Connect public, paid and private patent data with Google Patents Public Datasets

A method for detecting and quantifying analytes by means of scanning force microscopy

Info

Publication number
WO1996031775A1
WO1996031775A1 PCT/SE1996/000431 SE9600431W WO1996031775A1 WO 1996031775 A1 WO1996031775 A1 WO 1996031775A1 SE 9600431 W SE9600431 W SE 9600431W WO 1996031775 A1 WO1996031775 A1 WO 1996031775A1
Authority
WO
Grant status
Application
Patent type
Prior art keywords
surface
hsa
support
method
mica
Prior art date
Application number
PCT/SE1996/000431
Other languages
French (fr)
Inventor
Sven Oscarsson
Anna Bergman
Arjan Quist
Curt T. Reimann
Bo Sundqvist
Original Assignee
Sven Oscarsson
Anna Bergman
Arjan Quist
Reimann Curt T
Bo Sundqvist
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date

Links

Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01QSCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
    • G01Q60/00Particular types of SPM [Scanning Probe Microscopy] or microscopes; Essential components thereof
    • G01Q60/24AFM [Atomic Force Microscopy] or apparatus therefor, e.g. AFM probes
    • G01Q60/32AC mode
    • G01Q60/34Tapping mode
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y35/00Methods or apparatus for measurement or analysis of nanostructures
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay
    • G01N33/543Immunoassay; Biospecific binding assay with an insoluble carrier for immobilising immunochemicals

Abstract

Analytes are detected and quantified without the use of labels. A solution containing a substance capable of forming an immunocomplex is brought into contact with a support surface on which the other component of the immunocomplex to be formed is immobilized and the formed complex is detected and distinguished from substances which do not form part of the immunocomplex by means of scanning force microscopy.

Description

A method for detecting and quantifying analytes by means of scanning force microscopy

The present invention relates to a method for detecting and optionally quantifying analytes which are capable of forming immunocomplexes. More particularly the invention relates to such a method wherein no labelling of substances is used and wherein the immunocomplexes are formed on a sup¬ port surface and then detected and optionally quantified by means of scanning force microscopy. The most sensitive analytical techniques available today for detecting immunoassay complexes are radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) . Both of these techniques are used for example for testing for drug abuse. The RIA-test is very sensitive but has the disadvan- tage that a radioactive labelling must be used. In ELISA testing procedures detection is made by a simple colorimetric assay. The ELISA procedure is, however, usually less sensi¬ tive than the RIA procedure.

Scanning force microscopy (SFM) for three-dimensional imaging of macromolecules was introduced in 1986 and has since then found increasing interest and use in the investi¬ gation of biomolecules. The two modes of SFM, the contact mode (CM) and the more gentle tapping mode (TM) , have for example been used for investigation of the coiling of DNA, the structure of human serum albumin etc, adsorbed on support surfaces.

According to the present invention it has been found that it is possible to detect and optionally quantify analyte mo¬ lecules which are capable of forming immunocomplexes by form- ing the complexes on a support surface and then detecting and quantifying the formed immunocomplexes by means of SFM.

As mentioned above the RIA-procedure requires labelling with radioactive substances and in other biotechnical pro¬ cesses labelling of substances with e.g. gold or fluorescent compounds for detection is often used. The present invention is especially advantageous in that it provides an ultrasensi¬ tive technique for detecting immunocomplexes in which no labelling of substances is required. Extremely small amounts of substances can be detected, and even single analyte mole- cules .

The invention thus relates to a method as defined in the appended claims.

The present method is based on the finding that it is possible to detect and quantify analytes which are capable of forming immunocomplexes using SFM but without use of any special labelling for the detection.

The term "analyte" as used herein is intended to encompass the antigen or the hapten which is to be detected. The term "immunocomplex" as used herein is intended to encompass complexes formed by an antigen and an antibody or by a hapten and an antibody. Haptens are low molecular weight substances which can react with the effector cells of the immune response (humoral antibodies or stimulated T-lympho- cytes) . As some examples of haptens can be mentioned drugs and drug metabolites, hormones etc.

In the present method the immunocomplexes are formed by bringing a solution containing a substance capable of forming an immunocomplex into contact with a support surface on which the other component of the immunocomplex to be formed is im¬ mobilized. Hereby either analytes or antibodies against an analyte can be immobilized on the surface. In the first case, when analytes are immobilized, the solution which is brought into contact with the support surface thus contains anti- bodies. Usually, and preferably, antibodies will be immobi¬ lized on the support surface and a solution containing the analyte brought into contact with this. The solution can for example be a body fluid which is to be analyzed for the pres¬ ence of medicaments or drug abuse. Other instances wherein the present method can be used are for example in analysis of waste fluids, for example from industrial processes, which are to be investigated with regard to toxic substances such as phenol, styrene, acrylic acid, thiazoles etc.

One way of quantifying the analytes is to let the surface with the immobilized antibody directed to the analyte grad¬ ually pass through the sample. Depending on the amount of analyte in the liquid sample immunocomplexes will be formed over varying areas of the support surface or along different distances on this, which can be used for quantitative diag- nosis of the analyte.

The surface on which the detection is made is preferably organised in such a manner that it can accommodate a maximum number of immunocomplexes and this can for example be carried out by means of a multipoint applicator on a nanometer scale or by masking the surface to allow maximum number of com¬ plexes to be adsorbed on or bound to the surface.

When a sample is to be analyzed the solution containing the analyte is brought into contact with the surface, on which the other component of the immunocomplex to be formed is immobilised, in such a manner as to wet the surface. It might often be necessary to use very highly diluted samples. The actual operation of the scanning force microscopy in tapping mode can be carried out in air or a liquid cell can be used. In the latter operation manner the tip of the in¬ strument is inserted in the liquid cell which is then placed on the sample with a sealing O-ring between the cell and the sample substrate. As in the tapping mode in air the sample is mounted on top of the scanner. Since the fluid medium tends to damp the cantilevers normal resonant frequency, the entire liquid cell can be oscillated to drive the cantilever into oscillation. Further operation is similar to the operation of the tapping mode in air, which is well known to the man skilled in the art. The liquid cell is equipped with an inlet- and outlet tube which makes it possible to inject different solutions while operating the microscope.

For SFM-techniques it is important that the solid support surface is extremely smooth and plane to prevent the surface from interfering with the complexes to be analyzed. The support surface can be organic or inorganic. As examples of organic support surfaces can be mentioned teflon® and po¬ lystyrene. Usually inorganic supports are used. A support surface of glass can be used but usually smoother flat supp¬ ort surfaces of mica, graphite, gypsum, polished silicon, silicon wafers, or various forms of crystals which form an atomically flat surface are employed. Mica is a very suitable support surface. It has a surface roughness of « lA (Ang¬ strom) and a new clean surface can be exposed simply be peel¬ ing off a few layers of mica for example with Scotch tape. Generally speaking the surfaces used as supports in SFM-tech- niques have to be specially treated, for example by extremely careful washing, rinsing, drying etc, and often by chemical modification, in order that the desired imaging will be obtained on a level that allows separation of molecules, agg¬ regates, complexes etc.

It is possible to utilize the present method with support surfaces as above without derivatization, i.e. chemical modi¬ fication, whereby haptens, antigens or antibodies will be immobilized on the surface through adsorption. It is, how¬ ever, preferred that the support surfaces are derivatized so that a covalent bonding is obtained and thus a higher relia¬ bility in the detection and quantifying. It is likewise pre¬ ferred to use the tapping mode SFM for a higher reliability since there is a risk that the contact mode SFM will push away compounds and complexes, particularly of higher molecu¬ lar weight, from the support surface.

The support surfaces can be derivatized by per se known reagents as used for example for inorganic support materials for chromatography. A common method is silanization. The gen¬ eral structure of the silane reagent for covalent immobiliza¬ tion of proteins on inorganic surfaces is

Y X— Si - (CH2)n - R Y wherein

X and Y = Cl or X = Cl and Y = H; or

X and Y = alkyl or alkoxy groups with 1 or 2 carbon atoms; n = 1 - 8, preferably 3 and R is a functional group to which the naturally reactive groups of proteins can bind, such as amino or thiol groups of the protein or thiol groups introduced into the protein. Suitable functional groups R are -CH=CH2, an epoxy group, NH2, SH, pyridine or S-pyridine. Commercially available si- lanes can usually be chemically modified to change the func¬ tional group R to the in each case appropriate functional group, if required. Several protocols for silanization are known from the literature.

The reagent for derivatization of the surface can also be a hydrocarbon compound corresponding to the above given for¬ mula, ie of the same formula but with a carbon atom instead of the silicon atom. Another way of modifying the support surface is by covalent binding of organic polymers to the surface. Thus examples of immobilization of proteins via po¬ lymers such as polyethylene glycol and dextran are known from the literature. These known methods must usually be optimized and modified for use with SFM.

As mentioned above several protocols for silanization are known and also for other types of derivatization. Derivatiza¬ tion of mica can for example be carried out in liquid or in vapours. In one liquid process a piece of mica is peeled on both sides and put in a freshly prepared solution of the derivatizing reagent in a suitable solvent, such as for example toluene. The modified mica is then rinsed with the same solvent and dried, for example using a flow of nitrogen. Methods for derivatization in vapours for different reagents have been described. A piece of mica can for example be peeled on both sides and placed in a desiccator which also contains a small amount of the reagent and the desiccator is then placed under vacuum for a certain time. Example 1

In this experiment the formation of biocomplexes, or the lack thereof, between single human serum albumin molecules (HSA) and single or multiple antibodies, rabbit anti-human serum albumin (a-HSA) , and human immunoglobulin G (IgG) adsorbed on mica surfaces was investigated. SFM probing and studies were made with TM-SFM (Nanoscope III®, Digital Inst¬ ruments Inc., Santa Barbara, CA, USA) using tips with an end- radius of about 10 n .

HSA, a-HSA and IgG were dissolved in tris buffer (pH 7.4) at concentrations of 2.5 μg/ml, 13.4 μg/ l and 0.7 μg/ l re¬ spectively. The concentrations were selected to give roughly the same area density of molecules (40-60 per μm2) adsorbed on the surface. In each given exposure a 50 μl volume of the protein solution was placed on a freshly-cleaved mica surface (Muscovite green mica from Asheville-Schoonmaker Mica Co. Newport News VA, USA) . The solution was spread out over app¬ roximately 1 cm2, allowed to remain on the surface for 5 min- utes and subsequently rinsed away with 1 ml of tris buffer. The surface was then dried using a flow of nitrogen and probed with TM-SFM.

To study the antigen-antibody interaction the mica sur- faces were first exposed to one protein solution, rinsed with 1 ml tris buffer, dried and studied by TM-SFM. The surfaces were then exposed to the second protein solution (without recleaving) , rinsed, dried and studied again by TM-SFM. Each TM-SFM image covered an area of 0.5 μm x 0.5 μm. The height histograms drawn on basis of the different TM-SFM images showed large differences which made it possible to quantita¬ tively distinguish between different species on the surfaces. This is shown in the Table below which gives peak positions and full widths at half maximum (FWHM) . Table

Experiment 1st Peak pos. 2nd Peak pos.

± FWHM fnm ± FWHM (nm)

A HSA 0.62±0.28 B a-HSA 1.91±0.66

C IgG 1.75±0.60

D HSA exposed to IgG wθ.62 «1.75

E HSA exposed to a-HSA «0.62 3.0311.60

F IgG exposed to HSA «0.62 1.35±0.72 G a-HSA exposed to HSA «0.62 1.38«0.80

* Note: In experiments E, F and G the HSA contribution has been subtracted for the 2nd peak.

The following remarks are given to further clarify the results given in the Table above.

Although the height information obtained by TM-SFM is not totally topological, the height histogram showed large diff¬ erences which allowed quantitative distinction between the presence of different species on the surfaces. In experiment D, wherein HSA preadsorbed on the mica surface was exposed to IgG two distinct populations of molecules, within height ranges corresponding to those of separately adsorbed HSA and IgG were observed, and no interaction between HSA and human IgG could be observed. In experiment E, wherein HSA on mica was exposed to a-HSA, larger features, both with regard to lateral dimensions and height, were observed than with either HSA or a-HSA absorbed separately on mica. The image showed the expected complexing, since the a-HSA is raised in rabbit to specifically interact with HSA. In the results shown for experiment E the HSA contribution for the 2nd peak has been subtracted.

The experiments further showed that HSA binds sponta¬ neously and irreversibly to hydrophilic mica surfaces which were not derivatized, even though the net charge of HSA as well as the surface charge are negative. IgG did, however, bind more weakly to the mica and would thus preferably be immobilized on a derivatized surface.

Claims

Claims 1. A method for label-free detecting and optionally quantifying of analytes, characterized in that a solution containing a substance capable of forming an immunocomplex is brought into contact with a support surface on which the other component of the immunocomplex to be formed is im¬ mobilized and that the formed complex is detected and distin¬ guished from substances which do not form part of the immuno¬ complex by means of scanning force microscopy.
2. A method according to claim 1, characterized in that the two components forming the immunocomplex are antibodies and antigens.
3. A method according to claim 1 or 2, characterized in that the solution contains the substance to be detected and that this an antigen.
4. A method according to claim 1, characterized in that the solution contains the substance to be detected and that this a low molecular drug or a hormone.
5. A method according to any of the preceding claims, characterized in that the support surface is of glass, mica or polished silicon.
6. A method according to claim 5, characterized in that the support surface is chemically modified for immobilization by covalent binding.
7. A method according to claim 6, characterized in that the support surface is silanized.
8. A method according to any of the preceding claims, characterized in that tapping mode scanning force microscopy is used for the detection.
9. A method according to any of the preceding claims, characterized in that the scanning force microscope is oper¬ ated in air.
PCT/SE1996/000431 1995-04-04 1996-04-02 A method for detecting and quantifying analytes by means of scanning force microscopy WO1996031775A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
SE9501236-5 1995-04-04
SE9501236 1995-04-04

Publications (1)

Publication Number Publication Date
WO1996031775A1 true true WO1996031775A1 (en) 1996-10-10

Family

ID=20397833

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE1996/000431 WO1996031775A1 (en) 1995-04-04 1996-04-02 A method for detecting and quantifying analytes by means of scanning force microscopy

Country Status (1)

Country Link
WO (1) WO1996031775A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6716578B1 (en) 1999-03-08 2004-04-06 Bioforce Nanosciences, Inc. Method for solid state genome analysis
US6838292B1 (en) * 1999-04-19 2005-01-04 Alion Science And Technology Corporation Detection of biological warfare agents

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5156810A (en) * 1989-06-15 1992-10-20 Biocircuits Corporation Biosensors employing electrical, optical and mechanical signals
US5289004A (en) * 1990-03-27 1994-02-22 Olympus Optical Co., Ltd. Scanning probe microscope having cantilever and detecting sample characteristics by means of reflected sample examination light
US5322769A (en) * 1988-03-11 1994-06-21 Abbott Laboratories Methods for using CKS fusion proteins
US5363697A (en) * 1991-04-30 1994-11-15 Matsushita Electric Industrial Co., Ltd. Scanning probe microscope, molecular processing method using the scanning probe microscope and DNA base arrangement detecting method
US5372930A (en) * 1992-09-16 1994-12-13 The United States Of America As Represented By The Secretary Of The Navy Sensor for ultra-low concentration molecular recognition
US5412980A (en) * 1992-08-07 1995-05-09 Digital Instruments, Inc. Tapping atomic force microscope

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5322769A (en) * 1988-03-11 1994-06-21 Abbott Laboratories Methods for using CKS fusion proteins
US5156810A (en) * 1989-06-15 1992-10-20 Biocircuits Corporation Biosensors employing electrical, optical and mechanical signals
US5289004A (en) * 1990-03-27 1994-02-22 Olympus Optical Co., Ltd. Scanning probe microscope having cantilever and detecting sample characteristics by means of reflected sample examination light
US5363697A (en) * 1991-04-30 1994-11-15 Matsushita Electric Industrial Co., Ltd. Scanning probe microscope, molecular processing method using the scanning probe microscope and DNA base arrangement detecting method
US5412980A (en) * 1992-08-07 1995-05-09 Digital Instruments, Inc. Tapping atomic force microscope
US5372930A (en) * 1992-09-16 1994-12-13 The United States Of America As Represented By The Secretary Of The Navy Sensor for ultra-low concentration molecular recognition

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DIALOG INFORMATION SERVICES, File 73, EMBASE, Dialog Accession No. 9307864, Embase Accession No. 94262429, WARKENTIN P. et al., "Differential Surface Binding of Albumin, Immunoglobulin G and Fibrinogen"; & BIOMATERIALS, (UNITED KINGDOM), 1994, 15/10, (786-795). *
NATIONAL LIBRARY OF MEDICINE, File MEDLINE, Medline Accession No. 94146259, KARRASCH S. et al., "Covalent Binding of Biological Samples to Solid Supports for Scanning Probe Microscopy in Buffer Solution"; & BIOPHYS. J., Dec. 1993, 65(6), 2437-46. *
NUCLEIC ACIDS RESEARCH, Volume 22, No. 16, 1994, LIA I. PIETRASANTA et al., "Probing Specific Molecular Conformations with the Scanning Force Microscope. Complexes of Plasmid DNA and Anti-Z-DNA Antibodies", pages 3288-3292. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 92, February 1995, (USA), SANDOR DAMJANOVICH et al., "Structural Hierarchy in the Clustering of HLA Class I Molecules in the Plasma Membrane of Human Lymphoblastoid Cells", pages 1122-1126. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6716578B1 (en) 1999-03-08 2004-04-06 Bioforce Nanosciences, Inc. Method for solid state genome analysis
US6838292B1 (en) * 1999-04-19 2005-01-04 Alion Science And Technology Corporation Detection of biological warfare agents

Similar Documents

Publication Publication Date Title
Soukka et al. Supersensitive time-resolved immunofluorometric assay of free prostate-specific antigen with nanoparticle label technology
US4822566A (en) Optimized capacitive sensor for chemical analysis and measurement
US6498010B1 (en) Method for making a device for the simultaneous detection of multiple analytes
US5578577A (en) Method for storing labile proteins
US5372930A (en) Sensor for ultra-low concentration molecular recognition
US5879881A (en) Solid phase system for use in ligand-receptor assays
US20040115709A1 (en) Methods and devices for active bioassay
US5677133A (en) Dry chemistry cascade immunoassay and affinity assay
EP0312394A2 (en) Membrane-supported immunoassays
US5686315A (en) Assay device for one step detection of analyte
US6294342B1 (en) Magnetically assisted binding assays utilizing a magnetically responsive reagent
EP0200381A1 (en) A solid phase system for use in ligand-receptor assays
US5521102A (en) Controlled sensitivity immunochromatographic assay
US5569608A (en) Quantitative detection of analytes on immunochromatographic strips
US5474902A (en) Semi-permeable capillary assay device
US5998224A (en) Magnetically assisted binding assays utilizing a magnetically responsive reagent
US5565365A (en) Assay flow apparatus and method
US5405752A (en) Enzyme conjugate prepared with insoluble nonoparticle
US5081013A (en) Immunodiagnostic device and method
US6630309B2 (en) Determination of an analyte in a liquid medium
US6342396B1 (en) Method for isolating, in particular for detecting or quantifying an analyte in a medium
US6509196B1 (en) Compensation for non-specific signals in quantitative immunoassays
US4436826A (en) Tagged immunoassay
EP0874242A1 (en) Device and apparatus for the simultaneous detection of multiple analytes
US20060177873A1 (en) Method of adjusting the working range of a multi-analyte assay

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: CA