Note: Descriptions are shown in the official language in which they were submitted.
WO 92/15709PCl`/US92/01572
-1- ,'~.;.~.1.006~3
SCANNING PROBE MIC~O~COPY IMMU~OA~
Back~round of the Invention
.
5This invention relates generally to the detection of analytes, and
more parlicularly, relates to the detection of analytes by utilizing
scanning probe microscopy.
The two mo~t widely known and used scanning probe microscopy
1 0 (SPM) techniques involve the use of either the Scanning Tunnelling
Microscope (STM) or the Atomic Force Microscope (AFM), also known as
the Scan~ing Force Microscope (SFM). These techniques have been the
subject of many scientific publications and review articles. See, for
example, P. K. Hansma et al.,"Scanning Tunneling Microscopy and
1 5 Atomic Force Microscopy: Application to Biology and Technology" Science
242: 209 - 216 (1988); L. Feng et al., "Scanning Tunneling Microscopy of
Proteins on Graphite Surfaces" Scannine Microsco~r 3:399-410 (1989); J.
D. Baldeschwieler et al., "The Scanning Probe Microscope: A Powerfi~l
Tool For Visualiz;ing the Micro World" ~I.b~:34-3g
2 0 (February, 1991), as well as arti~les cited in t hese three publications.
STM and AFM instruments are commercially available from several
sources, such as Digital Instruments, Inc. (Santa Barbara, CA 93117),
TopoMetrix (Pasadena, CA 91106), and EG&G Princeton Applied ~ :
Research (l?rinceton, NJ 08~43).
2 5 -
In ScanI~ing Tunnelling Microscopy (STM), the oldest know~ SPM
method, the probe is electrical. At eacll X, Y coordinate position, the
sen~or, a ve~y fine wire drawn to an atomically sharp tip, is maintained a
constant distance above the surface by a piezoelect~c ceramic driver.
3 0 Iypically, a constant potential is maintained between the tip and the
surface to be esamined; hence, the requirement that the substrate be
conductive. Conductive substrates such as ~Iighly Oriented Pyrolyl;ic
Graphite (HOPG) (available from Digital Instruments, Santa Barbara
CA) and gold have been used. Crystals also can be examined, since they
3 5 are usually conductive. At each X, Y position, the Z coordinate at a
constant distance above the surface is recorded using the tunneling
cu~ent from ~e tip to the surface in a feedback circuit to keep the
tunneling current constant, and hence, the distance from ~e tip to
WO 92/1~;709 PCr/US92/01572
06~3 -2-
surface constant. The "map" resulting from such measurements
consists of the height, Z, of the surface at various X, Y coordinates. For
conductive substrates, this method allows the resolution of atoms in
crystalline latices (Hansma et al., 1988, .~upra). -
Originally, it was postulated that only conductive molecules could
be imaged via STM. Thus, for example, M. Amrein et al, ~cience, 240:
514 - 516 (1988) used STM to image a complex between DNA and recA, a
protein involved in recombination. However, the ~ample had been coated
10 with a conductive platinum, iridium and carbon film. More recently, T.
P. Beebe, Jr., et al., Science, 243: 370 - 372 (1989) imaged uncoated double-
stranded DNA deposited on a graphite surface with an STM. Moreover,
STM ha~ been used to image uncoated ZDNA on graphite, P. G. Arscott,
et al, N~,ture, 339: 484 - 486 (1989).
1 5
STM can be used to image samples immersed in an aqueous
solution. S. M. Lindsay et al, Science, 244: 1063 - 1064 (1989)
electrophoretically deposited double-s*anded DNA onto a gold surface
and observed the double helix conformation of the macromolecule in an
2 0 aqueous environment. The use of STM in ultra high vacuum has allowed
the imaging of DNA at the atomic level unth DNA dried onto HOPG, as ~ `
reported by R. J. Driscoll et al, ~j~ture, 346: 294 - 296 (1990).
Biological molecules other than DNA have been imaged by STM. L.
2 5 Feng et al."I~ sf Colloid and Interf~ce Science 126:650-653 (1988) have
imaged Human Serum Albumin adsorbed onto HOPG, and were able to
observe the three domains of the molecule. Also, R. D. Edstrom et al,
Biochemistrv: 28: 4939 - 4942 (1989) imaged Phosphorylase Kinase and
Phosphorylase b, two enzymes which are involved in energy utilization n
3 0 muscle. The proteins were dried from concentrateid solutions (1 mglmL)
onto graphite beforei scanning the dry samples at atmosphenc pressure.
Both Feng et al. and Edstrom et al~ were able to obser~e l~nown features of
the proteins ~hey were imagir,g.
3 5 Recenl Iy, howe~,rer, a caution to the interpretation of images of
materials deposited~on HOPG has been published. C.R. Clemmer and
T.P. Beebe, Jr.noted that there frequently are structures on HOPG which
may be misinterpreted as DNA or proteins adsorbed on the surface. C.R.
WO 92/15709 PCl`/US92/01572
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Clemmer and T.P. Beebe, Jr., "Graphite: A Mimic for DNA and Other
}3iomolecules in Scanning Tunneling Microscope Studies" Science
~51:640-642 (1991)
~;~
Atomic Force Microscopy (AFM) is usually performed by observing
the deflection of a microscopic spring as a probe is lowered to the surface
of the material to be examined. The spring ends in a ~ne tip and its
deflection by contact with the surface can be observed by monitoring a
beam of laser light reflected from a mirrored surface on the back of the ;~
1 0 probe. AFM does not require a conductive substrate, and further it doesnot require that the material to be imaged be conductive. The former
allows non-conductive materials such as plastic, glass, and silicon which
are compatible with various biochemical and biomedical processes to be
used. The latter allows the direct detection of various kinds of biological
1 5 molecules without the necessity of coating them with a conductive layer.
Unfortunately, the contact nature of AFM may perturb the sample itself, - -
thus allowing only a distorted image of ~he material on the surface. For
example, J. N. Lin et al., I.anemuir 6: 509-511 (1990) adsorbed a mouse
monoclonal antibody onto mica and monitored the reaction with AFM.
2 0 The dominant features observed were "aggregates" of antibody at the
initiation of the reaction which changed into "ridges" as the monitored
reaction proceeded. The authors reported that it was clear that the tip of
the probe had been "massaging" or pushing the molecules on the surface.
AFM also has been useful for studying conductive molecules at high
2 5 resolution. S. Manne et al., Science, 251: 183 - 186 (1991) have reported on
the imaging at atomic resolution of gold electrodes with copper deposited
on them.
Probes other than electron tunneling or atomic force can be used to
3 0 explore the surfaces of various substrates. For example, atomic scale
friction can be mèasured in ~a Scann~ng Force Microscope, as well as
surface "hardness" or "sof~nes~U. ~:lectrostatic interactions also provide
another probe of a surface; the seIIsor is essentially an AFM probe, but
with a charge (positive or negative) imparted to its surface. Electrostatic
3 5 repulsion or attraction is measured by deflection of the probe as it - -
approsches the surfa~ce inng a charge map of the surface.
WO 92/15709 PCl'/US92/01572
r~l nO68
Although the need for speci~cally and reproducibly presenting
samples to the scanning probe has been stated (for example, by P. K.
Hansma et al., supra), there previously has been no suggestion or
attempt at utilizing specific ligand-ligand interactions (such as antibody-
5 antigen or nucleic acid hyb~dization) to accomplish this end. K. Eric
Dre~ler and John S. Foster, Natur~ 343: 600 (1990) have mentioned the use
of ligand-ligand interactions in the context of SPM, but only as a
speculative proposal to effect a specific chemical reaction on a molecule at ~`
a selected site. Heretofore, there has there been no suggestion that SPM
1 0 methods such as Sll!~I or AFM can be useful in measuring the product of
a specific binding reaction (such as antibody-antigen or nucleic acid
hybridization). This invention provides novel methods which can be
utilized to accomplish these results, heretofore unknown. Also provided
in the present invention are means to firmly immobilize samples to the
1 5 substrate, thereby obviating the problems observed by Lin et al., su~ra, in
which the probe moved the sample making detailed structural
determination impossible.
Summarv of the Invention ;~
2 0 The present invention provides a first method for dete~nining the
presence and/or amount of an analyte in a test sample, which method
comprises contscting a test sample with a test piece to which an analyte
specific substance has been attached for a time and under conditio~s
sufficient to allow binding of the analyte to the test piece, and scanning
2 5 the test piece by scanning probe microscopy to determine the presence or
absence of the analyte. Quantitation of the amount of analyte present in
the test sample can be accomplished by counting the number of analyte
molecules in one or more fields of the test piece and determining the
concentration of analyte present in the test sample either by reference to
3 0 previously measured standards or mathematically. Scanning is
performed by monitoring the response of a microsensor as it is
systematically scanned~across a surface of the test piece in raster fashion
to define an X, Y plane such as to control the vertical displacement of the ~;;
microsensor thereby making a map of a fimction of t~e surface of the test
3 5 piece at various X, Y coordinates. Scanning ma~r be performed either by a
scanning tunnelling microscope or an atomic force microscope The
analyte spec fic substance is attached to the test piece by adsorption or by
covalent binding. The preferred solid phases of the test piece used for
WO 92/15709 PCr/US92/01572
i~10~fi8~ `
adsorption attachment are plastic or metal. The preferred solid phases of -
the test piece used for covalent attachment are derivatized plastic, metal,
glass and silicon. The analyte specific substance can be ~n antibody, an
antigen, an antigen analog, DNA, RNA, a lectin, a hapten, avidin, biotin,
folate binding protein, or intrinsic factor. ~;
The present invention also provides a second method of
determining the presence of an analyte which may be present in a test
sample, which method comprises (a) mixing the test sample with a
1 0 known amount of its specific binding partner to form a mi~ture; (b)
applying the mixture to the test piece to which the arlalyte or an analyte
analog has been attached, for a time and under conditions sufficient for a
reaction to occur; and (c) scanning the test piece by scann~ng probe
microscopy to determine the presence and/or amount of the specific
binding partner, wherein the presence and amount of the specific binding
partner bound is inversely proportional to the amount of analyte present
in the test sample. Quantitation of the amount of analyte present in the
test sample can be accomplished by counting the number of specific
binding partner molecules in one or more fields of the test piece and
2 0 determining the concentration of analyte present in the test sample either
by reference to previously measured standards or mathematically.
Scanning is performed by monitoring the response of a microsensor as it
is systematically scanned across a surface of the test piece in raster
~ashion to define an X, Y plane such as to control the vertical
2 5 displacement of the microsensor thereby making a map of a function of
the surface of the test piece at va~ious X, Y coordinates. The analyte
specific substance is attached to the test piece by adsorption or by covalent
binding. The preferred solid phases of the test piece used for adsorption
are plastic or metal. The preferred solid phases of the test piece used for
3 0 covalent attachment are derivatized plastic, metal, glass and silicon. The ~ ~
analyte specific substance can be an antibody, an antigen, an antigen ~ -;
analog, a hapten, DNA, RNA, a le~tin, avidin, biotin, folate binding ; -
protein or intrinsic factor. -
......
3 5 Also provided are test kits useful for determining the presence of ~ ~
an analyte suspected of being present in a test sample by scanning probe -;
microscopy, comprising a teist piece having bound thereto an analyte
specific substance, an analyte, or an analyte analog. The analytsi specific
: ':''.'.
WO 92~15709 PCI`/US92/01572
substance, analyte or analyte analog preferably is covalently attached to
the test piece of the test kit. The test piece of the test kit pre~erably
comprises a solid phase,the solid phase being selected from thé` group
consisting of derivatized plastic, glass, silicon and metal. The test piece
of the test kit most preferably comprises a silicon wafer.
~.
Brief Descripti~n ~f the l)rawings
FIG lA is an SPM image of an amine-activated silicon test piece.
FIG. lB is a graph of the profile of the /surface of an amine-
1 0 activated silicon test piece wherein height (in angstroms [~]) of the
surface of the test piece is plotted against surface leng~h (in nanometers
[nm]).
FIG. 2A is an SPM image of an activated silicon test piece to which
an analyte specific substance (anti-Hepatitis B surface antigen
monoclonal antibody) has been attached.
FIG. 2B is a graph of the profile of the surface of an amine-
activated silicon test piece to which an analyte specific substsnce (anti-
Hepatitis B surface antigen monoclonal antibody) has been attached,
wherein height (in angstroms tA]) of the surface of the test piece is plotted
2 0 against surface length ~in nanometers [nm]).
FIG. 3A is an SPM image of an activated silicon test pieces to
which an snalyte specific substance (anti Hepatitis B surface antigen
monoclonal antibody) has been attached, after reaction wîth an analyte (r
Hepatitis B surface antigen).
2 5 FIG. 3B is a graph of the profile of the surface of an amine-
activated silicon test piece to which an analyte specific substance (anti-
Hepatitis B surface antigen monoclonal antibody) has been attached, afl;er
reaction with an analyte (r Hepatitis B surface antigen), wherein height
(in nanometers [nm]) of 1 he surface of the test piece is plotted against
surface length (in nanonieters ~nm]).
FIG. 4 is an SPM image of an amine-act*ated silicon test piece to
which an analyte specific substan~ (anti-Hepatitis B surface antigen
monoclonal antibody) has been attached, af~er reaction with an analyte (r
Hepatitis B surface antigen).
D~tailed Descri~tion of the Invention
The present in~rention provides a means for specifically
binding various analytes to a surface and a molecule by molecule
wo 92/15709 rcr/usg21ols72
-7 ~ 1, 0 ~ 3
determination of the presence or absence of the analyte in question using
Scan~ing Probe Microscopy techniques. The present invention thus
provides the application of a powerful new analytical tool to the problem of
determining the presence or absence of specific molecules. The
5 molecules are typically, though not limited to, molecules having
biological properties such as antibodies, antigens, drugs, and the like.
The molecules can be nucleic acids such as DNA and RNA. Or,
supramolecular structures consisting of assemblages of macromolecules
such as virus coat protein particles, viruses, bacteria, membranes or
1 0 membrane fragments, cells or fragments of cells can be the subject of the
current invention. The determination of the presence of the molecule of
interest also can res~t in the quantitation of the amount of analyte
present, if desired.
1 5 The analytical tool used in this invention is termed Scanning Probe
~Iic -~scopy (SPM) and consists of a related family of techniques which
can determine the properties of a surface at the molecular and even
atomic level. In this assay technique, a micro-sensor is rapidly and
systematically scanned across a surface in (for example) the X direction.
2 0 Upon the completion of one pass, the sensor is advanced a small distance
in the Y direction~ and another trace is made back in the X direction,
thereby defining an X, Y plane. The response of the sensor is monitored
as it i8 rastered (i.e., systematically scanned3 across the surface.
Customarily> the sensor response is utilized in a feedback loop to control
2 5 the Z position of the sensor. In this fashion, a complete map is made of a -
function of the surface at various X, Y coordinates. If an STM is used,
the microsensor detects cu2Tent flowing across the gap between the
surface and the microsensor. The current flow is used in a feedback loop
to control the height of the senor above the surface so as to keep the
3 0 current flow at a preselected value. West et al., U. S. Patent No. 4,~52,857,
which is incorporated herein by reference, report an improved STM -
which utilizes an adaptive feedba~ control circuit. If an AFM is used,
the microsensor physicslly contacts the surface, snd the displacement of
the sensor is used in a feedback loop to control the height of the sensor
3 5 above the surface.
.
The present invention provides an immunoassay which utilizes ~;
specific binding members. A "specific binding member," as used herein, -~
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210`0683 ~
is a member of a specific binding pair. That is, two dif~erent molecules
where one of the molecules through chemical or physical means
specifically binds to the second molecule. Therefore, in addition to
antigen and antibody specific binding pairs of common immunoassays,
5 other specific binding pairs can include biotin and avidin, carbohydrates
and lectins, complementary nucleotide sequences, ef~ector and receptor
molecules, cofactors and enzymes, enzyme inhibitors and enzymes, and
the like. Furthermore, specific binding pairs can include members that
are analogs of the original specific binding members, for example, an
10 analyte-analog. Immunoreactive specific binding members include
antigens, antigen fragments, antibodies and antibody fragments, both
monoclonal and polyclonal, and complexes thereof, including those
formed by recombinant DNA molecules. The term "hapten", as used
herein, refers to a partial antigen or non-protein binding member which
1 5 is capable of binding to an antibody, but which is not capable of eliciting ~ -
antibody formation unless coupled to a carrier protein. -
"Analyte," as used herein, is the substance to be detected which
may be present in the test sample. The analyte can be any substance for
20 which there exists a naturally occurring specific Wnding member (such
as, an antibody), or for which a specific binding member can be prepared. ;
Thus, an analyte is a substance that can bind to one or more specific
binding members in an assay. "Analyte" also includes any antiger~ic
substances, haptens, antibodies, and combinations thereof. A.s a member
2 5 of 8 specific binding pair, the analyte can be detected by means of
naturally occurring specific binding partners (pairs) such as the use of
intrinsic factor protein as a member of a specific binding pair for the
determination of Vitamin B12, the use of folate-binding protein to
determine folic acid, or the use of a lectin as a member of a specific
3 0 binding pair for the determination of a carbohydrate. The analyte can
include a protein, a peptide, an amino acid, a hormone, a steroid, a
vitamin, a drug including those ad~inistered for therapeutic purposes as
well as those administered for illi~it purposes, a bacterium, a virus, and
metabolites of or antibodies to any of the above substances. The details for
3 5 the preparation of such antibodies and the suitability for use as speciSc
binding members àre well known to those skilled in the art.
wo 92/15709 PCr/US92/01~72
The types of molecules and supra-molecular structures to be
assayed by this invention are those customanly measured by ligand-
ligand interactions. Macromolecular antigens most of~en have been
measured in "sandwich" immunoassay measurement~ which utilize one
5 antibody to "capture" the analyte and another antibody having a
measurable signal generating compound (measurable detectable label) to
allow detection of the reaction by determining the signal produced.
Signal generating compounds (labels) have included radioisotopes,
fluorophores, chemiluminescent compounds and enzymes, whose
1 0 presence can be determined by the conversion of appropriate substrates to
chromogens, fluorophores, or light~
Low molecular weight antigens conventionally have been detected ;
by competitive binding immunoassays such as Radioimmunoassay
15 (RIA), Fluorescence Polarization Immunoassay (FPLA), and Enzyme
Multiplied Immunoassay Technique (EMIT). Nucleic acids have been
detected by various hybridization techniques frequently following an
amplification procedure such as the Polymerase Chain Reaction (PCR) or
the Ligase Chain Reaction (LCR).
2 0
Supra-molecular structures frequently have been determined by
binding specific binding agents such as antibodies with a measurable
signal generating compound (measurable detectable label) to surface
markers and observing the measurable bound label after removal of
2 5 excess reagent. Altematively, features internal to the structure could be
stained allowing observation of the particle. Specificity in the latter case `
can be imparted by immunologically trapping the structure using specific
binding agents such as antibodies against surface markers. Thus,
supramolecular analytes such as cells, malignant cells, blood cells,
3 0 sperm, and microorganisms including bacteria, viruses, parasites,
rickettsia, fungi, mycobacterium, mycoplasma, may be analyzed! by the
method of this invention.
Test samples which may be tested by the methods of the invention
3 5 includes both liquid and solid substances of biological and non-biological -
origin. Liquid andlor solid samples include those of biological origin, for
example, human and animal body fluids such as whole blood, serum~
plasma, cerebrospinal fluid, urine, biological fluids such as cell culture
WO 92/15709 PCr/US92/01~i72
n~s3 -1~ "
supe~atants, tissue specimens and cell specimens, and the like. Tissue
specimens and cell specimens do not need to be fixed in order to be - `:
assayed by the method of the invention. Also contemplated are liquid and -
solid non-biological specimens including soil samples, water samples, oil ~-
5 samples, and the like.
A test piece comprising a solid phase may be used according to the
method of the invention. A "solid phase", as used herein, refers to any
material which is insoluble, or can be made insoluble by a ~ubsequent
1 0 reaction. The solid phase can be chosen for its intrinsic ability to attractand immobilize the capture reagent. Alternatively, the solid phase can
retain an ~dditional receptor which has the aWity to at~ract and
immobilize the capture reagent. The additional receptor can include a
charged substance that is oppositely charged with respect to the capture
1 5 reagent itself or to a charged substance cor~ugated to the capture reagent.
As yet another alternative, the receptor molecule can be any specific
binding member which is immobilized upon (attached to) the solid phase
and which has the ability to immobilize the capture reagent through a
specific binding reaction. The receptor molecule enables the indirect
2 0 binding of the capture reagent to a solid phase material before the
performance of the assay or during the performance of the assay. The
solid phase thus can be a plastic, deri~ratized plastic, metal, glass or
silicon surface of a test tube, microtiter well, sheet, bead, microparticle,
chip, and other configurations known to those of ordinary skill in the ar~.
It is contemplated and within the scope of the invention that the test
piece comprising a solid phase also can comprise any suitable porous
material. By "porous" is meant that the material is one through which
the test sample can easily pass. In the present invention, the solid phase
3 0 can include NucleoporeTM membranes (available from VWR Scientific,
Chicago, I1) and other porous surfaces such as woven nylon
polyethylene and polypropylene me4h membranes made by Spectrum
Medical Industries, Inc., Los An~eles, CA) or other porous or open pore
materials well known to those skilled in the art (e.g., polyethylene sheet
3 5 matenal). The solid phase can also comprise polymeric or glass beads,
microparticles, tubes~ sheets, plates, slides, wells, tapes, test tubes, or the
like.
WO 92/15709 PCI/US92/01572
21OOfi~3 ~
Although the details provided hereîn are focused on an
immunological procedure, the extension to other specific ligand-ligand
interactions is contemplated and within the scope of the invention. Also
contemplated and within the scope of the invention is the use of surface - ~;
5 probes other than Electron Tunnelling or Atomic Force, such as atomic
friction, scanning magnetic, scanning thermal or other scanning
micromechanical instruments, when used in a scanning mode.
The use of SPM to monitor specific binding reactions can occur in
1 0 many ways. In one embodiment of the invention, one member of a
specific binding partner (analyte specific substance) is attached to a
surface suitable for scanning. The attachment of the analyte specific
substance may be by adsorption to a test piece which comprises a solid
phase of a plastic or metal surface, following methods known to those of
1 5 ordinary skill in the art. Or, covalent attachment of a specific binding
partner (analyte specirSc substance) to a test piece which test piece
comprises a solid phase of derivatized plastic, metal, silicon, or glass ma~r
be utilized. Covalent attachment methods are known to those skilled in
the art and include a vanety of means to irreversibly link specific binding
20 partners to the test piece. If the test piece is silicon or glass, the surface
must be activated prior to attaching the specific binding partner.
Activated silane compounds such as triethoxy amino propyl silane
(available from Sigma Chemical Co., St. Louis, MO), triethoxy vinyl '
silane (Aldrich Chemical Co., Milwaukee, WI), and (3-mercapto-propyl~
2 5 trimethoxy silane (Sigma Chemical Co., St. Louis, MO) can be used to
introduce reactive groups such as amino-, vinyl, and thiol, respectively.
Such activated surfaces can be used to link the binding partner directly
(in the cases of amino or thiol) or the activated surface can be further
reacted with linkers such as glutaraldehyde, bis (succinimidyl) suberate,
3 0 SPPD 9 succi~imidyl 3-[2-pyridyldithio] propionate), SMCC (succinimidyl-
4-tN-maleimidométhYI] cyclohexane-1-carboxylate), SLAB (succiniimidyl
[4-iodoacetyl] aminobenzoate), ~nd SMPB (succinimidyl 4-[1-
maleimidophenyl] butyrate) to separate ~e binding partner from the
surface. The vinyl group can be oxidized to provide a means for covalent
3 5 attachment. It also can be used as an anchor for the polymenzation of
various polymers such as poly acrylic acid, which can provide multiple ~;
attachment points for specific binding partners. The amino surface can
be reacted with oxidized dextrans of various molecular weights to provide
WO 92/15709 PCI`/US92~01572
~t()~J683 -12-
hydrophilic linkers of dif~erent size and capacity. Examples of oxidizable
dextrans include Dextran T-40 (molecular weight 40,000 daltons),
Dextran T-110 (molecular weight 110,000 daltons), Dextran T-500 ~
(molecular weight 500,000 daltons), Dextran T-2M (molecular weight ; -;
5 2,000,000 daltons) (all of which are available from Pharmacia,
LOCATION), or Ficoll (molecular weight 70,000 daltons (available from
Sigma Chemical Co., St. Louis, MO). Also, polyelectrolyte interactions
may be used to immobilize a specific binding partner on a surface of a test
piece by using techniques and chemistries described by pending U. S.
1 0 Patent applications Serial No. 150,278, filed January 29, 1988, and Serial
No. 375,029, filed July 7, 1989, each of which enjoys common ownership
and each of which is incorporated herein by reference. The preferred
method of attachment is by covalent means.
Following attachment of a specific binding member, the surface
may be further treated with materials such as serum, proteins, or other
blocking agents to minimize non-specific binding. The surface also may
be scanned either at the site of manufacture or point of use to verify its
suitability for assay purposes. The scanning process is not anticipated to
2 0 alter the specific binding properties of the test piece.
It is preferred that the analyte to be tested be bound to a test piece.
Test pieces contemplated include surfaces of glass, plastic, metal and
silicon. Most preferably, the test piece comprises a silicon wafer. The
2 5 wafer can be of varying thickness. Therefore, silicon wafers from about
10 to about 30 mils can be used as the test piece. Preferably, the silicon
wafer will be of a thickness of about 18 to about 22 mils. 17~e silicon wafer
is sliced from a large crystal and can have an orientation of (100) or (111).
Most preferably, a silicon wafer such as described ~ be a polished,
30 monocrystalline silicon slice.
,
To perform an assay, tt~e te~-t piece to which an analyte specific
substance has been attached is contacted with a test sample for an
adequate period of time under conditions sufficient to allow the binding of
3 5 the analyte specific binding partner, if present in the test sample, to the
analyte specific substance. Upon completion of the reaction, the test piece
is scanned by scanning probe microscopy methods described herein to - -
determine the presence or absence of the analyte. A micro-sensor iæ ``
WO 92/15709 PCI/US92/01572
-13- l~l 00~83 ~
rapidly and systematically scanned across a surface in (for example) the
X direction. Upon the completion of one pass, the sensor i8 advanced in a
small direction in the Y direction, and another trace is made back in the
X direction. The response of the sensor is monitored as it is rastered (i.e.,
5 systematically scanned) across the surface. Customarily, the sensor
response is utilized in a feedback loop to control the Z position of the ~;
sensor. In this fashion, a complete map is made of a function of the
surface at various X, Y coordinates.
1 0If quantitation of the analyte is desired, the number of molecules in
a particular field is counted and, either by reference to previously
measured standards or mathematically, the concentration i~ the sample
is determined. Multiple fields on the same test piece may be examined to
extend the sensitivity and accuracy of the method. Before the sa~ple is
t 5 scanned, the test piece may be washed and dried or washed and scanned
wet. Optionally, the test piece may be scanned with the sample still in
place. The probe will sense only those molecules tightly bound at the
surface of the test piece; therefore, weakly bound molecules unll not
interfere. It can be anticipated that the probe itself will sweep the surface - -
2 0 clean of lightly bound interfering molecules.
Alternately, at the co~venience of the practitioner of the invention, ;
the sample containing the analyte may be mi~ed with a known amount of
its specific binding partner and the mixture applied to the test piece to
2 5 which the analyte or an analyte analog has been attached. Thi8 amount
of specific binding partner can be determined prior to beginning the
assay. This so-formed mixture is incubated for a time and under
conditions sufficient for a binding reaction to occur. Again, upon
completion of the reaction, the test piece is scanned as previously
3 0 described for the presence and amount of the specific binding partner.
The presence and amount of the specific binding partner bound will be
inversely proportional to the amount of analyte in the sample. As
previously stated, the sensitivity and accuracy of the measurement
process may be enhanced by scanning multiple fields of the same test -
3 5 piece.
~ .
If the analyte is a polynucleic acid such as DNA or RNA, the `;
appropriate specific binding partner is a polynucleic acid complementary -~
WO 92/1~709 PCr/US92/01572
~l no6s3 -1~
to one or more target sequences suspected of being present in the test
sample. Such methods are well known in the art. See, for example,
Falkow et al., U.S. Patent No. 4, 358,535 and Ranki et al., U.S. Patent No.
4, 486,539, both of which are incorporated herein by reference
5 Attachment of the capture sequence o~to the test piece may be achieved
covalently as, for example, described by Stabinsky, U.S. Patent No.
4,751,177, which is incorporated herein by reference Alternatively, the
capture sequence may be derivatized with a hapten, which in turn may be
bound to the surface of the test piece through an antibody or the like as
1 0 described by Snitman et al., published as International Patent Publication
WO 86/07387, which is incorporated herein by reference. The hapten
derivatized capture sequence may be mixed with the sample prior to
exposure to the test piece or it may be pre-bound to the antibody activated
test piece. The antibody may be attached as described above. Prior to its
1 5 exposure to the test piece, the target sequence may be amplified by use of
the polymerase chain reaction (PCR) or ligase chain reaction (LCR) such
as to lessen the number of fields which need to be scanned to determine
the presence or absence of analyte.
2 0 U. S. Patents No. 4,683,195 and 4,683,202 teach a method of
amplifying DNA sequences by using PCR. Both of these patents are
incorporated herein by reference. Briefly, in PCR, two complementary
polynucleotide strands are amplified by treating the strands with two
oligonucleotide primers such that an extension product of each primer is ~
2 5 synthesized which is complementary to each nucleic acid strand. The
primers are selected such that the extension product of one p~imer forms
a template fior the synthesis of an extension product from the other
primer once the extension product of the one primer is separated from the
template. A chain reaction is maintained by a cycle of denaturing the
3 0 primer extension products from their templates, treating the single- `
stranded molecule generated with the same primers to re-annea~, and ~
allowing the primers to form further extension products. The cycle is ~ `
repeated for any many times as it takes to increase the target nucleic acid
segments to a concentration where they can be detected. The amplified
3 5 target sequence can be detected by denaturing the double-stranded `-
products formed by PCR, and treating those products with one or more `~
reporter probes which hybridîze with the extension products. -
WO92/15709 ~ 3 Pcr/US92/01572
The Ligase Chain Reaction (LCR) amplifies sections of pol~nucleic
acid by copying the section of polynucleic acid, and copying the copies of
that section of polynucleic acid, many times over. This method is
described in European Patent Application No. 0 320 308 published June
14, 1989, which is inco~porated herein by reference. In this procedure,
two probes (for example, A and B) complementaly to immediately
adjacent regions of a target sequence are hyb~idized and ligated. This
ligated probe then is denatured away from the target, after which it is
hybridized with two additional probes (A' and B') of sense opposite to the
1 0 ;nitial probes A and B. The secondary probes are themselves then ligated.
Subsequent cycles of denaturation/hybridization/ligation create the
formation of double-length probes of both sense (+) and antisense (-).
:
In LCR, the nucleic acid of the sample is provided either as single
1 5 stranded or as double-stranded polynucleic acid which is denatured to
separate the strands. Four probes are utilized: the first two probes (A ~ -
and B) are the so-called primary probes, and the second two probes (A'
and B') are the so-called secondary probes. The first probe (A) is a single
strand capable of hybridizing to a first segment of the primary strand of
2 C the target nucleol~ide sequence. The second probe (b) is capable of
hybridizing to a second segment of the primaly strand of the target
nucleotide sequence. The 5' end of the first segment of the primary
strand of the target is positioned relati~e to the 3' end of the second
segment of the primary strand of the target to enable joi~ing of the 3' end
2 5 of the first probe to the 5' end of the second probe, when the probes are
hybridized to the primary strand of the target nucleotide sequence. The
third probe (A') is capable of hybridizing to the first probe, and the fourtb
probe (B') is capable of hybridizing to the second probe (B). The hybridized
probes are ligated to form reorganized fused probe sequences. Then, the
3 0 polynucleic acid in the sample is denatured to separate ligated probes ;
from sample polynucleic acid. Successive cycles wherein the ligated
probes and target polynucleic àcid undergo the above-described process
are performed to increase the amount of detectable pol~nucleic acid in the
sample. The amount of cycles performed is dependent upon the sequence
3 5 used and the sensitivity required of the test. Usually, tbe cycle can be
repeated from 15 to 60 times.
',
' - .
WO 92/15709 ~ ~ PCr/US92J01572
) b ~
The sensitivity of SPM immunoaSsays may be enhanced by use of
standard immunological me1;hods appropriately adapted to the type of
probe used. For example, if the antigen captured by ~pecific antibody
binding has an epitope available for binding a second antibody, a second
5 antibody labelled with materials such as colloidal metals, colloidal non-
metals, small latex particles, charged polymers, liposomes, fixed cells,
and the like can be used for enhanced detection of the analyte. For
competitive assays when the analyte or an analog is attached to the
surface, the specific binding ligand used in limlted amounts may be
1 0 appropriately labelled. For polynucleic acid analytes, in addition to the
complementary capture sequence, a complementary, non-overlapping,
label sequence can be used. In addition to the labels mentioned above,
long sequences of double stranded DNA, linear or circular, may be used
to enhance the sensitivity of the method.
1 5
By proper selection of the specific binding partner, a high degree of
specificity can be imparted to a Scanning Probe Microscope Assay. That
is, if an antibody, monoclonal or polyclonal, with very low cross-reactivity,
is used to bind analyte to the surface of the test piece, it is anticipated that2 0 by proper combin~tion of washings and probe manipulations a high
degree of specificity will be exhibited by the assay. That is, only the
desired analyte will be specifically immobilized on the surface of the test
piece and so the detection of any material bound incremental to the
specific binding partner originally attached to the surface will constitute
2 5 a positive binding event. Additional specificity can be built into the assayif an addit;onal requirement for a binding eve~t to be scored as a positive
result is used. Since the scanning probe microscope affords information
on the size, shape and other charactelistics can be used to provide ;~
additional specificity requirements for the assay result to be regarded as `
3 0 positive. For example, in an assay for Carcino-Embryonic Antigen
(CEA), if material from the sample which is bound to the test piece does
not conform to the established parameters of the CEA molecule (a multi~
domain, heavily glycosylated molecule of appro~mately 180,000 daltons),
then the test result will be scored as negative, thus avoiding a "false
3 5 positive" result. Methods snd algorithms for automatically analyzing
images with regard to shape and size are well known in the field of image `
analysis. Neural network methods may be advantageous for the
identification of analytes on the basis of their size and shape in -
WO 92/15709 PCr/US92/01572
-17~ 3
comparison to known samples. Scanning Probe Microscopy
Immunoassay (SPMIA) of~ers both necessary (binding to surface) and
suf~icient (size, shape, other characteristics) conditions to make the
assays very resistant to erroneous results.
In the case of polynucleic acid assays, i.e., for DNA or RNA,
stringency conditions will be used which limit the non-specific binding of
target to capture probe. Commonly used st2ingen~y co~ditions include -
high temperature, denaturing surfactants, organic ~olvents, high ionic
1 0 strength buf~ers and the like. If a capture probe is used, it must be of
sufficient length to allow efficient binding under conditions of high
stringency. It is anticipated that there will be limited information
available from the size and shape of the captured target sequence relevant
to establishing sufficiency conditions to allow additional specificity in the
1 5 assay. However, as pointed out by Driscoll et al. (sub~ra), SPM allows
atomic resolution of double stranded nucleic acids, thereby allowing
verification of the "fit" of the target to capture sequence as ~ell as
confirmation that the sequence is that which is expected .
2 0 Further, it is contemplated that means of accelerating the binding
reaction will provide shorter assay time. Temperature changes wherein
the temperature used to af~ix the analyte specific substance to the test
piece, as well as, the use of polyethylene glycol, ultrasonic exposure and
other means, each may contribute either alone or in combination to the
2 5 acceleration of the reaction. Reactions of macromolecular arld ~
supramolecular analytes can be accelerated by conducting the reaction ;
in an ultracentrifuge wherein the analyte is sedimented onto the surface
of the test piece at high gravitational force. Non-bolLnd material can be ; -
removed by washing, and the specifically bolmd material analyzed by
3 0 SPM. These accelerating means are contQmplated to be within the scope
of the present invention. ~-
If quantitation of the an~lyte is desired, the number of molecules in
a particular field is counted and, either by reference to pr~iously
3 5 measured standards or mathematically, the concentration in the sample '~
is dete~nined. Thus, if a reaction is at equilibrium with bigh affinity
binders on the surface, it is possible to calculate the number of antigens -
which on average wil~be in each field. For example, if a 50 ~L sample of
WO 92/15709 P~/US92/01572
. ! t n n fi 8 3 --18
Hepatitis B Surface Antigen at 1 ng/mL is applied to a 8 mm x 8 mm test
piece, there w~ll be appro~cimately 15 x 10 6 particles (~V= 2 x 106 daltons)
per test piece. On average, there will be 0.23 particle~ per 1~ x 1 ,u field.
By counting multiple fields and applying known statistical methods such
5 as the binomial distribution, it is possible to accurately determine the
concentration of analyte. It is contemplated that algonthms can provide
a means for counting objects in the field, thereby automating quantita1ion -
of the number of objects in the field.
1 0 The present invention will now be described by way of Examples,
which are meant to illustrate, but not to limit, the spirit and scope of the
in~rention.
EXAMPLES
Examplè 1
~eparation of ~mine Activated Test Pie~es
Polished monocrystalline silicon slices 0.5 mm thick (100)
orientation and n type (obtained from Unisil Corporation) were diced into
8 mm squares. The test pieces were immersed in a 10æ (v/v) solution of
2 0 triethoxy amino propyl silane (Sigma Chemical Co., St. Louis, MO) in
water for 15 minutes at room temperature. The pieces then were washed
with a stream of distilled water from a laboratory squeeze bottle for ~ `
appro2imately five (5) seconds, and blown dry unth a nitrogen stream.
The drying was accomplished by gripping a test piece with tweezers at
2 5 one corner and then directing a gentle stream of nitrogen across the
surface of the test piece aimed toward the tweezers. Drying was
continued until all visible drops of water were removed from the sur~ace
which then exhibited a shiny, highly reflective surface with no visible
residue.
Example 2
Coating of Test Pieces With Antibodv
A solution of murine mo~oclonal antibody directed against
Hepatitis B Surface Antigen (anti-HBsAg) (IgG1 subtype) (available from
3 5 Abbott Laboratories, Abbott Park, IL in the commercially availabl¢ kit
AUZYME~ MONO)~ was prepared in 2-tN-mo2pholino] ethsne sulfonic
acid (MES) (Sigma Ghemical Co., St. Louis, MO) buffer (50mM, pH 7.0) at
aconcentratîonof40~g/mL. Asolutionofl-ethyl-3-(3-
WO 92/15709 PCI/US92/01572 ~
~-1Q0~83
dimethylaminopropryl) carbodiimide (EDAC) (Aldlich Chemical Co., -~
Milwaukee, WI) also was prepared in 50 mM ~ES, pH 7.0, at a
concentration of 500 ~Iglm~., Equal volumes of the two reagents then were
mixed to form a solution and immediately, 50 ~L of the solution were
5 applied to each silicon wafer, which had been activated following t~e
procedure described in Example 1. The samples were allowed to react at '
room temperature for about 15 minutes and then were rinsed with ' "'
distilled water and blown dry with a nitrogen stream as described in
Example 1. The dry, highly reflective surface showed no ~isible residue.
1 0
Example 3
Reaction With rHBsA~
Test pieces with anti-HBsAg covalently attached and prepared as -:
described in Example 2, were covered with 50 llL of a solution containing
1 5 200 ~lgimL of recombinant Hepatitis B Surface Antigen subtype Ay
(rHBsAg) (available from Abbott Laboratories, Abbott Park, IL in the ~'
commercially available l~it Abbott IMx~E9 AUSAB~)) in 50 mM MES buffer
pH 7.0, and allowed to react for approximately 20 minutes at room
temperature. After reaction, the test pieces were washed with distilled
2 0 water and dried with a stream of nitrogen gas as described in Example 1. " ''
Again, the dry, highly reflective surface appeared ~ree of any visible '~
residue. '
Example ~ ;
2 5 ElliP~ometric Examination of Test Pieces '"
A test piece carried through the sequences described in Examples 1
and 2 was measured in a Rudolph Ellipsometer (model Auto EL, available "~
from Rudolph Research, Flanders, NJ). Using a refractive index of 1.46, '"'
a film thickness of 37.7 Angstroms was obtained. Then, a test piece
3 0 carried through the sequences described in Examples l, 2, and 3 was - '
measured in the Rudolph Ellipsometer.' Again using a refracti~e index of
1.46, a thickness of 65.1 Angstroms was obtained. The thickness increase '' '
of 27.4 Angstrnms was due to binding of the rHBsAg to the antibody coated ~'
surface of the test piece.
;'
wo 9~/15709Pcr/uss2/ol572
-20-
2~ no683
Example 5
Atomic Force Microscopy Examination of Test Pieces
An Atomic Force Microscope, built by TopoMetrix (Pasadena, CA)
was used to scan the surfaces of the various test pieces produced
5 according to Examples 1, 2 and 3 detailed hereinbelow in points a through
d, as follows. A force of approximately 6 nN (nanoNewtons) was applied
by the sensor which scanned across the surface at 500 nm/s (nanometers
per second) in the X direction. Upon the completion of one pass, the
sensor was advanced in a small direction in the Y direction, and another
1 0 trace was made back in the X direction. The response of the sensor was
monitored as it was rastered (i.e., systematically scanned) across the
surface. The sensor response was utilized in a feedback loop to control
the Z position of the sensor. In this fashion, a complete map was made of
a function of the surface at various X, Y coordinates. Two hundred or
15 four hundred line images were obtained.
.,
a. A bare silicon test piece, which contained no antibody on it, was -~
scanned. The surface appeared smooth and featureles~.
. . ~ . .
b. A test piece prepared as described in Example 1 was scanned.
FIG. lA is an SPM image of the surface of .,he activated silicon test piece.
Referring to FIG. lA, the image shows a smooth, featureless surface. ,
Three different lines (1, 2 and 33 were traced across the X, Y plane.
Referring to FIG. lB, which traces the apparent elevation of the probe
2 5 above the surface across the X, Y plane at three different lines (1, 2 and 3)
, the traces approximate a smooth catenary. The app~rent increase in
thickness at the edges of the trace are thought to be artifact.
c. A test piece prepared according to Example 2 was scanned. -
3 0 FIG.2A is an SPM image of the silicon test piece coated with an analyte ~ `~
specific substance (antibo~y). Referring to FIG. 2A, the image shows ~a
smooth, essentially featureless surface. Three dif~erent lines (1, 2 and 3)
were traced across the X, Y plane. FIG. 2B, which traces ~he apparent
elevation of the probe across the X, Y plane at three dif~erent lines (1, 2
3 5 and 3) above the surface, the traces approximate a smooth catenary. The ~ -
apparent increase in~thick~ess at the edges of the traces are thought to be `~;
artifact. ~
WO 92/1~709 PCI`/US9Z/01~72
-21~ lflO683
d. A test piece prepared according to Example 3 was scanned and
multiple fields were examined. FIG. 3A is an SPM image of a test piece
- coated w~th analyte-specific substance (antibody directed against
- Hepatitis B Surface Antigen [anti-HBsAg3) reacted with analyte
(recombinant Hepatitis B Surface Antigen [rHBsAg~). Referring to FIG.
3A, the figure shows multiple antigen particles immobilized on the
surface of the test piece. One major cluster of particles i~ ~een
approximately in the center with other particles in contact with another
distributed around the image. Three different lines (1, 2 and 3) were
1 0 traced across the X, Y plane. FIG. 3B gives three traces across the field.
The upper trace (1) shows essentially constant elevation above the
surface, while traces 2 and 3 traverse the large grouping of antigen
particles. The artifact at the edges of the trace are still present. FIG. 4,
which provides a different projection of a field, shows the image of
1 5 multiple globular objects, crowded together, others presenting as isolated
hemispheres when not in contact with other objects.
Thus, the method of the invention described herein can be used to
assay for analytes which may be present in a test sample~ It will be
2 0 appreciated by those skilled in the art that many of the concepts of the ~;
present invention are equally applicable to other types of binding assays.
The embodiments described and presented herein are intended as
examples rather than as limitations. Thus, the description of the
invention is not intended to limit the invention to the particular
2 5 embodiments disclosed, but it is intended to encompass all equi~ralents
and subject matter within the spirit and scope of the invention as
described and contemplated above, and as set forth in the following ;~
claims.
