JP2000275180A - Organic luminescent semiconductor nano-crystal probe for biological use and process for producing and using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the subject probe formed from a luminescent semiconductor nano-crystal compd. capable of being bonded to affinity molecules and capable of emitting electromagnetic radiation with a narrow wavelength band upon excitation, having a wide absorbing band without the presence of a red radiation tail part and capable of detecting an absorption change. SOLUTION: A luminescent semiconductor nano-crystal compd. forming this probe and bondable to affinity molecules can generate luminescent, absorption, scattering and diffraction when it is excited by an electromagnetic radiation source or corpuscular beam and can show a detectable absorption change and a semiconductor nano-crystal capable of emitting, scattering and diffracting radiation with a narrow wavelength band is pref. This probe contains a connecting agent connected not only to this semiconductor nano-crystal but also to affinity molecules. That is, by bonding the luminescent semiconductor nano-crystal compd. to affinity molecules bondable to a detectable substance in a material, an org. luminescent semiconductor nano-crystal probe can be formed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The invention described in the present specification is:
Between the U.S. Department of Energy and the University of California, th
e Ernest Orlando Lawrence
Berkeley National Labora
Contract No. DE-AC03-SF for tory business
It was made in or below the process of 00998. The United States Government may have rights in the invention.

[0002] The present invention relates to organic luminescent semiconductor nanocrystal probes for biological applications, wherein the probes emit and / or absorb and / or scatter or excite when excited by radiation or particle beams. Contains a plurality of semiconductor nanocrystals that can diffract.

[0003]

BACKGROUND OF THE INVENTION Fluorescent labeling of biological systems is a well-known analytical tool used in modern biotechnology and analytical chemistry. Applications of such fluorescent labels include medical (and non-medical) fluorescence microscopy, histology, flow cytometry, fluorescence in situ hybridization (medical assays and research), DNA sequencing, immunoassays, binding assays, separations, etc. Such techniques are mentioned.

[0004] Traditionally, such fluorescent labels have involved the use of organic dye molecules attached to a moiety, which in turn binds selectively to a particular biological system and causes its presence. Is then confirmed by exciting it to cause it to fluoresce. There are a number of problems with such an analysis system. First, the emission of visible wavelength light from excited dye molecules is usually characterized by the presence of a broad emission tail as well as a broad emission spectrum on the red side of this spectrum. . That is, the overall emission spectrum is fairly broad (broad). As a result, due to the broad spectrum emission and emission tail of this labeled molecule,
Strict constraints on the number of different colored organic dye molecules that can be used simultaneously or sequentially in analysis, as it is difficult to detect or distinguish the presence of many different detectable substances, either simultaneously or even at different times There is.
Another problem is that most dye molecules have a relatively narrow absorption spectrum and therefore, for multiple wavelength probes, multiple excitations used either in tandem or continuous. For continuous excitation of a beam or otherwise a series of probes, each individually excited at a different wavelength, requires either a broad spectrum excitation source used continuously with different filters .

[0005] Another problem frequently encountered in the presence of dye molecule labels is that of photostability. Available fluorescent molecules fade or irreversibly emit after repeated absorption / emission excitation (10 ⁴ -10 ⁸ ) cycles. These problems are often overcome by minimizing the amount of time that the sample is exposed to light and by removing oxygen and / or other radical species from the sample.

In addition, due to electron microscopy techniques, the probe tools used to study these systems are quite different from the probes used to study by fluorescence. Therefore, it is not possible to label a material with a single type of probe for both electron microscopy and fluorescence.

[0007]

Thus, without the presence of the large red emitting tail characteristic of the dye molecule, the broad (wi)
de) can have an absorption band and exhibit a detectable change in absorption or can emit radiation in a narrow wavelength band (so that a plurality of such probe materials can be used simultaneously; It is desirable to provide stable probe materials for biological applications that can emit light in different narrow wavelength bands) and / or scatter or diffract radiation. It also provides a single stable probe material that can be used to image the same sample by both optical and electron microscopy.
Similarly desirable.

[0008]

SUMMARY OF THE INVENTION The compounds of the present invention are luminescent semiconductor nanocrystal compounds that can be linked to affinity molecules and emit electromagnetic radiation in a narrow wavelength band when excited. Containing the following a) and b): a) a semiconductor nanocrystal capable of emitting light in a narrow wavelength band when excited; and b) linked to the semiconductor nanocrystal and to the affinity molecule At least one linking agent.

[0009] In one embodiment, the semiconductor nanocrystal is capable of absorbing energy over a wide bandwidth.

In one embodiment, the linking agent is capable of linking to the affinity molecule via a further linking agent that can link to both the glass coating and the affinity molecule.
The glass coating on the semiconductor nanocrystal is included.

[0011] In one embodiment, the glass coating on the semiconductor nanocrystal contains a coating of silica glass.

[0012] In one embodiment, the linking agent contains a first moiety linked to the semiconductor nanocrystal and a second moiety linkable to the affinity molecule.

In one embodiment, the one or more linking agents comprises a glass coating on the semiconductor nanocrystal and a linking material, wherein the linking material comprises
It contains a first portion linked to a glass coating on the semiconductor nanocrystal and a second portion that can link to the affinity molecule.

The probe of the present invention is an organic light emitting semiconductor nanocrystal probe capable of binding to a detectable substance and emitting electromagnetic radiation in a narrow wavelength band when excited, capable of binding to the detectable substance. Contains a luminescent semiconductor nanocrystal compound linked to an affinity molecule.

In another aspect, the probe of the present invention is an organic luminescent semiconductor nanocrystal probe capable of binding to a detectable substance and emitting electromagnetic radiation in a narrow wavelength band when excited, wherein the probe comprises: Treating the material with the organic light-emitting semiconductor nanocrystal probe and determining the presence of the detectable substance in the material, comprising: a), b) and c) Subsequent exposure of the material to the excitation energy excites the semiconductor nanocrystals in the organic luminescent semiconductor nanocrystal probe bound to the detectable substance, causing emission of narrow wavelength band electromagnetic radiation, Means that the detectable substance bound to the organic light emitting semiconductor nanocrystal probe is present in the material: a) A) a semiconductor nanocrystal capable of emitting a narrow wavelength band of electromagnetic radiation when excited; b) at least one linking agent linked to the semiconductor nanocrystal and having a second moiety linkable to an affinity molecule. A species linking agent; and c) an affinity molecule that links to the second portion of the linking agent and is capable of selectively binding to the detectable substance.

In one embodiment, the linking agent comprises a glass coating on the semiconductor nanocrystal.

In one embodiment, the material treated with the organic light emitting semiconductor nanocrystal probe to determine the presence of the detectable substance contains a biological material.

In one embodiment, the material treated with the organic light emitting semiconductor nanocrystal probe to determine the presence of the detectable substance contains an organic material.

In one embodiment, the material treated with the organic light emitting semiconductor nanocrystal probe to determine the presence of the detectable substance contains an inorganic material.

The process of the present invention is a process for forming a luminescent semiconductor nanocrystal compound that can be linked to an affinity molecule and emit, when excited, a narrow wavelength band of electromagnetic radiation. Linking together a semiconductor nanocrystal capable of emitting a narrow wavelength band of electromagnetic radiation and a linking agent having a first portion linked to said semiconductor nanocrystal and a second portion linked to an affinity molecule. I do.

[0021] In one embodiment, the method further comprises forming a glass coating on the semiconductor nanocrystal and then treating the glass with a linking agent capable of linking with affinity molecules.

In another aspect, the process of the present invention is a process for forming an organic light emitting semiconductor nanocrystal probe capable of binding to a detectable substance and emitting electromagnetic radiation in a narrow wavelength band when excited. Linking an affinity molecule capable of binding to a detectable substance with a luminescent semiconductor nanocrystal compound.

In another aspect, the process of the present invention is a process for forming an organic light emitting semiconductor nanocrystal probe capable of binding to a detectable substance and emitting, when excited, a narrow wavelength band of electromagnetic radiation, The process includes the following steps: a) linking a semiconductor nanocrystal capable of emitting a narrow wavelength band of electromagnetic radiation when excited to a first moiety linked to the semiconductor nanocrystal and an affinity molecule; Linking the linking agent to a linking agent having a second moiety, and b) linking the linking agent to an affinity molecule capable of binding to the detectable substance.

In one embodiment, the step of linking the semiconductor nanocrystal and the linking agent together is performed before the step of linking the linking agent and the affinity molecule together.

In one embodiment, the step of linking the linking agent and the affinity molecule together is performed before the step of linking the semiconductor nanocrystal and the linking agent together.

In one embodiment, the step of linking the semiconductor nanocrystal and the linking agent together further comprises the step of coating the semiconductor nanocrystal with glass, and then the step of coating the glass-coated semiconductor nanocrystal. And treating with a linking agent capable of linking to the affinity molecule.

In another aspect, the process of the present invention is a process for treating a material to determine the presence of one or more detectable substances in the material, the process comprising the following steps: ), C) and d): a) electromagnetic radiation in a first narrow wavelength band, when excited, capable of binding the material, if present, to a first detectable substance in the material; Contacting with a first organic light-emitting semiconductor nanocrystal probe that is capable of emitting, wherein the first organic light-emitting semiconductor nanocrystal probe contains the following i), ii) and iii): i) wide A first semiconductor nanocrystal capable of being excited over a bandwidth and emitting electromagnetic radiation of the first narrow wavelength band when excited; ii) an affinity capable of selectively binding to the detectable substance. And iii) a linking agent linked to the first semiconductor nanocrystal and also linked to the affinity molecule; b) from the material, the first of the first organic light emitting semiconductor nanocrystal probes Removing the portion that is not bound to the detectable substance; and c) exposing the material to energy capable of exciting the first semiconductor nanocrystal to cause the first detectable substance within the material Emitting electromagnetic radiation in the first narrow wavelength band, indicative of the presence of: and d) in the first organic light-emitting semiconductor nanocrystal probe, the radiation emitted by the first semiconductor nanocrystal. Detecting the electromagnetic radiation in a first narrow wavelength band.

In one embodiment, the method comprises the additional step of treating the material with at least a second organic light emitting semiconductor nanocrystal probe capable of binding to a further detectable substance in the material, and An organic light emitting semiconductor nanocrystal probe comprising a second semiconductor nanocrystal capable of being excited with a wide bandwidth and emitting electromagnetic radiation of a second narrow wavelength band different from the first narrow wavelength band; Thereby, exposing the material to energy that can excite both the first and second nanocrystals causes any of the first or second semiconductor nanocrystals present in the material to be exposed. Emits electromagnetic radiation in different narrow wavelength bands, whereby the presence or absence of more than one detectable substance in the material is reduced by a single excitation energy It can be detected simultaneously using a source.

In one embodiment, at least one further organic light emitting semiconductor nanocrystal probe is used to treat said material, each of said organic light emitting semiconductor nanocrystal probes being selective for a different detectable substance. And each of the organic light emitting semiconductor nanocrystal probes can be excited over a wide wavelength range and emit different narrow wavelength bands of electromagnetic radiation, so that multiple detectable substances are Using an excitation source, they can be analyzed simultaneously in the material.

In one embodiment, prior to the step of removing a portion of the first organic light emitting semiconductor nanocrystal probe that is not bound to the first detectable substance from the material, the material comprises: Treated with all of the organic light emitting semiconductor nanocrystal probes, and the removing step comprises:
The method further includes removing a portion of the organic light-emitting semiconductor nanocrystal probe that is not bound to a detectable substance in the material.

In one embodiment, exposure of the material to light of a selected wavelength is used to selectively excite one or more, but not all, of the organic light emitting semiconductor nanocrystal probes. Thus, it is possible to identify the presence of a particular labeled detectable substance or a subset of different labeled detectable substances in the material.

In one embodiment, the material comprises a biological material.

In one embodiment, exposing the material to energy capable of exciting the first semiconductor nanocrystals to emit electromagnetic radiation further comprises:
Exposing the material to an electromagnetic radiation source capable of emitting broad or narrow spectrum of photons.

In one embodiment, the step of exposing the material to energy capable of exciting the first semiconductor nanocrystal to emit electromagnetic radiation further comprises:
Exposing the material to an electron beam.

In another aspect, the process of the present invention is a process for treating a material to determine the presence of a detectable substance in the material, the process comprising the following steps: a), b), c A) an organic light-emitting semiconductor nanocrystal probe capable of binding the material, if present, to a first detectable substance in the material and absorbing energy when excited. Contacting, wherein the organic light-emitting semiconductor nanocrystal probe comprises the following i), ii) and iii): i) can be excited over a wide bandwidth and can absorb energy when excited A first semiconductor nanocrystal; ii) an affinity molecule that can selectively bind to the detectable substance; and iii) linked to the first semiconductor nanocrystal and the affinity B) removing from the material a portion of the organic light-emitting semiconductor nanocrystal probe that is not bound to the first detectable substance; and c) removing the material from the material. Exposing the first semiconductor nanocrystal to an excitable energy to absorb energy and indicate the presence of the first detectable substance in the material; and d) the detectable in the material. Detecting a change in absorbed energy that is indicative of the presence of the organic light emitting semiconductor nanocrystal probe bound to a substance.

In one embodiment, the method further comprises treating the material with at least a second organic light emitting semiconductor nanocrystal probe capable of binding to a further detectable substance in the material, The luminescent semiconductor nanocrystal probe contains a second semiconductor nanocrystal that can be excited with a wide bandwidth, resulting in a detectable change in absorbance, whereby the first and second nanocrystals Exposure of the material to energy that can excite both causes the first or second semiconductor nanocrystals present in the material to absorb electromagnetic radiation of different wavelength bands, thereby The presence or absence of more than one detectable substance within can be detected simultaneously using a single excitation energy source.

In one embodiment, at least one further organic light emitting semiconductor nanocrystal probe is used to treat said material, each of said organic light emitting semiconductor nanocrystal probes being selective for a different detectable substance. And each of the organic light-emitting semiconductor nanocrystal probes can be excited over a wide wavelength range and can absorb electromagnetic radiation, so that multiple detectable substances can be combined using a single excitation source. hand,
It can be analyzed simultaneously in the material.

In one embodiment, the step of exposing the material to energy capable of exciting the first semiconductor nanocrystal to emit electromagnetic radiation further comprises the step of exposing the material to a broad or narrow spectrum. Exposing to a source of electromagnetic radiation capable of emitting photons.

In one embodiment, exposing the material to energy capable of exciting the first semiconductor nanocrystals and emitting electromagnetic radiation further comprises exposing the material to an X-ray source. It includes doing.

In another aspect, the process of the present invention is a process for treating a material to determine the presence of a detectable substance in the material, the process comprising the following steps: a), b), c A) an organic light-emitting semiconductor nanocrystal that can bind the material, if present, to a first detectable substance in the material and, when excited, can scatter or diffract energy Contacting with a probe, wherein the organic light-emitting semiconductor nanocrystal probe comprises the following i), ii) and iii): i) a light-emitting semiconductor nanocrystal probe capable of scattering or diffracting over a wide bandwidth having a characteristic cross-section. One semiconductor nanocrystal; ii) an affinity molecule that can selectively bind to the detectable substance; and iii) linked to the first semiconductor nanocrystal and the parent. B) removing from the material a portion of the organic light emitting semiconductor nanocrystal probe that is not bound to the first detectable substance; and c) removing the material from the material. Exposing the first semiconductor nanocrystal to excitable energy to scatter or diffract energy to indicate the presence of the first detectable substance in the material; and d) scattered or diffracted energy Detecting a change in the organic light-emitting semiconductor nanocrystal probe bound to the detectable substance in the material.

In one embodiment, the method further comprises treating the material with at least a second organic luminescent semiconductor nanocrystal probe capable of binding to a second detectable substance in the material, The organic light-emitting semiconductor nanocrystal probe of the invention also contains a second semiconductor nanocrystal that can scatter or diffract energy, resulting in a detectable scattering cross-section change, whereby the first Exposure to energy that can scatter or diffract both of the first and second nanocrystals can result in a particular organic light emitting semiconductor nanocrystal being present in either the first and second semiconductor nanocrystals present in the material. The scattering cross section characteristic of the probe scatters or diffracts energy, thereby reducing the presence of more than one detectable substance in the material. The absence can be detected simultaneously using a single excitation energy source.

In one embodiment, at least one further organic light emitting semiconductor nanocrystal probe is used to treat said material, each of said organic light emitting semiconductor nanocrystal probes being selective for a different detectable substance. And each of the organic light-emitting semiconductor nanocrystal probes can exhibit a different scattering cross-section and scatter or diffract energy so that multiple detectable substances can be combined using a single excitation source. ,
It can be analyzed simultaneously in the material.

In one embodiment, the step of exposing the material to energy capable of exciting the first semiconductor nanocrystal to scatter or diffract the energy further comprises: exposing the material to an electron beam or other Exposure to a particle beam.

In one embodiment, exposing the material to energy capable of exciting the first semiconductor nanocrystal to scatter or diffract the energy further comprises: exposing the material to an X-ray source. Exposure.

In one embodiment, exposing the material to energy capable of exciting the first semiconductor nanocrystal to scatter or diffract the energy;
And the step of detecting the scattering or diffraction of energy is both performed using a transmission electron microscope.

In one embodiment, exposing the material to energy capable of exciting the first semiconductor nanocrystal to scatter or diffract the energy;
And the step of detecting the scattering or diffraction of the energy is both performed using a scanning electron microscope.

In another aspect, the compound of the present invention is a luminescent semiconductor nanocrystal compound capable of linking to an affinity molecule and absorbing energy in a narrow wavelength band when excited, comprising: a) and b) containing: a) a semiconductor nanocrystal capable of absorbing energy in a narrow wavelength band when excited; and b) a linking agent linked to the semiconductor nanocrystal and the affinity molecule. At least one linking agent that can be linked to

In another aspect, the compound of the present invention is a luminescent semiconductor nanocrystal compound capable of linking to an affinity molecule and scattering or diffracting energy in a narrow wavelength band when excited, wherein the compound is A) below
And b) containing: a) a semiconductor nanocrystal capable of scattering or diffracting energy in a narrow wavelength band when excited; and b) a linking agent linking the semiconductor nanocrystal and the affinity molecule. At least one linking agent that can be linked to

In another aspect, the probe of the present invention is an organic luminescent semiconductor nanocrystal probe capable of binding to a detectable substance and absorbing energy in a narrow wavelength band when excited. It contains a luminescent semiconductor nanocrystal compound linked to an affinity molecule that can bind to a detectable substance.

In another aspect, the probe of the present invention is an organic light-emitting semiconductor nanocrystal probe capable of binding to a detectable substance and absorbing energy in a narrow wavelength band when excited. A), b) and c), whereby the treatment of a material with the organic light emitting semiconductor nanocrystal probe and the treatment to determine the presence of the detectable substance in the material Subsequent exposure of the material to the excitation energy excites the semiconductor nanocrystals in the organic light-emitting semiconductor nanocrystal probe bound to the detectable substance, causing absorption of a narrow wavelength band of energy, Within the material means the presence of the detectable substance bound to the organic luminescent semiconductor nanocrystal probe: B) at least one type of linking agent that links the semiconductor nanocrystals and that has a second moiety that can link to an affinity molecule; A linking agent; and c) an affinity molecule that links to the second portion of the linking agent and is capable of selectively binding to the detectable substance.

In another aspect, the probe of the present invention is an organic light emitting semiconductor nanocrystal probe capable of binding to a detectable substance and scattering or diffracting energy in a narrow wavelength band when excited. It contains a luminescent semiconductor nanocrystal compound linked to an affinity molecule that can bind to the substance.

In another aspect, the probe of the present invention is an organic light-emitting semiconductor nanocrystal probe capable of binding to a detectable substance and scattering or diffracting energy in a narrow wavelength band when excited. , Comprising the following a), b) and c), whereby the treatment of the material with the organic light-emitting semiconductor nanocrystal probe and the treatment for determining the presence of the detectable substance in the material: Subsequent exposure of the material to be excited to the excitation energy excites the semiconductor nanocrystals in the organic luminescent semiconductor nanocrystal probe bound to the detectable substance, causing scattering or diffraction of narrow wavelength band energy; This indicates the presence of the detectable substance in the material bound to the organic light emitting semiconductor nanocrystal probe. A) a semiconductor nanocrystal that can scatter or diffract energy in a narrow wavelength band when excited; b) a linking agent that links the semiconductor nanocrystal and that can link to an affinity molecule. At least one linking agent having two moieties; and c) an affinity molecule that links the second portion of the linking agent and is capable of selectively binding to the detectable substance.

In another aspect, the process of the present invention is a process for forming a luminescent semiconductor nanocrystal compound that can be linked to an affinity molecule and, when excited, can absorb energy in a narrow wavelength band. Is
Includes: a linkage having a semiconductor nanocrystal capable of absorbing energy in a narrow wavelength band when excited, a first portion linked to the semiconductor nanocrystal and a second portion linked to an affinity molecule. Connecting together the agents.

In another aspect, the process of the present invention is a process for forming a luminescent semiconductor nanocrystal compound that can be linked to an affinity molecule and that, when excited, can scatter or diffract energy in a narrow wavelength band, The process includes: a semiconductor nanocrystal capable of scattering or diffracting energy in a narrow wavelength band when excited, and a first moiety linked to the semiconductor nanocrystal and a second moiety linked to an affinity molecule. Linking together a linking agent having two parts.

In another aspect, the process of the present invention is a process for forming an organic light emitting semiconductor nanocrystal probe that can bind to a detectable substance and, when excited, absorb energy in a narrow wavelength band. Linking the luminescent semiconductor nanocrystal compound to an affinity molecule capable of binding to the possible substance.

In another aspect, the process of the present invention is a process for forming an organic light emitting semiconductor nanocrystal probe capable of binding to a detectable substance and absorbing energy in a narrow wavelength band when excited. The process includes the following steps: a) A semiconductor nanocrystal that can absorb energy in a narrow wavelength band when excited can be linked to a first moiety linked to the semiconductor nanocrystal and to an affinity molecule. Linking to a linking agent having a second moiety; and b) linking the linking agent to an affinity molecule capable of binding to the detectable substance.

In another aspect, the process of the present invention is a process for forming an organic light emitting semiconductor nanocrystal probe that can bind to a detectable substance and, when excited, can scatter or diffract energy in a narrow wavelength band. Linking the luminescent semiconductor nanocrystal compound with an affinity molecule capable of binding to the detectable substance.
In another aspect, the process of the present invention is a process for forming an organic light emitting semiconductor nanocrystal probe that can bind to a detectable substance and, when excited, can scatter or diffract energy in a narrow wavelength band, the process comprising: Comprises the following steps: a) linking a semiconductor nanocrystal that can scatter or diffract energy in a narrow wavelength band when excited to a first moiety linked to the semiconductor nanocrystal and to an affinity molecule; Linking the resulting linking agent with a second moiety; and b) linking the linking agent to an affinity molecule capable of binding to the detectable substance.

[0058]

DETAILED DESCRIPTION OF THE INVENTION (Summary of the Invention) The present invention is capable of emitting and / or absorbing and / or scattering or diffracting when excited by an electromagnetic radiation source (wide or narrow bandwidth) or particle beam. To form organic light emitting semiconductor nanocrystal probes that can exhibit detectable changes in absorption when excited and / or emit radiation in a narrow wavelength band and / or scatter or diffract. Luminescent semiconductor nanocrystal compounds that can bind to affinity molecules. The luminescent semiconductor nanocrystal compound preferably contains: (1) luminescence and / or absorption and / or scattering or when excited by a (wide or narrow bandwidth) electromagnetic radiation source or particle beam. A semiconductor nanocrystal that can diffract and exhibit a detectable change in absorption when so excited and / or emit and / or scatter or diffract radiation in a narrow wavelength band; and (2) the semiconductor nanocrystal. A binding agent having a first moiety linked to a crystal and a second moiety capable of linking to an affinity molecule.

The present invention further includes an organic light emitting semiconductor nanocrystal probe formed by bonding the light emitting semiconductor nanocrystal compound to an affinity molecule capable of binding to a detectable substance in the material. As a result, the organic light-emitting semiconductor nanocrystal probe can, in one embodiment, absorb or scatter or diffract energy from either a particle beam or a source of electromagnetic radiation (wide or narrow bandwidth). Can emit electromagnetic radiation in a narrow wavelength band when excited.
In contrast, in another embodiment, the amount of such absorbed or scattered or diffracted energy from either a particle beam or a source of electromagnetic radiation (wide or narrow bandwidth) is detectable, , Absorption, scattering or diffraction changes can be detected.

Thus, treatment of the material with the organic light emitting semiconductor nanocrystal probe and the (particle beam or broad or narrow bandwidth of the material to be processed to determine the presence of the detectable substance in the material Subsequent exposure to excitation energy (from one of the sources of electromagnetic radiation) excites the semiconductor nanocrystals in the organic light-emitting semiconductor nanocrystal probe bound to the detectable substance and emits a narrow wavelength band of electromagnetic radiation. And / or cause a detectable change in the amount of absorbed and / or scattered or diffracted energy, which means the presence of a detectable substance in the material bound to the organic light emitting semiconductor nanocrystal probe.

The present invention also provides a process for producing the luminescent semiconductor nanocrystal compound and an organic luminescent semiconductor nanocrystal probe containing the luminescent semiconductor nanocrystal compound linked to an affinity molecule capable of binding to the detectable substance. Process. The organic light-emitting semiconductor nanocrystal probes of the present invention are stable with respect to repeated light excitation or exposure to oxygen or other radicals. The invention further includes a process for treating a material, such as a biological material, to determine the presence of a detectable substance in the material, the process comprising contacting the material with an organic light emitting semiconductor nanocrystal probe. Removing the portions of the organic light emitting semiconductor nanocrystal probe that are not bound to the detectable substance from the material, and then removing the material from either an electromagnetic radiation source (wide or narrow bandwidth) or a particle beam. Exposing to activation energy derived from crab. The presence of the detectable substance in the material is then determined by measuring the absorbance of energy by the organic light emitting semiconductor nanocrystal probe, and / or detecting the emission of narrow wavelength band radiation by the organic light emitting semiconductor nanocrystal probe, and And / or by detecting scattering or diffraction by the organic light emitting semiconductor nanocrystal probe, which (in each case)
The presence of an organic light emitting semiconductor nanocrystal probe bound to a detectable substance in this material is shown.

DETAILED DESCRIPTION The present invention is directed to a method for detecting a change in absorption that can be bound to an organic molecule and detectable when excited by either an electromagnetic radiation source (wide or narrow bandwidth) or a particle beam. Includes luminescent semiconductor nanocrystal compounds that can exhibit and / or emit narrow wavelength band electromagnetic radiation and / or scatter or diffract. The luminescent semiconductor nanocrystal compound, in turn, contains: (1) may exhibit a detectable change in absorption when excited by either a (wide or narrow bandwidth) electromagnetic radiation source or a particle beam. And / or a semiconductor nanocrystal capable of emitting a narrow wavelength band of electromagnetic radiation; and (2) a first portion linked to the semiconductor nanocrystal and a second portion linked to an organic affinity molecule, respectively. One or more binders.

The present invention also includes the above-described light-emitting semiconductor nanocrystal compound linked to an organic affinity molecule (via a linking agent) to form an organic light-emitting semiconductor nanocrystal probe. May be capable of binding to a detectable substance and exhibit a detectable change in absorption when excited by either an electromagnetic radiation source (either broad or narrow bandwidth) or a particle beam and / or exhibit a narrow wavelength band. It can emit electromagnetic radiation and / or scatter or diffract. Processing of a material (typically a biological material) with an organic light emitting semiconductor nanocrystal probe and subsequent to the excitation energy of the processed material to determine the presence of a detectable substance in the material Exposure excites the semiconductor nanocrystals in the organic light-emitting semiconductor nanocrystal probe bound to a detectable substance to detect the absorption and / or emission and / or scattered or diffracted energy of electromagnetic radiation in a narrow wavelength band Cause significant changes,
(In each case), these imply the presence of a detectable substance in the material bound to the organic light-emitting semiconductor nanocrystal probe.

The present invention also provides a process for producing a luminescent semiconductor nanocrystal compound and a process for producing an organic luminescent semiconductor nanocrystal probe containing a luminescent semiconductor nanocrystal compound linked to an affinity molecule capable of binding to a detectable substance. Is included.

The invention further includes a process for processing a material, such as a biological material, to determine the presence of a detectable substance in the material, the process comprising: (1) Contacting the material with an organic light-emitting semiconductor nanocrystal probe; (2) removing the portion of the organic light-emitting semiconductor nanocrystal probe that is not bound to a detectable substance from the material; Exposing the crystal to energy that can excite the crystal (eg, the electromagnetic energy source or particle beam) to cause a detectable change in absorption and / or emission and / or scattering or diffraction of electromagnetic radiation in a narrow wavelength band. This, in each case, indicates the presence of an organic luminescent semiconductor nanocrystal probe bound to a detectable substance in the material. And it has, and (4) the semiconductor nanocrystals in organic light-emitting semiconductor nanocrystal probe, absorbed energy or radiation to detecting changes or scattering or diffraction of electromagnetic radiation was.

A. Definitions The use of the term "nanometer crystal" or "nanocrystal" herein refers to an average cross section of only about 20 nanometers (nm) or 20 x ^10-9 meters (200 Angstroms), preferably About 10n
nanocrystals having an average cross section of only m (100 Angstroms) and having a minimum average cross section of about 1 nm, but in some cases smaller average cross sections, ie, About 0.5
As small as nm (5 Angstroms) are acceptable. Typically, nanocrystals are about 1 nm (1
It has an average cross-section ranging in size from about 0 Angstroms to about 100 Angstroms.

The use of the term “semiconductor nanocrystal” refers to a group II-V which can emit electromagnetic radiation when excited.
Nanometer crystals of Group I and III-V semiconductor compounds, i.e. nanocrystals, are used, but the use of Group IV semiconductors (e.g. germanium or silicon) or the use of organic semiconductors is subject to certain conditions. Below, it may be viable.

The use of the term “narrow wavelength band” refers to the emission of electromagnetic radiation from semiconductor nanocrystals of about 40 nm.
refers to a wavelength band of radiation not exceeding nm, and preferably not exceeding about 20 nm, and symmetric about the center, which is about 100 nm for the typical dye molecule. Are contrasting, this dye molecule has a red tail that can further extend the bandwidth to the order of 100 nm. The stated bandwidth is determined by measuring the emission width at half peak height (FWHM) and is between 200 nm and 200 nm.
It should be noted that a range of 0 nm is appropriate.

The use of the term “broad absorption band” refers to a continuously increasing absorption with respect to the electromagnetic radiation absorption of a semiconductor nanocrystal from the start, which is near the “narrow wavelength band” of this radiation. Happens at slightly higher energies. This is in contrast to the "narrow absorption band" of the dye molecule, which occurs near the high energy emission peak but falls off rapidly from that wavelength.

The use of the term “detectable substance” refers to the presence or absence of a substance (eg, a biological material) that can be ascertained by use of the organic light emitting semiconductor nanocrystal probe of the present invention. Means an entity or a group.

The use of the term “affinity molecule” is part of the organic light-emitting semiconductor nanocrystal probe of the present invention, wherein a detectable substance (if any) within the material to be analyzed (eg, biological material). , If present).

The use of the term "linking agent" means a material that can be linked to semiconductor nanocrystals and also to affinity molecules.

The terms “link” and “linking” refer to an affinity molecule, either directly or through a moiety identified herein as a linking agent. To state the adhesion between the and the semiconductor nanocrystal. Adhesion may include any type of bond, including but not limited to covalent, ionic, or hydrogen bonds, Van der Waals forces or mechanical bonds.

The terms “bond” and “bonding” are meant to describe the adhesion between an affinity molecule and a detectable substance. Adhesion may include any type of bond, including but not limited to covalent, ionic, hydrogen, Van der Waals or mechanical bonds.

The term “luminescent semiconductor nanocrystal compound” as used herein refers to defining a semiconductor nanocrystal that is linked to one or more linking agents and can link to affinity molecules. Intend. On the other hand, the term "organic light emitting semiconductor nanocrystal probe" is intended to define a light emitting semiconductor nanocrystal compound linked to an affinity molecule.

The term “glass” as used herein refers to one or more oxides of silicon, boron and / or phosphorus or mixtures thereof, as well as one or more metal silicates, borane It is meant to include the optional inclusion of a metal acid salt or metal phosphate.

B. Semiconductor Nanocrystals Semiconductor nanocrystals useful in practicing the present invention include Group II-VI semiconductors (eg, MgS, MgS
e, MgTe, CaS, CaSe, CaTe, SrS,
SrSe, SrTe, BaS, BaSe, BaTe, Z
nS, ZnSe, ZnTe, CdS, CdSe, CdT
e, HgS, HgSe and HgTe) nanocrystals; and III-V semiconductors (eg, GaAs).
s, InGaAs, InP and InAs). As mentioned above, the use of Group IV semiconductors (eg, germanium or silicon) or the use of organic semiconductors may also be feasible under certain conditions.

The formation of group III-V semiconductor nanometer crystals has been described in co-pending and commonly assigned U.S. patent application Ser. No. 08/08, Alivisatos et al.
No. 235,265, which also describes Group II-VI semiconductor nanocrystals, which is published Nov. 1, 1991.
Filed on April 29, 1994 as a FWC application filed on the 1st of Application No. 07 / 796,246. And US Pat. No. 5,262,357 to Alivisatos et al., Which is also assigned to the assignee of the present invention. They also describe the use of crystal growth terminators to control the size of the semiconductor nanocrystals during formation. Al
Application No. 08 / 235,265 to ibisatos et al.
And U.S. Pat. No. 5,2of Alivisatos et al.
The teachings of 62,357 are each specifically incorporated herein by reference.

In a preferred embodiment, the nanocrystals are used in a core / shell configuration, wherein the first semiconductor nanocrystal is, for example,
Forming nuclei with diameters ranging from about 20 Angstroms to about 100 Angstroms, and another sheath of semiconductor nanocrystal material overlying the nuclear nanocrystals, e.g.
It grows to the thickness of a monolayer.
For example, a CdS sheath with a thickness of 1-10 monolayers may be CdS
When grown epitaxially on Se nuclei, the quantum yield of photoluminescence at room temperature increases significantly. The formation of such nucleus / sheath nanocrystals has been described by one of the present inventors and another and is described in "Epitaxial G".
row of Highly Luminescence
nt CdSe / CdS Core / Shell Na
nocrystals with Photostab
ility and Electronic Access
ssibility "(Peng, Schlamp, K
adavanich, and Alivasatos, J
ownnal of the American Ch
eical Society, Vol. 119, No. 30
No., 1997, pp. 7019 to 7029), the contents of which are specifically incorporated herein by reference.

The semiconductor nanocrystals used in the present invention have the ability to emit light in a narrow wavelength band of about 40 nm or less, preferably about 20 nm or less, so that a broad emission line (eg, about 100 nm ) And has a wide radiating tail (eg, about 100 nm further)
Is different from the use of a dye molecule having the red side of the spectrum), a plurality of differently colored organic light-emitting semiconductor nanocrystal probes having different semiconductor nanocrystals without overlapping (or a small amount of overlapping) wavelengths of emitted light. It allows for simultaneous use and therefore allows for the simultaneous detection of multiple detectable substances.

C. Affinity Molecules Certain affinity molecules that form part of the organic light emitting semiconductor nanocrystal probes of the present invention are specific detectable substances (eg, whose presence or absence in a biological material should be confirmed). Are selected based on their affinity for Basically, an affinity molecule can include any molecule that can be linked to a luminescent semiconductor nanocrystal compound that can specifically recognize a particular detectable substance. Generally, any affinity molecule useful in the prior art, in combination with a dye molecule, to provide specific recognition of a detectable substance, will be useful in forming the organic light emitting semiconductor nanocrystal probes of the present invention. I know. Such affinity molecules include, for example, monoclonal and polyclonal antibodies, nucleic acids (both monomeric and oligomeric),
Types of substances include proteins, polysaccharides, and small molecules (eg, saccharides, peptides, drugs and ligands). A list of such affinity molecules can be found in published literature, eg, “Handbook of Fluo”.
resumeProbes and Research
h Chemicals "(6th edition) (RP Ha
Molecular Probe by ugland
s, Inc. Available from).

D. Linking Agents The organic luminescent semiconductor nanocrystal probes of the present invention are typically used for the detection of one or more detectable substances in an organic material, in particular for the detection of one or more detectable substances in a biological material. Knows its usefulness. This is because the affinity molecule or moiety that binds the organic light-emitting semiconductor nanocrystal probe to the detectable substance is such that the presence of the detectable substance in the organic / biological material can be subsequently confirmed. Need to be present in the probe. However, since semiconductor nanocrystals are inorganic, they may not bind directly to organophilic molecules. Therefore, in these cases, some types of linking agents that can form linkages not only with inorganic semiconductor nanocrystals, but also with organic affinity molecules in organic light-emitting semiconductor nanocrystal probes, include organic light-emitting semiconductor nanocrystal probes. Must be within.

One form in which the semiconductor nanocrystal can be linked to the affinity molecule via a linking agent is to use the linking agent (eg, a substituted silane such as 3-mercaptopropyl-trimethoxysilane) to form the semiconductor nanocrystal. To a glass (for example, silica (SiO _x , where x = 1 to
By coating with a thin layer of 2)) and connecting the nanocrystals to the glass. The glass-coated semiconductor nanocrystal can then be further treated with a linking agent, for example, an amine such as 3-aminopropyl-trimethoxysilane, which links the glass-coated semiconductor nanocrystal to an affinity molecule. Acts to be.
That is, the glass-coated semiconductor nanocrystals can then be linked to affinity molecules. It is within the contemplation of the present invention that the initial luminescent semiconductor nanocrystal compound can also be chemically modified after manufacture to effectively link to the affinity molecule. Various publications summarize the standard class of chemical materials that can be used for this purpose, in particular, "Handbook of F.
fluoresce Probes and Res
EARCH CHEMICALS "(6th edition) (RP
Molecular Pr by Haugland
obes, Inc. Available from
Conjugate Technologies "(Academ by Greg Hermanson
ic Press, available from New York).

When the semiconductor nanocrystals are coated with a thin layer of glass, the glass may be, for example, about 0.5 nm
Silica glass (SiO _x , having a thickness in the range of about 10 nm, preferably in the range of about 0.5 nm to about 2 nm)
Here, x = 1 to 2) may be included.

The semiconductor nanocrystal is prepared by first coating the nanocrystal with a surfactant (eg, tris-octyl-phosphine oxide), and then coating the nanocrystal coated with the surfactant with a linking agent (eg, 3- Dissolved in a basic methanol solution of mercaptopropyltrimethoxysilane, followed by partial hydrolysis, followed by a glass-affinity molecular linking agent such as aminopropyltrimethoxysilane; (Which functions to form a linkage with the affinity molecule) is coated with a thin glass coating such as silica.

If the linking agent does not involve the use of a glass coating on the semiconductor nanocrystal, it can include a number of different materials, depending on the particular affinity molecule, which in turn can be analyzed Depends on the type of detectable substance. Although individual linking agents can be used to link individual semiconductor nanocrystals, one or more linking agents may be linked to the same semiconductor nanocrystal, and vice versa. It should also be noted that it is within consideration.

The following table illustrates a few examples of the types of linking agents that can be used to link both semiconductor nanocrystals (or glass coatings on nanocrystals) and organophilic molecules in a probe. But it is understood that this is not all the list:

[0088]

Embedded image

It should further be noted that multiple polymerizable linking agents can be used together to form an encapsulated net or link around individual nanocrystals (or groups of nanocrystals). This is especially important when certain linking agents cannot form a strong bond with the nanocrystal. Examples of linking agents that can bind together in this manner and surround the nanocrystals with a network of linking agents include, but are not limited to, diacetylene, acrylate, acrylamide, vinyl, styryl. And the silicon oxides, boron oxides, phosphorus oxides, silicates, borates and phosphates.

E. Probe Excitation and Emission / Absorption Detection As mentioned earlier, the organic light emitting semiconductor nanocrystal probes of the present invention, in contrast to the dye molecules used in the prior art, can excite over a wide bandwidth and have a narrow 4 shows the emission in the wavelength band. Therefore, electromagnetic radiation with wavelengths ranging from X-rays to ultraviolet, visible, and infrared light can be used to excite the luminescent semiconductor nanocrystals in the probe. In addition, light-emitting semiconductor nanocrystals
It can be excited from the impact of a particle beam (eg, an electron beam (e-beam)). Furthermore, due to the wide bandwidth over which the luminescent semiconductor nanocrystals can be excited, several probes, ie,
For the simultaneous excitation of several probes emitting radiation at different frequencies, a common excitation source can be used and therefore the simultaneous excitation and detection of the presence of several probes (for example, Indicating the presence of several detectable substances within a given material).

Thus, for example, to excite a first organic light emitting semiconductor nanocrystal probe capable of emitting radiation of a second frequency (eg, red light), a laser radiation source of a predetermined frequency (eg, , Blue light) can be used, thereby indicating the presence of a first detectable substance in the irradiated material to which an organic light emitting semiconductor nanocrystal probe emitting a particular red light is bound. at the same time,
The same blue light laser source can also excite a second organic light emitting semiconductor nanocrystal probe (in the same material) that can emit a third frequency of radiation (eg, green light);
This indicates the presence of a second detectable substance in the irradiated material to which an organic light emitting semiconductor nanocrystal probe emitting a specific green light is bound. Therefore, unlike the prior art (due to the wide bandwidth over which the organic light-emitting semiconductor nanocrystal probes of the present invention can be excited), there is no need to use multiple excitation sources, and each probe has a specific semiconductor The narrow emission band of the nanocrystals makes it possible to eliminate sequencing and / or fine filtration for detecting the emitted radiation.

With respect to the energy absorption by the probe of the present invention, when the excitation source is an electron beam or an X-ray source, the presence of the organic luminescent semiconductor nanocrystal probe in the material being analyzed bound to the detectable substance of interest is It can be ascertained using a commercially available energy absorption or scattering or diffraction detection system, wherein the absorption or scattering cross section of the material being analyzed is measured.
on) or a change in diffraction can be detected, which indicates the presence of the probe in the material, which in turn indicates the presence of the detectable substance to which the probe is bound in the material being analyzed. In addition, to detect the presence of an organic luminescent semiconductor nanocrystal probe bound to a detectable substance by using a conventional detection system of visible light emission to observe the narrow emission wavelength visible radiation of the probe,
It may be possible to use an electron or X-ray source.

The following examples not only further illustrate the formation of the organic light emitting semiconductor nanocrystal probes of the present invention, but also use them to detect the presence of detectable substances in materials such as biological materials. It serves to further illustrate its use.

[0094]

EXAMPLE 1 To illustrate the formation of a luminescent semiconductor nanocrystal compound (containing a semiconductor nanocrystal linked to a linking agent), (CH ₃ ) ₄ NOH.5H ₂
Using O, 20 ml of a 5 mM solution of (4-mercapto) benzoic acid at pH 10 was prepared. To this solution was added Tris-octylphosphine oxide coated CdSe.
/ CdS (nucleus / sheath) nanocrystal 20mg,
Then, the mixture was stirred until it was completely dissolved. Heating the resulting nanocrystal / linker solution at 50-60 ° C. for 5 hours,
Then, it was concentrated to a few ml by evaporation. Then, an equal volume of acetone was added and the nanocrystals precipitated uniformly from the solution. The precipitate may then be washed with acetone, dried, and then stored.

The prepared luminescent semiconductor nanocrystal compound can be linked to a suitable affinity molecule and the organic luminescent compound of the present invention for processing biological material to determine the presence or absence of a detectable substance. A semiconductor nanocrystal probe can be formed. That is, the luminescent semiconductor nanocrystal compound prepared above can be linked to, for example, avidin (as an affinity molecule) or streptavidin,
Biological materials can be processed to form organic luminescent semiconductor nanocrystal probes to confirm the presence of biotin. Alternatively, the prepared luminescent semiconductor nanocrystal compound can be linked to anti-digoxiginene, and the biological material can be processed to form an organic luminescent semiconductor nanocrystal probe to confirm the presence of digoxiginene.

(Example 2) (Glass core connected to a linking agent)
Luminescent semiconductor (containing coated semiconductor nanocrystals)
To illustrate the formation of somatic nanocrystal compounds, meta
Aqueous solution of 25% by volume dimethyl sulfoxide in ethanol
In 40 ml, add 3-mercaptopropyl-trimethoxy
50 μl of silane are added and the pH is adjusted to (CH_Three)_FourN
OH ・ 5H _TwoIt adjusted to 10-11 using O. Next
In this solution, tris-octylphosphine oxide
Coated CdSe / CdS (core / sheath) particles (this
Is the Peng, Schlamp, Kadavani
ch and Alivisatos
Dissolve 10 mg (prepared by surgery) and add several hours
While stirring. The solution was washed with (CH_Three)_FourNOH ・ 5H_TwoO and p
Dilute with 40 ml of methanol adjusted to H10.
And heated at 69 ° C. for 1 hour. The solution was stirred for 1 hour,
0% by volume methanol / 9.89% by volume H_TwoO / 0.1
Volume% trimethoxysilylpropyl urea / 0.01 volume
% Aminopropyl-trimethoxysilane solution (this is
40 ml) (stirred for at least one hour) and add
And stirred for 2 hours. Subsequently, the reaction was allowed to proceed for 15 minutes.
Over time, heated to 69 ° C. and then cooled.
10% by volume chlorotrimethylsilane solution in methanol
(This is (CH_Three)_FourNOH ・ 5H_TwoPH using O
 Add 10 ml (adjusted to 10) and stir for 2 hours
And then heated to 60 ° C. and then under vacuum,
Partially concentrated. Once evaporate all methanol
Solution containing the luminescent semiconductor nanocrystal compound
As an oily product to be precipitated with acetone. Glow
Semiconductor nanocrystal compounds are then
Can be redissolved in a buffer solution of
Link to affinity molecules to process biological material for detection
Of the present invention to determine the presence or absence of an active substance.
For forming electroluminescent semiconductor nanocrystal probes
Prepare.

Thus, the present invention provides that when excited by either an electromagnetic radiation source (wide or narrow bandwidth) or a particle beam, it can emit narrow wavelength band electromagnetic radiation and / or absorb energy. Provided is an organic light emitting semiconductor nanocrystal probe comprising a semiconductor nanocrystal which can scatter or diffract the obtained and / or excitations. This allows the simultaneous use of multiple such probe materials that emit different wavelengths of electromagnetic radiation, thereby simultaneously detecting the presence of multiple detectable substances within a given material. Becomes possible. The probe material is stable in the presence of light or oxygen, can be excited with a wide spectrum of energy, and has a narrow emission band, so that multiple detectables in materials such as biological materials Improved materials and processes for simultaneous and / or sequential detection of substances are obtained.

[0098]

According to the present invention, the present invention has a wide absorption band and exhibits a detectable change in absorption or a narrow wavelength band without the presence of the large red emission tail characteristic of dye molecules. Can emit radiation (which allows multiple such probe materials to be used simultaneously, each emitting light in a different narrow wavelength band) and / or can scatter or diffract the radiation, A stable probe material for biological applications is provided. Also, both light and electron microscopy provide a single, stable probe material that can be used to image the same sample.

In summary, described herein are luminescent semiconductor nanocrystal compounds that can bind to affinity molecules. This compound contains: (1)
Can emit (emit) narrow wavelength band electromagnetic radiation and / or absorb energy when excited by an electromagnetic radiation source (wide or narrow bandwidth) or particle beam, and / or electromagnetic radiation And (2) at least one linking agent having a first portion linked to the semiconductor nanocrystal and a second portion linked to an affinity molecule. When excited by an electromagnetic radiation source (either broad or narrow bandwidth) or a particle beam, the luminescent semiconductor nanocrystal compound can bind to a detectable substance in the analyzed material by binding to an affinity molecule. And can emit organic radiation of narrow wavelength width and / or form organic light emitting semiconductor nanocrystal probes that can absorb, scatter, or diffract energy. Probes are stable to repeated exposure to light in the presence of oxygen and / or other radicals.

The treatment of the material with the organic luminescent semiconductor nanocrystal probe and subsequent exposure of the treated material to the excitation energy to determine the presence of a detectable substance in the material bound to the probe is detected. Exciting the semiconductor nanocrystals in the probe bound to the possible substance, causing emission of narrow wavelength bands of electromagnetic radiation and / or detectable absorption and / or scattering or diffraction of energy, in each case ,
This implies the presence of a detectable substance in the material bound to the organic light emitting semiconductor nanocrystal probe. Since the semiconductor nanocrystals in the probe can be excited over a wide bandwidth of energy and emit electromagnetic radiation in a narrow bandwidth, a single energy source can be used to simultaneously emit electromagnetic radiation in different wavelength bands. Exciting multiple such probes to emit,
It is possible to analyze multiple detectable substances in the substance to be analyzed simultaneously.

Further, a process for making a light emitting semiconductor nanocrystal compound and for making an organic light emitting semiconductor nanocrystal probe comprising a light emitting semiconductor nanocrystal compound linked to an affinity molecule capable of binding to a detectable substance. Is described. A process for using a probe to determine the presence of a detectable material in a material is also described.

[Brief description of the drawings]

FIG. 1 is a block diagram of a light emitting semiconductor nanocrystal compound of the present invention.

FIG. 2 is a block diagram of an organic light emitting semiconductor nanocrystal probe of the present invention.

FIG. 3 is a block diagram showing an affinity between an organic light emitting semiconductor nanocrystal probe of the present invention and a detectable substance.

FIG. 4 is a flow sheet illustrating a process for forming the organic light emitting semiconductor nanocrystal probe of the present invention.

FIG. 5 is a flow sheet illustrating an exemplary use of an organic light emitting semiconductor nanocrystal probe of the present invention in detecting the presence of a detectable substance in a material such as a biological material.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/53 G01N 33/53 S 33/543 541 33/543 541Z 33/552 33/552 // C12Q 1 / 68 C12Q 1/68 Z (72) Inventor Marcel Bruches Jr. United States of America California 94706, Albany, Hillside Avenue 816 (72) Inventor Paul Alivisatos United States of America 94611, Auckland, Estates Drive 5941 F-term (reference) 2G043 AA01 BA16 CA03 DA02 EA01 GA07 GB28 KA02 KA09 2G045 AA40 BA13 BB01 BB07 BB46 BB48 BB51 FA11 FA12 FA13 FA14 FA16 FA25 FA26 FA27 FA29 FB07 GC11 GC15 GC30 2G054 AA06 AB07 CA21 EA05 FA28 GA01 GA02 GA03 GA04 4B06QAQ3 QRAQ3 QRAQ3

Claims (51)

[Claims] 1. A luminescent semiconductor nanocrystal compound which can be linked to an affinity molecule and emits electromagnetic radiation in a narrow wavelength band when excited, said compound comprising the following a) and b): To: a) a semiconductor nanocrystal that can emit light in a narrow wavelength band when excited; and b) at least one linking agent that can be linked to the semiconductor nanocrystal and linked to the affinity molecule. 2. The light-emitting semiconductor nanocrystal compound according to claim 1, wherein the semiconductor nanocrystal is capable of absorbing energy over a wide bandwidth. 3. The linking agent contains the glass coating on the semiconductor nanocrystal, which can link to the affinity molecule via a further linking agent that can link both the glass coating and the affinity molecule. , Claim 1
3. The light-emitting semiconductor nanocrystal compound according to item 1. 4. The luminescent semiconductor nanocrystal compound of claim 1, wherein said glass coating on said semiconductor nanocrystal comprises a coating of silica glass. 5. The luminescent semiconductor nanocrystal compound according to claim 1, wherein the linking agent contains a first portion linked to the semiconductor nanocrystal and a second portion capable of linking to the affinity molecule. 6. The method of claim 1, wherein the one or more linking agents include a glass coating on the semiconductor nanocrystal and a linking material, wherein the linking material is linked to a glass coating on the semiconductor nanocrystal. A moiety and a second moiety capable of linking to the affinity molecule,
The light-emitting semiconductor nanocrystal compound according to claim 1. 7. An organic light-emitting semiconductor nanocrystal probe capable of binding to a detectable substance and emitting electromagnetic radiation in a narrow wavelength band when excited, comprising an affinity molecule capable of binding to the detectable substance. A probe containing a linked luminescent semiconductor nanocrystal compound. 8. An organic light-emitting semiconductor nanocrystal probe capable of binding to a detectable substance and emitting, when excited, a narrow wavelength band of electromagnetic radiation, the probe comprising the following a), b) and c) thereby treating the material with the organic light-emitting semiconductor nanocrystal probe to determine the presence of the detectable substance in the material, and subsequent to the excitation energy of the treated material Exposure excites the semiconductor nanocrystals in the organic light-emitting semiconductor nanocrystal probe bound to the detectable substance, causing emission of a narrow wavelength band of electromagnetic radiation, which in the material, Means the presence of the detectable substance bound to the organic light emitting semiconductor nanocrystal probe: a) When excited, a narrow wavelength band of A) a semiconductor nanocrystal capable of emitting magnetic radiation; b) at least one linking agent linked to said semiconductor nanocrystal and having a second moiety capable of linking to an affinity molecule; and c) said linking agent. An affinity molecule that binds to the second portion of the linking agent and is capable of selectively binding to the detectable substance. 9. The organic light emitting semiconductor nanocrystal probe according to claim 8, wherein the linking agent comprises a glass coating on the semiconductor nanocrystal. 10. The material treated with the organic light emitting semiconductor nanocrystal probe to determine the presence of the detectable substance, comprises a biological material.
2. The organic light emitting semiconductor nanocrystal probe according to item 1. 11. The organic light emitting semiconductor nanocrystal probe according to claim 8, wherein the material treated with the organic light emitting semiconductor nanocrystal probe to determine the presence of the detectable substance comprises an organic material. 12. The organic light emitting semiconductor nanocrystal probe of claim 8, wherein the material treated with the organic light emitting semiconductor nanocrystal probe to determine the presence of the detectable substance comprises an inorganic material. 13. A process for forming a luminescent semiconductor nanocrystal compound that can be linked to an affinity molecule and emit electromagnetic radiation in a narrow wavelength band when excited, comprising a narrow wavelength band when excited. A process comprising linking together a semiconductor nanocrystal capable of emitting electromagnetic radiation and a linking agent having a first portion linked to said semiconductor nanocrystal and a second portion linked to an affinity molecule. 14. The process of claim 13, further comprising forming a glass coating on the semiconductor nanocrystal and then treating the glass with a linking agent capable of linking with affinity molecules. . 15. A process for forming an organic light emitting semiconductor nanocrystal probe capable of binding to a detectable substance and emitting electromagnetic radiation in a narrow wavelength band when excited, comprising an affinity capable of binding to the detectable substance. Sex molecules,
A process comprising linking a light-emitting semiconductor nanocrystal compound. 16. A process for forming an organic light emitting semiconductor nanocrystal probe capable of binding to a detectable substance and emitting, when excited, a narrow wavelength band of electromagnetic radiation, the process comprising the following steps: Includes: a) a semiconductor nanocrystal capable of emitting a narrow wavelength band of electromagnetic radiation when excited, having a first portion linked to the semiconductor nanocrystal and a second portion linked to an affinity molecule. Linking to a linking agent; and b) linking the linking agent to an affinity molecule capable of binding to the detectable substance. 17. The process of claim 16, wherein linking the semiconductor nanocrystal and the linking agent together occurs before linking the linking agent and the affinity molecule together. 18. The step of linking the linking agent and the affinity molecule together is performed before the step of linking the semiconductor nanocrystal and the linking agent together,
The process of claim 16. 19. The step of linking the semiconductor nanocrystal and the linking agent together further comprises: coating the semiconductor nanocrystal with glass; and then bonding the glass-coated semiconductor nanocrystal to the affinity layer. 17. The process of claim 16, comprising treating with a linking agent capable of linking the molecule. 20. A process for treating a material to determine the presence of one or more detectable substances in the material, the process comprising the following steps: a), b), c) and d).
A) a first material capable of binding the material, if present, to a first detectable substance in the material and emitting electromagnetic radiation of a first narrow wavelength band when excited; Contacting with an organic light emitting semiconductor nanocrystal probe, wherein the first organic light emitting semiconductor nanocrystal probe comprises the following i), ii) and iii):
i) a first semiconductor nanocrystal that can be excited over a wide bandwidth and, when excited, emits the first narrow wavelength band of electromagnetic radiation; ii) selectively binds to the detectable substance. Iii) a linking agent linked to the first semiconductor nanocrystal and also linked to the affinity molecule; and b) from the material the first organic light-emitting semiconductor nanocrystal probe from the material. Removing a portion that is not bound to a first detectable substance; and c) exposing the material to energy capable of exciting the first semiconductor nanocrystal to cause the first semiconductor nanocrystal to be exposed to the first semiconductor nanocrystal. Emitting said first narrow wavelength band of electromagnetic radiation indicating the presence of a detectable substance; and d) said first semiconductor nanocrystals in said first organic light emitting semiconductor nanocrystal probe. The step of detecting the electromagnetic radiation of more emitted said first narrow wavelength band. 21. An additional step of treating said material with at least a second organic light emitting semiconductor nanocrystal probe capable of binding to a further detectable substance in said material, and comprising: The crystal probe contains a second semiconductor nanocrystal that can be excited with a wide bandwidth and emit electromagnetic radiation in a second narrow wavelength band different from the first narrow wavelength band, thereby Exposure of the material to energy capable of exciting both the first and second nanocrystals comprises:
Either the first or second semiconductor nanocrystals present in the material emit electromagnetic radiation of a different narrow wavelength band, whereby the presence of more than one detectable substance in the material 21. The process of claim 20, wherein the absence can be detected simultaneously using a single excitation energy source. 22. At least one additional organic light emitting semiconductor nanocrystal probe is used to process said material, each of said organic light emitting semiconductor nanocrystal probes being capable of selectively binding to a different detectable substance. And each of the organic light emitting semiconductor nanocrystal probes can be excited over a wide wavelength range,
22. The process of claim 21, wherein electromagnetic radiation of different and narrow wavelength bands can be emitted, such that multiple detectable substances can be analyzed in the material simultaneously using a single excitation source. 23. The method of claim 19, wherein the material is removed from the material prior to the step of removing a portion of the first organic light emitting semiconductor nanocrystal probe that is not bound to the first detectable substance. Treating with all of the nanocrystal probes, and the removing step further comprises removing a portion of all of the organic light emitting semiconductor nanocrystal probes that is not bound to a detectable substance in the material. A process according to claim 21. 24. Exposure to light of a selected wavelength of the material is thereby used to selectively excite one or more, but not all, of the organic light emitting semiconductor nanocrystal probes, and therefore 21. The method of claim 21, wherein the identification of the presence of a particular labeled detectable substance or a subset of different labeled detectable substances in the material is enabled.
24. The process according to 2 or 23. 25. The process for treating a material according to claim 20, wherein said material comprises a biological material. 26. The step of exposing the material to energy capable of exciting the first semiconductor nanocrystals to emit electromagnetic radiation, further comprising: emitting the material with broad or narrow spectrum of photons. 21. The process for treating a material of claim 20, comprising exposing to a source of electromagnetic radiation. 27. Exposing the material to energy capable of exciting the first semiconductor nanocrystal to emit electromagnetic radiation further comprises exposing the material to an electron beam. A process for treating a material according to claim 20. 28. A process for treating a material to determine the presence of a detectable substance in the material, the process comprising the following a), b), c) and d): a) contacting the material with an organic light-emitting semiconductor nanocrystal probe capable of binding to a first detectable substance in the material, if present, and absorbing energy when excited; The organic light-emitting semiconductor nanocrystal probe contains the following i), ii) and iii): i) a first semiconductor nanocrystal that can be excited over a wide bandwidth and, when excited, can absorb energy; ii. A) an affinity molecule capable of selectively binding to the detectable substance; and iii) a linking agent linked to the first semiconductor nanocrystal and also linked to the affinity molecule; Removing from the sample the portion of the organic light emitting semiconductor nanocrystal probe that is not bound to the first detectable substance; and c) converting the material to an energy capable of exciting the first semiconductor nanocrystal. Exposing to absorb energy and indicating the presence of the first detectable substance in the material; and d) detecting the organic light emitting semiconductor nanocrystal probe bound to the detectable substance in the material. Detecting a change in absorbed energy that is indicative of its presence. 29. The process of claim 28, further comprising treating the material with at least a second organic light emitting semiconductor nanocrystal probe capable of binding to a further detectable substance in the material. Wherein the second organic light-emitting semiconductor nanocrystal probe comprises a second semiconductor nanocrystal that can be excited with a wide bandwidth, resulting in a detectable absorbance change, whereby the first Exposure of the material to energy that can excite both the first and second nanocrystals causes electromagnetic radiation in different wavelength bands to be applied to either the first or second semiconductor nanocrystals present in the material. Absorb,
A process whereby the presence or absence of more than one detectable substance in a material can be detected simultaneously using a single excitation energy source. 30. At least one additional organic light emitting semiconductor nanocrystal probe is used to treat said material, each of said organic light emitting semiconductor nanocrystal probes being capable of selectively binding to a different detectable substance. And each of the organic light emitting semiconductor nanocrystal probes can be excited over a wide wavelength range and can absorb electromagnetic radiation, so that multiple detectable substances can be incorporated into the material using a single excitation source. 30. The process of claim 29, which can be analyzed simultaneously. 31. The step of exposing the material to energy capable of exciting the first semiconductor nanocrystals to emit electromagnetic radiation further comprises emitting the material with broad or narrow spectrum of photons. 29. The method of claim 28, comprising exposing to a source of electromagnetic radiation.
Process for processing materials. 32. The step of exposing the material to energy capable of exciting the first semiconductor nanocrystal to emit electromagnetic radiation further comprises exposing the material to an X-ray source. 29. The process for treating a material according to claim 28. 33. A process for treating a material to determine the presence of a detectable substance in the material, the process comprising the following a), b), c) and d): a) contacting the material with an organic light emitting semiconductor nanocrystal probe capable of binding to a first detectable substance in the material, if present, and capable of scattering or diffracting energy when excited. The organic light emitting semiconductor nanocrystal probe comprises the following i), ii) and iii): i) a first semiconductor nanocrystal that can scatter or diffract over a wide bandwidth with a characteristic cross section; ii) An affinity molecule capable of selectively binding to the detectable substance; and iii) a linking agent linked to the first semiconductor nanocrystal and also linked to the affinity molecule; b) Removing from the material a portion of the organic light-emitting semiconductor nanocrystal probe that is not bound to the first detectable substance; and c) exposing the material to energy capable of exciting the first semiconductor nanocrystal. Scattering or diffracting energy to indicate the presence of the first detectable substance in the material; and d) detecting a change in the scattered or diffracted energy and Indicating the presence of the organic light emitting semiconductor nanocrystal probe bound to the detectable substance. 34. The method further comprising treating the material with at least a second organic light emitting semiconductor nanocrystal probe capable of binding to a second detectable substance in the material, wherein the second organic light emitting semiconductor The nanocrystal probe also contains a second semiconductor nanocrystal that can scatter or diffract energy, resulting in a detectable scattering cross-section change, whereby the first or second of the material Exposure to energy that can scatter or diffract both of the nanocrystals is characteristic of a particular organic light emitting semiconductor nanocrystal probe in either the first and second semiconductor nanocrystals present in the material. Scatters or diffracts energy at different scattering cross-sections, such that the presence or absence of more than one detectable substance in the material is a single 34. The process of claim 33, which can be detected simultaneously using a source of excitation energy. 35. At least one additional organic light emitting semiconductor nanocrystal probe is used to process said material, each of said organic light emitting semiconductor nanocrystal probes being capable of selectively binding to a different detectable substance. And each of the organic light-emitting semiconductor nanocrystal probes exhibits a different scattering cross-section and can scatter or diffract energy, so that multiple detectable substances can be combined within the material using a single excitation source. 35. The process of claim 34, which can be analyzed simultaneously. 36. The step of exposing the material to energy capable of exciting the first semiconductor nanocrystal to scatter or diffract the energy further comprises exposing the material to an electron beam or other particle beam. 34. The process for treating a material according to claim 33, comprising exposing. 37. The step of exposing the material to energy capable of exciting the first semiconductor nanocrystal to scatter or diffract energy further comprises exposing the material to an X-ray source. 34. The process for treating a material according to claim 33, comprising. 38. The step of exposing the material to energy capable of exciting the first semiconductor nanocrystal to scatter or diffract energy and the step of detecting the scatter or diffraction of energy comprises: 34. The process for processing a material according to claim 33, performed using a transmission electron microscope. 39. The step of exposing the material to energy capable of exciting the first semiconductor nanocrystal to scatter or diffract energy and the step of detecting scatter or diffraction of the energy comprises: ,
34. The process for processing a material according to claim 33, wherein the process is performed using a scanning electron microscope. 40. A luminescent semiconductor nanocrystal compound capable of linking to an affinity molecule and absorbing energy of a narrow wavelength band when excited, said compound comprising the following a) and b): A) a semiconductor nanocrystal capable of absorbing energy in a narrow wavelength band when excited; and b) at least one linking agent linking the semiconductor nanocrystal and linking the affinity molecule. Linking agent. 41. A light-emitting semiconductor nanocrystal compound capable of linking to an affinity molecule and scattering or diffracting energy in a narrow wavelength band when excited, wherein the compound has the following a) and b): Contains: a) a semiconductor nanocrystal that can scatter or diffract energy in a narrow wavelength band when excited; and b) a linking agent that links to the semiconductor nanocrystal and can link to the affinity molecule. At least one linking agent; 42. An organic luminescent semiconductor nanocrystal probe capable of binding to a detectable substance and absorbing energy in a narrow wavelength band when excited, said probe comprising an affinity capable of binding to said detectable substance. A probe containing a luminescent semiconductor nanocrystal compound linked to a hydrophilic molecule. 43. An organic light emitting semiconductor nanocrystal probe capable of binding to a detectable substance and absorbing energy in a narrow wavelength band when excited, said probe comprising the following a), b) and c: ), Whereby the treatment of the material with the organic luminescent semiconductor nanocrystal probe and the excitation energy of the treated material to determine the presence of the detectable substance in the material Exposure excites the semiconductor nanocrystals in the organic luminescent semiconductor nanocrystal probe bound to the detectable substance, causing absorption of a narrow wavelength band of energy, which within the material, Implies the presence of the detectable substance bound to a luminescent semiconductor nanocrystal probe: a) energy in a narrow wavelength band when excited B) at least one linking agent that links the semiconductor nanocrystal and has a second moiety that can link to an affinity molecule; and c) the linking. An affinity molecule that is linked to the second part of the agent and is capable of selectively binding to the detectable substance. 44. An organic light emitting semiconductor nanocrystal probe capable of binding to a detectable substance and scattering or diffracting energy in a narrow wavelength band when excited, wherein the affinity molecule is capable of binding to the detectable substance. A probe comprising a luminescent semiconductor nanocrystal compound linked to a probe. 45. An organic light emitting semiconductor nanocrystal probe capable of binding to a detectable substance and scattering or diffracting energy in a narrow wavelength band when excited, said probe comprising the following a), b) And c) thereby treating the material with the organic light emitting semiconductor nanocrystal probe and the excitation energy of the treated material to determine the presence of the detectable substance in the material. Subsequent exposure excites the semiconductor nanocrystals in the organic luminescent semiconductor nanocrystal probe bound to the detectable substance, causing the scattering or diffraction of energy in a narrow wavelength band, which causes Means the presence of the detectable substance bound to the organic light emitting semiconductor nanocrystal probe: a) When excited A semiconductor nanocrystal capable of scattering or diffracting energy in a narrow wavelength band; b) at least one type of linking agent that links the semiconductor nanocrystal and has a second moiety that can link to an affinity molecule And c) an affinity molecule that links to the second portion of the linking agent and is capable of selectively binding to the detectable substance. 46. A process for forming a luminescent semiconductor nanocrystal compound that can be linked to an affinity molecule and, when excited, absorb energy in a narrow wavelength band, the process comprising: A semiconductor nanocrystal capable of absorbing energy in a narrow wavelength band when it is formed, and a linking agent having a first portion linked to the semiconductor nanocrystal and a second portion linked to an affinity molecule are linked together. Process. 47. A process for forming a luminescent semiconductor nanocrystal compound that can be linked to an affinity molecule and, when excited, scatter or diffract energy in a narrow wavelength band, the process comprising: A semiconductor nanocrystal capable of scattering or diffracting energy in a narrow wavelength band when excited, and a linking agent having a first portion linked to the semiconductor nanocrystal and a second portion linked to an affinity molecule. Joining together. 48. A process for forming an organic light emitting semiconductor nanocrystal probe capable of binding to a detectable substance and absorbing energy in a narrow wavelength band when excited, comprising an affinity capable of binding to the detectable substance. In the molecule,
A process comprising linking a light emitting semiconductor nanocrystal compound. 49. A process for forming an organic light emitting semiconductor nanocrystal probe capable of binding to a detectable substance and absorbing energy in a narrow wavelength band when excited, said process comprising the steps of: A) a linking agent having a first portion linked to a semiconductor nanocrystal capable of absorbing energy in a narrow wavelength band when excited, and a second portion linked to an affinity molecule; And b) linking the linking agent with an affinity molecule capable of binding to the detectable substance. 50. A process for forming an organic light emitting semiconductor nanocrystal probe capable of binding to a detectable substance and scattering or diffracting energy in a narrow wavelength band when excited, wherein the probe may bind to the detectable substance. A process comprising linking a luminescent semiconductor nanocrystal compound with an affinity molecule. 51. A process for forming an organic light emitting semiconductor nanocrystal probe capable of binding to a detectable substance and scattering or diffracting energy in a narrow wavelength band when excited, said process comprising the following steps: A) a semiconductor nanocrystal capable of scattering or diffracting energy in a narrow wavelength band when excited, a first portion linked to the semiconductor nanocrystal and a second portion linked to an affinity molecule. And b) linking the linking agent with an affinity molecule capable of binding to the detectable substance.