EP1664736A1 - Detection de nanoparticules - Google Patents

Detection de nanoparticules

Info

Publication number
EP1664736A1
EP1664736A1 EP04765113A EP04765113A EP1664736A1 EP 1664736 A1 EP1664736 A1 EP 1664736A1 EP 04765113 A EP04765113 A EP 04765113A EP 04765113 A EP04765113 A EP 04765113A EP 1664736 A1 EP1664736 A1 EP 1664736A1
Authority
EP
European Patent Office
Prior art keywords
nanoparticles
sensor
peptides
biomolecules
signal
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP04765113A
Other languages
German (de)
English (en)
Inventor
Daniel Hoffmann
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Stiftung Caesar Center of Advanced European Studies and Research
Original Assignee
Stiftung Caesar Center of Advanced European Studies and Research
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
Application filed by Stiftung Caesar Center of Advanced European Studies and Research filed Critical Stiftung Caesar Center of Advanced European Studies and Research
Publication of EP1664736A1 publication Critical patent/EP1664736A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N15/0606—Investigating concentration of particle suspensions by collecting particles on a support
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons
    • G01N21/554—Attenuated total reflection and using surface plasmons detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance

Definitions

  • the invention relates to a method for the detection of particles in aqueous solution, in particular inorganic particles or particles which, like soot, cannot be termed “biological” and whose size is in the range of nanometers (nanoparticles), the detection not by means of a specific binding to proteins and
  • the invention also relates to a device for carrying out the method.
  • nanotechnology is a young field, it represents a dynamically developing scientific and economic branch.
  • the main focus is the controlled production of structures on the length scale of a few nanometers, with inorganic nanoparticles often being used as building blocks.
  • These nanoparticles are inorganic and other “non-biological” particles, ie nanoparticles that do not have any biological macromolecules.
  • nanotechnology it is still a major problem to quantitatively detect the nanoparticles that are produced Methods such as transmission electron microscopy to detect nanoparticles on a laboratory scale, however, there is no known inexpensive method with which the occurrence of nanoparticles can be monitored on a larger scale.
  • BESTATIGUNGSKOPIE It is therefore an object of the present invention to provide a method for the detection of such nanoparticles, which are not to be referred to as biological, which can be implemented with simple means and which allows fast and reliable measurements of the concentration of the nanoparticles. It is also an object of the invention to provide a device for implementing the method.
  • biomolecules in particular peptides
  • detection of these nanoparticles takes place through the binding of the nanoparticles to the peptides on a sensor and the computational evaluation of the sensor signal with a view to determining the quantity and size distribution of the nanoparticles Detection does not take place by means of a specific binding to proteins and / or nucleic acid in the nanoparticles.
  • the areas of application for the invention are diverse: for example, it can be assumed that nanoparticles are taken up by humans and then, similar to what is suspected for soot, trigger harmful biochemical and biological processes.
  • the extent of the biological effect depends on the amount and type of particles.
  • suitable protective measures such as the elimination of leaks
  • these two variables should be monitored in the vicinity of potential sources of nanoparticles, which enables the invention.
  • the monitoring of nanoparticles in aqueous solution is particularly interesting, which corresponds to a simple model of the situation on mucous membranes.
  • the mucous membranes form a biological structure through which nanoparticles can be absorbed into the body.
  • detection for quality control purposes in production is also of economic interest. For example, it may be desirable to produce certain size monodisperse nanoparticles. A measurement of the size distribution can then be used to control particle production.
  • peptides for predetermined nanoparticles are selected using methods of directed evolution, for example the “phage display”, which bind these nanoparticles firmly and specifically.
  • the peptides are then immobilized on the surface of a sensor before the actual detection takes place.
  • the solution is then applied to the sensor surface by means of a fluid, where the nanoparticles are specifically bound by the immobilized peptides and detected by the sensor.
  • the level of the sensor signal is recorded in equilibrium, but the increase in the signal upon binding of the nanoparticle and the fall of the signal when rinsing with a particle-free solution are also recorded determined by calculation.
  • the measurement on the solution carrying the particles is compared with a standard, for example with a reference measurement on a sensor of the same design on a particle-free solution or on solutions with a known particle content.
  • a reference measurement on a sensor of the same design on a particle-free solution or on solutions with a known particle content By rinsing with a particle-free solution, bound nanoparticles are removed again before the sensor is available for further measurements.
  • Figure 1 shows schematically a detector
  • Figure 2 schematically shows the surface of a detector
  • the peptide molecules P are coupled via their amino end to polyethylene glycol (PEG) or alkyl chains, which in turn are bound to the gold surface of the sensor chip via SH functions.
  • the following embodiment shows how the method according to the invention for the detection of GaAs particles by means of microbalance sensors can be used.
  • peptides that specifically bind GaAs are selected using the phage display method. This step, which is known per se, leads to peptides such as, for example, the peptide with the sequence RLELAIPLQGSG, which binds specifically to GaAs (100) surfaces (Whaley et al. 2000 Nature 405: 665).
  • This peptide is synthesized in and coupled on a microbalance to at least two sensor elements, one for the actual measurement and one for reference.
  • the microbalance has a gold surface which is prepared by covering with “soap-assembled monolayers” made of bifunctional alkyl chains or with a layer of bifunctional polyethylene glycol ( PEG).
  • PEG polyethylene glycol
  • One of the two functions is an SH function, the other an OH or carboxyl function.
  • the alkyl chains react with the gold surface via the SH group.
  • the above-mentioned peptide is via its amino end coupled with the carboxyl groups of the PEG or alkyl chains
  • Figure 2 shows a schematic representation of the surface of the sensor after immobilization of the peptides.
  • GaAs nanoparticles are now passed in aqueous solution through a flow cell (Liss et al. 2002 Anal. Chem. 74: 4488) over the sensor. They are bound by the peptides on the sensor surface.
  • the microbalance signal is accumulated over a period of time, the length of which depends on the concentration of the nanoparticles.
  • the flow cell is decoupled from the flow of the particle-containing solution and rinsed with particle-free solution. The drop in the signal is registered and is used to calculate the distribution of the nanoparticle sizes, as described below:
  • s 0 for the microbalance is proportional to the total mass of the bound nanoparticles, which results from the mass m of the individual particle and the amount N. So ⁇ N m (2)
  • m const pa 3/2 , where a is the interaction surface between the nanoparticle egg and the substrate.
  • the distribution of interest N (a) of the particle sizes can be calculated numerically from Eq. 5 on the basis of the measured curve of s (t) and after determining the constant on the right-hand side by means of calibration measurements or model calculations.
  • the numerical evaluation can be carried out by discretization at intervals of a followed by a "singular value decomposition".
  • the measurement signal s (t) or the distribution N (a) calculated therefrom can then be used for monitoring purposes.
  • optical methods can also be used for the detection.
  • the optical detection described below is less suitable for the detection of nanoparticles that interfere with the fluorescence of a group in a peptide that is used for the detection.
  • peptide A a peptide which specifically binds the nanoparticles to be detected.
  • Peptide A is immobilized as described.
  • peptide B a further peptide is created, which must meet the following conditions: it must also bind the nanoparticle specifically, it must not bind the surface on which peptide A is immobilized and it must contain a fluorophore.
  • the same protocol can be used as for the creation of peptide A, in particular a phage display with a selection for the binding of the desired nanoparticle egg.
  • an additional step is built into the selection: only those phages are amplified that do not bind to a surface, as shown in Fig. 2.
  • the fluorophore in peptide B can be realized, for example, by subsequent modification with suitable fluorophores (Dansyl, Alexa TM, Cascade Blue, etc.) or by a tryptophan, which must be contained in all elements of the phage library. In the latter case, the fulfillment of the first two Conditions tested after modification. If necessary, further modifications are carried out until all three conditions are met.
  • the nanoparticle-containing solution is then passed over the immobilized peptides A by means of a flow cell, as shown above.
  • the detection is again carried out by a microbalance or by surface plasmon resonance.
  • Peptides B are used to further increase the sensitivity of the method.
  • a solution containing peptides B is also passed into the flow cell for this purpose.
  • Peptides B bind to the nanoparticles that have already been bound by the immobilized peptides A. Then it is rinsed with a solution that contains neither nanoparticles nor peptides B. With the start of this rinsing process, the fluorescence of the peptides is optically excited via direct irradiation or surface plasmons and also optically detected. In this way, nanoparticles can be detected even at lower concentrations.

Landscapes

  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Nanotechnology (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Dispersion Chemistry (AREA)
  • Molecular Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de détection de particules se trouvant dans une solution aqueuse, dont la taille est de l'ordre du nanomètre (nanoparticules), cette détection ne se faisant pas par l'intermédiaire d'une liaison spécifique à des protéines et/ou acides nucléiques dans les nanoparticules. Selon l'invention, on obtient des biomolécules, en particulier des peptides, qui se fixent de façon spécifique à la substance et ainsi aux nanoparticules. Les biomolécules sont immobilisées à la surface d'un capteur qui peut détecter une grandeur dépendant de la masse, la solution aqueuse contenant les nanoparticules étant amenée sur la surface du capteur et le signal émis par celui-ci étant enregistré. La surface du capteur est rincée avec une solution standard, une modification du signal est enregistrée, et, à partir de l'allure du signal émis par le capteur, la quantité N(a) de nanoparticules est déterminée.
EP04765113A 2003-09-24 2004-09-13 Detection de nanoparticules Withdrawn EP1664736A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2003144515 DE10344515A1 (de) 2003-09-24 2003-09-24 Detektion von Nanopartikeln
PCT/EP2004/010191 WO2005033674A1 (fr) 2003-09-24 2004-09-13 Detection de nanoparticules

Publications (1)

Publication Number Publication Date
EP1664736A1 true EP1664736A1 (fr) 2006-06-07

Family

ID=34398945

Family Applications (1)

Application Number Title Priority Date Filing Date
EP04765113A Withdrawn EP1664736A1 (fr) 2003-09-24 2004-09-13 Detection de nanoparticules

Country Status (3)

Country Link
EP (1) EP1664736A1 (fr)
DE (1) DE10344515A1 (fr)
WO (1) WO2005033674A1 (fr)

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6579726B1 (en) * 1999-07-30 2003-06-17 Surromed, Inc. Instruments, methods and reagents for surface plasmon resonance
EP1364201A4 (fr) * 2001-02-06 2005-01-05 Univ Auburn Dispositifs de detection de ligands et utilisation de ces dispositifs
DE10128093A1 (de) * 2001-06-11 2003-03-27 Christof M Niemeyer Verfahren zum Nachweis von Substanzen und Artikel zur Durchführung dieser Verfahren
FI118061B (fi) * 2001-09-24 2007-06-15 Beanor Oy Menetelmä ja bioanturi analyysiä varten
WO2003074548A2 (fr) * 2001-11-07 2003-09-12 Auburn University Capteurs de ligands de bacteriophages et leurs utilisations

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2005033674A1 *

Also Published As

Publication number Publication date
WO2005033674A1 (fr) 2005-04-14
DE10344515A1 (de) 2005-04-28

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