, there exists a need for a sensing device that selec-
tively responds to the presence of specific small molecule ligands dissolved in an
20 aqueous medium. Furthermore, it is important to have a sensing device that
produces an output that varies according to the concentration of the dissolved
substance. It is also important that the sensor be able to respond to a wide range
of concentrations and that it be capable of functioning in a continuous mode.
Finally, the device should be long-lived and be capable of operating under rela-
25 tively harsh conditions. It should be small, easily manufactured and it should
retain its selectivity ~nd sensitivity over the span of its useful life.
Summary of the Invention
The invention consists of a cycling biochip sensor having a semi-
conductor substratum, and a binding protein capable of reversibly complexing a
30 ligand in aqueous solution when immobilized on the semiconductor substratum.
The binding protein is attached to the semiconductor substratum such that
complexing the ligand with the binding protein perturbs the semiconductor
substratum in a detectable manner. The binding protein is capable of forming a
high affinity binding complex with a small molecule ligand upon contacting the
~3~ I 31 621~
small molecule ligand in aqueous solution. The binding protein is denatured uponheating to temperatures of about 75C, releasing the small molecule ligand into
solution. The binding protein then renatures upon cooling, regenerating the
biochip sensor. The cycling biochip sensor also is equipped with a signal pro-
5 cessor coupled to the sensor for producing a signsl that varies according to thesmall molecule ligand and binding protein forming a complex.
The cycling biochip sensor has a protein immobilized on the
semiconductor surface wherein the protein is selected from appropriate ligand-
binding proteins, protein fragments or synthetic analogs thereof. In a preferred0 embodiment of the invention, the protein is selected from periplasmic binding
proteins, and mutant periplasmic binding proteins having an altered amino acid
sequence. Examples of suitable periplasmic binding proteins include those
isolated from Escherichia coli and Salmonella typhimurium, which form a high
affinity binding complex with arginine, ornithine, Iysine, oligopeptides, cystine,
lS glutamine, glutamate, aspsrtate, histidine, leucine, isoleucine, valine, threonine,
arabinose, galactose, glucose, maltose, ribose, xylose, B-methylgalactoside,
c;trate, phosphate, glycerol-3-phosphate, sulfate, vitamin BI2, thiamine, and
cadmium.
The cycling biochip sensor has a semiconductor substratum made
2~ from Si, Ge, Ga As, SiC, and silicon on saphire, at least a portion of the surface
being oxidized or covered with a metal and/or metal oxide. Alternatively, those
skilled in the art will appreciate that the substratum can be a metal or
metal/metal oxide system, to which a biologically active molecule can be
immobilized. Examples of such metal or metal/metal oxide systems include
25 antimony or antimony/antimony oxide, or palladium or palladiumlpalladium
oxide. The cycling biochip sensor further consists of a linking compound
covalently bonding the binding protein to the semiconductor substratum. The
linking compound reacted with the oxidized semiconductor substratum consists
of a compound with the structural formu]a:
H2 N(CH2)n-Si(O-(cH2)m-cH3)3
where:
1316212
n is an integer from 2 to 7
m is an integer from 0 to 4
and a homobifunctionsl cross-linking compound or a heterobifunctional cross-
5 linking compound, selected from dimethyl adipimidate, dimethyl-3,3'-dithiobis-propionimidate, dimethyl pimelimidate, dimethyl suberimidate, bis-12-(succin-
imidooxycarbonyloxy)ethyl]sulfone, disuccinimidylsuberate, disuccinimidyl-
tartrate, dithiobis-(succinimidylpropionate), 3,3'-dithiobis(sulfosuccinimidylpro-
pionate), ethyleneglycolbis-(succinimidylsuccinate), bis-12-sulfosuccinimidooxy-
lO carbonyloxy)ethyl]sulfonate and the heterobifunctional cross-linking compound is
selected from those having functional groups independently selected from
N-hydroxysuccinimidyl, maleimidyl, pyridyl, N-hydroxy-sulfosuccinimidyl, alkyl
c~-keto halides, benzyl halides alkyl or aryl ~-haloamide, aryl isothiocysnate and
azidophenyl.
Also provided for in the present invention is a kit for detecting a
smsll molecule ligand in aqueous solution made by covalently linking a cycling
biochip sensor to a semiconductor substratum. The binding protein is capable of
reversibly complexing a small molecule li~and in agueous solution, which per-
turbs the semiconductor substratum in a detectable manner. A sterile trans-0 parent package containing the cycling biochip sensor is also provided.
Detailed Description of the Invention
A class of proteins termed "binding proteins" resides in the space
between the inner and outer membranes (periplasmic space) of gram-negative
prokaryotes. These proteins are involved in transporting nutrients across the
25 plasma membrane. These binding proteins bind their respective nutrients with
high specificity and affinity. In addition, these binding proteins are resistant to
proteases and heat. Further, these proteins bind their respective substrates
through a broad range of pH values and ionic strengths. These special propertiesmake this class of proteins especially adaptable for use in cycling column adsor-
30 bers for removal of specific small molecules from aqueous environments~ and asbinding components of "biochip" sensors.
X-ray crystallographic studies of several of these binding proteins
show that they are bilobate in shape, the space between each lobe defining a
concave cleft where the ligand binds to the protein. The molecùle is flexible and
1 31 62 41 '2
62839-lOb9
upon ligand binding, the two lobes close down on the ligand in a
"Venus fly-trap~ type blndlng mechanism. Thus the protein ligand
blnding causes a conformatlonal change ln the proteln (Ames,
G.F.L., Ann. Rev. Brochure 198~ 55:397-425) resultlng ln a hl~h
afflnlty blndlng complex. The blndlng afflnlty (KD) for many
saccharlde ligands ls from about 0.1 ~ M to about 1.0 ~ M, for
amlno aclds is from about 0.01 ~ M to about 50 ~ M, and for anlons
about 0.02 ~ M to about 30 ~ M. (Furlong, C.E., Methods In
Enzymology 125: 279-289 (1986)).
The small KDs make these blndlng proteins excellent
candldates for small molecule llgand adsorbers as well as for
elements of sensors when they are immoblllzed on the surface of an
lnsulated-gate fleld-effect tr~ns~stor (IGFET). However, the hlgh
llgand afflnlty ls dlrectly related to a low dlssoclatlon rate
requlrlng that these protelns be "encouraged" to release thelr
llgand by external stlmulus to prevent the sensor from remalnlng
saturated. It is known, for example, that lsolated blndlng
protelns often carry llgands complexed wlth thelr blndlng sltes
(Amanuma, H.M. et al., J. Blol. Chem., 79, 1167 (1976)). It has
been dlscovered ln accordance wlth the present lnventlon that
prokaryote perlplasmlc blndlng protelns whlch have formed a hlgh
afflnlty blndlng complex wlth a llgand wlll release the llgand
upon heatlng to temperatures above 75C and that the blndlng
protein wlll renature to lts orlglnal conformatlon upon coollng to
amblent temperature. In addltlon to hlgh temperature stablllty,
prokaryote substrate blndlng protelns are stable to drylng con-
dltlons, protease treatment, and other denaturlng condltlons such
as guanldlne-HCl treatment (Mlller, P.M., Olson, J.S., Pflugrath,
1316212
5a 62839-1069
J.W. and Qulocho, F.A., J. Biol. Chem., 258: 13665-13672 (1983)).
Thus, these blnding protelns may be sultably employed in a sensor
for c~etectlng low concentratlons (near the KD) of small molecule
llgands ln aqueous solution, and they may be cycled by heating and
coollng or other reverslbly denaturlng condltions such as organic
solvents and chaotroplc agents to release the bound ligand and
regenerate the actlve sensor. These sensors belng useful for both
quantltative and qualltatlve measurement of llgands ln aqueous
solutlon. Alternatively, those skllled in the art will appreclate
that the substratum can be metals or metal/metal oxide materlals
such as antlmony or palladlum as metals and antimony/antimonyoxide
or palladlum/palladlum oxlde as metal/metal oxldes.
1316212
In accordance with the present invention a cycling biochip sensor
has a semiconductor base or substratum. Suitable semiconductors include, Si,
Ge, Ga As, SiC, and si}icon on sapphire. A particularly useful semiconductor is
the insulated~ste field-effect transistor (IGFET) having one or more insulated
5 gate regions. When the amount of charge near these insulated gste regions
either increases or decreases, a physical property of the FET, for example its
conductance, varies accordingly. This variation in a physical property of the
IGFET can be used to produce a signal that varies according to the amount of
ligand binding complex formed. This signal may be processed according to
10 techniques well known in the electronics art. In the case of an IGFET, binding of
a ligand to a binding protein immobilized near the surface of the insulated gateregion is the event that causes the conductance to vary according to the amount
of substrate binding that has occurred.
In one configuration of the present invention, the cycling biochip
15 sensor is a remote sensor connected by wire leads to electronic circuitry suitable
for processing signals produced by the remote biochip sensor, and providing
information to the user regarding the presence and relative amounts of ligands in
aqueous solution. In this configuration, these cycling biochip sensors may be
replaced from time to time with new sensors having the same or different bind-
20 ing proteins immobilized on the insulated gate regions. These remote cyclingbiochip sensors and wire leads are provided in kit form contained in sterile
transparent packages.
In another embodiment of the present invention the substrate is a
charged species. Periplasmic binding proteins having specificity for anions such25 as citrate, phosphate, glycerol-3-phosphate sulfate can be immobilized upon the
insulated gate region of the IGFET. As the number of these anions bound to
their respective binding proteins increases, the current from source to drain
varies due to the increased charge sequestered near the insulated gate region.
The increased charge sequestered near the insulated gate is due in part to the
30 charged ligand and in part to the conformational change of the protein that
occurs upon ligand binding.
In still another embodiment of the invention, uncharged or neutral
ligands complexed with binding proteins immobilized at the insulated gate regionof the IGFET produce a similar change in conductance according to the number
1 31 62 1 2
7 62839-1069
of ligand molecules bound. It ls believed that conductance change
ln thle IGFET upon blndlng neutral llgands is due to conformatlonal
chancle lnduced upon ligand bindlng whlch alters the surface charge
of the blndlng protelns. Examples of sultable perlplasmlc blndlng
protelns havlng hlgh speciflclty for neutral llgands lnclude pro-
telns whlch blnd arablnose, galactose, glucose, maltose, rlbose,
xylose, ~-methylgalactoslde, vltamln B12, and thlamine.
Zwltterlons complexed wlth blndlng protelns on the lnsu-
lated gate region of an IGFET are also capable of producing a
change in conductance proportlonal to the number of lons bound.
Examples of sultable perlplasmlc blndlng protelns havlng hlgh
afflnlty and specificlty for zwltterions lnclude bindlng protelns
of: cystlne, glutamlne, glutamate, aspartate hlstidlne, leuclne,
lsoleuclne, vallne, and threonlne.
Other blnding protelns obtained from Escherlchla coli
and Salmonella tvPhimurlum whlch are sultable for use ln accor-
dance wlth the present lnventlon lnclude those descrlbed by
Furlong, C.E., ln Methods ln Enzymology 125: 279-289 (1986),
hereln lncorporated by reference. Blndlng protelns from these
prokaryotes are partlcularly useful slnce they can be easlly
produced ln large quantltles ln a bloreactor. Stlll other pro-
telns sultable for use lnclude those descrlbed by Copeland, B.R.,
et al., J. Blol. Chem., 257: 15065-15071 (1982). In addltlon,
two cadmium binding proteins from E. Coli have been descrlbed by
Khazaell, M.B., Appl. Environ. Mlcroblol. 41: 46, (1981). These
latter bindlng protelns are suitable for use in a cycllng cadmlum
adsorber.
In general, for a blologlcally active molecule to be
,.,:, ~-,
1316212
7a 62839-1069
useful as a llgand adsorber or senslng element of a blochlp sensor
lt need only bind its llgand wlth an afflnlty and speclflclty
approprlate to the partlcular appllcatlon. A general procedure
for identlfylng and lsolatlng protelns ls descrlbed by Copeland,
B.R., et al., vlda suPra. The entlre proteln may not be necessary
lf an actlve blndlng domaln of the proteln can be lsolated and lf
the domaln can be reverslbly denatured.
For a blndlng proteln or other protein to be useful as a
senslng element for a blochlp sensor, lt ls preferred that the
blndlng proteln have a KD near the concentratlon of the solutlon
belng measured. If the concentratlon of llgand ln solutlon ls
hlgher than that of the KD for a glven blndlng proteln, the
1316212
-8-
measuring system can employ a feedback controlled dilution system to adjust,
cslculate, and control the necessary dilution of the original solution to bring it
within measurement range for a given binding protein attached to the IGFET. In
general, a binding protein is suitable for use in detecting the corresponding
ligand at a concentration of from about KD/20 to about 2KD. For example, the
phosphate bind;ng protein from E. Coli has a KD of 0.8 ~. M, therefore, this peri-
plasmic binding protein would be suitable for use in a biochip sensor for
measuring phosphate concentrations from about 40 ~, M to about 1.6 ~ M. In
many instances it is useful to measure phosphate concentration above this
range. For example, normal ranges for serum phosphate are between 0.74 and
3.1 mM (Teitz, N.W. Textbook of Clinical Chemistry, W.B. Saunders Co. (1986))
or about 1000 times higher than the useful range of the "wild type" E. Coli phos-
phate-binding protein. Mutant binding proteins having an altered amino acid
sequence and therefore KDs different from the wild type KD can be produced by
site specific oligonucleotide-mutagenesis (see for example Schultz, S. C. and
Richards, J.H., Proc. Natl. Acad. Sci USA 83, 1588-1592 (1986), and references
cited therein). In this way a spectrum of phosphate binding proteins can be
produced each with its own KD. A mutant phosphate binding protein can then be
selected for a given applicat;on.
In an alternative embodiment of the invention, a biochip sensor
effective in measuring a broad range of ligsnd concentrations is produced by
immobilizing binding proteins having different KDs selected from among the
various mutant binding proteins or other proteins described above, onto different
insulated gate regions of the IGFET. By selectively choosing binding proteins for
a particular ligand with KDs separated from one another by a factor of about 100or less, a broad range cycling biochip sensor ;s produced.
In another embodiment of the invention binding proteins for
different ligands are immobilized on different insulated gate regions of the
IGFET to produce a multiple substrate cycling biochip sensor. For example,
normal serum sulfate and citrate concentrations for adults are 25 to 40 mM and
80 to 160 mM respectively. (Montgomery, R., et al., Biochemistry 3rd Ed., C.V.
.~losby Co. 1980.) Thus selecting mutant binding proteins for sulfate, citrate and
phosphate with KDs of about 30 mM, 100 mM, and 1.5 mM respectively and
immobilizing these proteins on diîferent insulated gate regions of a IGFET would
1316212
produce a sulfate-citrate-phosphate cyc1in~ biochip sensor suitable for contin-
uously measuring serum levels of these three anions. Alternatively, the "wild
type" binding proteins from E. Coli or S. Typhimirium could be used if a feedback
controlled dilution system as previously described is employed.
S It will be appreciated by those skilled in the art that a broad
range multiple substrate biochip sensor can be produced by immobilizing an arrayof binding proteins having a broad spectrum of KDs for each ligand on separate
insulated gate regions of an IGFET.
The use of periplasmic ligand-binding proteins or other
appropriate proteins as elements of a biochip sensor has the advantage of
forming a stable high affinity complex with a substrate without altering the
ligand, unlike the case with enzymes. However, in order to make a sensor from a
binding protein useful for more than one concentration measurement, these
binding proteins must be encouraged to release their ligand under conditions that
do not alter either the specificity or affinity for a particular ligand.
A variety of methods and substances are suitable for reversing
ligand binding to a binding protein. These include the use of "denaturing" sub-
stances such as guanidine-HCI, urea, organic solvents, and chaotropic agents.
Other methods suitable for inducing ligand release from these binding proteins
include incubation in a substrate free aqueous solution.
The preferred method for inducing the release of ligand from an
immobilized binding protein is to heat the protein to a temperature sufficient to
cause the prote;n to release its ligand in a relatively short period of time, the
time and temperature determined by the particular application. In the case of
periplasmic binding proteins, it is critical that the proteins be immobilized on a
surface prior to heating, otherwise the protein coagulates irreversibly. Thus,
heat is used as a "denaturant" only when periplasmic binding proteins are
immobilized.
There are many advantages of using heat instead of ionic or
molecular denaturants to facilitate ligand release. First, the use of denaturants
such as guanidine-HCI to remove ligands from binding prote;ns necessarily re-
quires removal of the sensor from the test medium. This may be undesirable for
continuous concentration measurements. Furthermore, guanidine-HCl must be
completely removed from the sensor prior to its reuse, a time-consuming pro-
-lo- 1 31 621 2
cedure. Secondly, hest is easily generated by a number of methods including
microwave irrsdiation, resistive heating from the semiconductor itself, and
infrared irradiation. The removal of ligands by heating the protein to a point
that it may be reversibly renatured allows the ligand to be released into a stream
of desired composition, such as deionized water, for example, where the ligand
may be concentrated in the absence of salts or other molecules. The use of heat
avoids the introduction of other materials, which may be difficult to remove.
Tl~e procedure is general, in that many different proteins may be used in this
process. The only requirement is that the protein has the required specificity
and affinity for the ligand of interest and that the protein be reversibly de-
natured by heat.
Thirdly, the temperature and its duration can be accurately and
automatically reproduced or varied according to a particular application withoutthe introduction of time-consuming, nonstandard procedures, the reproducibility
of which may depend on the skill of the technician.
In applications where the biochip sensor is to be used intermit-
tently, simply immersing the sensor in hot water is effective to induce release of
l;gand from the protein. For example, a biochip sensor of periplasmic phosphate
binding prote;n immobilized on a silicon semiconductor exposed to 10 1I M
K3PO4/Tris-HCI buffer pH 7.4 becomes saturated with phosphate within 10
seconds. Immersion of the biochip sensor in 80C water for 15 to 3û seconds
causes the release of greater then 85% of the bound phosphate without
measurable loss of binding affinity or specificity.
In applications where the biochip sensor is to be used continuously
or where removal of the sensor is impractical, heat is applied to the sensor by
resistive means, i.e., passing current through the semiconductor. The amount of
current and its duration in order to effect ligand release will depend on the
particular application and can be empirically determined.
The means for immobilizing a binding protein on a semiconductor
insulated gate surface depends on the composition of the semiconductor. In the
case of a doped silicon semiconductor, 8 portion of the surface is cleaned and athin silicon dioxide layer approximately 500-1000 A thick is thermally grown on
the cleaned surface under an atmosphere of dry oxygen by the method described
in Sze, S.M., Physics of Semiconductor Devices, 2nd Ed., John Wiley ~ Sons
1316212
11 62839-1069
1981). The sillcon dioxide surface is then preferably derlvatized
usinS~ vapor phase depositlon of sllane compounds having structural
Formula I:
H2N(CH2)n-$l(0-(CH2)mCH3)3
where
n is an lnteger from 2 to 7
m ls an lnteger from 0 to 4.
A preferred compound of structural Formula I ls 3-amino-
propyltrlethoxysllane (APT~S). Vapor phase depositlon of a com-
pound of structural Formula I deposlts a chemlcally actlve layerof the silane compound covalently bonde~ to the S102 surface.
A blndlng proteln, selected from those prevlously des-
crlbed, ls then immoblllzed on the derlvatlzed surface of the
semlconductor by a covalent bond. Bonding the proteln to the
derivatizsd SiO2 surface must be conducted under conditions that
preserve the biological activity of the binding protein. This may
be achieved ln a number of ways. For example, the derivatized
semiconductor base has a free primary amlno group whlch can form
an adduct wlth a blndlng proteln whlch has been ltself derlvatlzed
2~ wlth a Michael-type acceptor, e.g., malelmlde. Alternatlvely, the
free amino group of the derivatlzed glass can be derivatlzed by
convertlng the amino group lnto a maleimide whlch ln turn may form
an adduct wlth a nucleophillc group on the protein. Still another
method of llnklng the bindlny proteln to the derlvatlzed semi-
conductor ls to bond the bindlng proteln through blfunctlonal
cross-llnkers, of the type dlsclosed ln the 19~-87 catalog of the
t3~6212
lla 62839-10~9
Pelrce Chemlcal Company, Rockfork, Illlnois, pages 312-340. These
cross-llnkers lnclude but are not llmlted to: homob~functlonal
cross-llnklng compounds selected from dlmethyl adlplmldate, dl-
methyl-3,3'-dlthlobls-proplonlmldate, dlmethyl plmellmldate, dl-
methyl suberlmldate, bls-[2-sulfosucclnlmldooxycarbonyloxy~-
ethyl]sulfone, dlsucclnlmdyl suberate, dlsucclnimldyl tartrate,
dlthlobls-(succlnlmldyl proplonate), dlthlobls-(3,3'-dlthlobls-
(succlnlmldylsucclnate), bls-[2-(sulfosucclnlmldooxycarbonyl-
oxy)ethyl~ sulfonate and heteroblfunctlonal cross-llnklng
compounds
. .
-la- 131621 2
selected from those having functional groups independently selected from N-
hyrdroxysuccinimidyl, maleimidyl, pyridyl, N-hydroxy-sulfoxysucinimidyl, alkyl
~-keto halides, benzyl halides alkyl or aryl -haloamide, aryl isothiocyanate and
azidophenyl.
For other semiconductors such as Si3N~, immobilization of the
binding protein is achieved by the same procedures described above, since Si3N4
forms ~ thin surface oxide layer of several A providing sites for attachment forcompounds of structural Formula 1. Alternatively, avidin or streptavidin may be
used to couple biotinylated binding protein to a biotin derivatized surface.
It will be appreciated that the sensitivity and effectiveness of the
biochip sensor will depend on the number of binding proteins immobilized per
unit area or dens;ty of the cycling biochip surface. In the case of a silicon based
IGFET, it has been found that suitable binding protein densities are on the order
of 1013 protein binding sites/cm2. Quiocho, F.A. and Pflugrath, J.W., J. Biol.
Chem., ass 6559 (1980). The maximum density being determined by the protein
molecular diameter. The lower limit is determined by minimum signal
detection. The preferred protein densities range from 101 to 1013 protein
binding sites/cm2.
Those skilled in the art will recognize that the embodiments
disclosed herein are exemplary in nature and that various changes can be made
therein without departing from the scope and spirit of the invention. Numerous
configurations of the sensor can also be provided to enhance the utility of a
measuring instrument with respect to a particular application. Because of the
above and numerous other variations and modifications that will occur to those
skilled in the art, the following claims should not be limited to the embodiments
illustrated and discussed.
