Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1999-03-30
2001-05-22
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C427S002130, C427S002220, C427S212000, C427S213300, C427S215000, C427S216000, C427S220000, C427S414000, C428S402000, C428S403000, C436S524000, C436S525000
Reexamination Certificate
active
06235540
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to methods of making semiconductor nanoparticle compounds useful in the analysis of blood cell populations, and particularly to compounds which contain amino-derivatized polysaccharides.
BACKGROUND OF THE INVENTION
Multiplex labeling of cells for analysis of biological samples, e.g., mixed cell populations, has been described. However, known methods have limitations which are dictated by the finite number of fluorescence emission colors of known organic fluorophores which can be squeezed into the visible, near-ultraviolet (UV), near-infrared (IR) spectral regions in which conventional measurements are made, e.g., by flow cytometry. These limitations include the widths of emission bands, the spectral overlap between these emission bands, and the excitation wavelength requirements.
Two examples of labels for cells are CdSe core nanoparticles which have been used for biological staining and observation with a fluorescence microscope [Bruchez J. M. et al., Science 281, 2013 (1998) and Chan, W. C. W. and Nie, S., Science 281, 2016 (1998)].
As the upper limit in the number of usable colors was reached, other methods, based on fluorescence intensity differences have been developed. For example, mutually exclusive pairs of targeted white blood cell populations with widely different, intrinsic numbers of receptors per cell can be labeled by a single color marker and analyzed by flow cytometry [U.S. Pat. No. 5,538,855].
Several non-radioactive gene probes, oligos with attached fluorescent dye that hybridize or bind to sample DNA have been described [L. M. Smith et al.,
Nature
, 321:674-679 (1986) and L. M. Smith et al,
Nucl. Acids Res
., 13:2399-2412 (1985)] and are being used for labeling of biological samples. Automated DNA sequencers use four fluorescent dyes with non-overlapping emission bands, one per nucleotide base. However, electrophoretic mobilities of the fluorescent dye-oligo primer conjugates need to be similar for all four conjugates. Also the molecular weight of the conjugates cannot be too high, otherwise they will not move through the polyacrylamide or agarose gel used in the electrophoresis.
The need for increased sensitivity of probes used in automated analysis by attaching multiple marker molecules per oligonucleotide primer were recognized as early as 1986 [L. M. Smith et al, cited above]. However, only a limited degree of fluorescence enhancement has been possible for dye-oligo conjugates that are constrained to low molecular weight for separation by gel electrophoresis.
Aminodextrans have been used as reducing and/or protective agents in the preparation or coating of monodispersed colloidal dispersions of magnetic ferrite [U.S. Pat. No. 5,240,640], metal [U.S. Pat. No. 5,248,772], polystyrene [U.S. Pat. No. 5,466,609; U.S. Pat. No. 5,707,877; U.S. Pat. No. 5,639,620; U.S. Pat. No. 5,776,706], and polystyrene-metal [U.S. Pat. No. 5,552,086; U.S. Pat. No. 5,527,713] particles. Aminodextran of sufficiently large molecular weight can accommodate multiple protein molecules. Complexes containing such aminodextrans conjugated to a ligand and a selected fluorescent marker or label have been described. [See, Smith, C., et al, “Detection of Low-Density Surface Markers Using Novel Amplified Fluorochrome-Conjugated Antibodies”, Cytometry, Suppl. 9, p. 56, presented at XIX Congress of International Society for Analytical Cytology, Mar. 3-7, 1998; R. Mylvaganam, et al., “Seven Markers, Four Colors, Single Laser Flow Cytometry Using Amplified Fluorochrome Conjugated Antibodies”,
Cytometry
, Suppl. 9, p. 117 (1998), as presented at XIX Congress of International Society for Analytical Cytology, Mar. 3-7, 1998.] However, there continues to be a need for probes which permit increased assay sensitivity, by providing narrower fluorescence bandwidths and enhanced intensities, decreased probe size and increased probe stability.
SUMMARY OF THE INVENTION
The present invention advantageously provides particles which are of a smaller size than previously described labeled aminodextran complexes. The nanoparticles of the invention are firther coated with an aminodextran of high degree of substitution to provide higher luminescence intensity than was previously possible in a single small particle.
Thus, in one aspect, the present invention provides a semiconductor nanoparticle for the analysis of fluid samples. The semiconductor nanoparticle contains a water soluble amino derivative of a polysaccharide having a molecular weight from approximately 3,000 to 3,000,000 Da, a size in diameter of less than approximately 150 nanometers, and a degree of substitution of total number of primary and secondary amino groups in the polysaccharide molecule ranging from 1/150 to 4/1. To form the semiconductor nanoparticle, this aminopolysaccharide is linked to at least one nanoparticle of the formula:
(XY)
n
wherein X is selected from the group comprising Cd
2+
, Hg
2+
, and Zn
2+
and combinations thereof, and Y is selected from the group comprising S
2−
, Se
2−
and Te
2−
and combinations thereof, and n=approximately 50 to 1000.
In another aspect, the present invention provides a semiconductor nanoparticle useful for the analysis of biological samples which is bound to an aminodextran having a molecular weight from approximately 3,000 to 500,000 Da, has a size in diameter of 2 to about 10 nanometers, and a degree of substitution of total number of primary and secondary amino groups in the dextran molecule ranging from 1/150 to 4/1. The aminodextran is covalently bound to at least one nanoparticle, which is defined as above.
In yet another aspect, the present invention provides a method of making a semiconductor nanoparticle. This method involves the steps of reacting an amino derivative of a polysaccharide having a molecular weight from approximately 3,000 to 3,000,000 Da with a Periodic Table Group lIB water soluble salt and a Group VIA salt to form a semiconductor nanoparticle. In this method, the semiconductor nanoparticle is a complex of the amino derivative of a polysaccharide and a nanoparticle. The aminopolysaccharide has a diameter of less than approximately 150 nanometers and a degree of substitution of total number of primary and secondary amino groups in the polysaccharide molecule ranging from 1/150 to 4/1. The Group IIB salt having a cation selected from the group consisting of Cd
2+
, Hg
2+
, and Zn
2+
and combinations thereof; and an anion selected from the group consisting Cl
−
, ClO
4
−
, NO
3
−
and SO
4
2−
; said Group VIA water soluble salt having a cation selected from the group consisting of Li
+
, Na
+
, K
+
, Rb
+
, Cs
+
, Sr
2+
, and Ba
2+
and an anion selected from the group consisting of S
2−
, Se
2−
and Te
2−
and combinations thereof; such that the anion selected for the Group IIB salt does not precipitate with the cation of the Group VIA salt. In this method, the presence of the reducing sugar in the polysaccharide retards photo-oxidation of the nanoparticle in the formed polysaccharide semiconductor nanoparticle complex.
In yet a flirther aspect, the present invention provides a method of making a semiconductor nanoparticle useful in the analysis of biological samples. This method involves mixing an amino derivative of a polysaccharide having a molecular weight from approximately 3,000,000 Da, has a size in diameter of less than approximately 150 nanometers, and a degree of substitution of total number of primary and secondary amino groups in the polysaccharide molecule ranging from 1/150 to 4/1, a water soluble first salt having a cation selected from the group consisting of Cd
2+
, Hg
2+
, and Zn
2+
and combinations thereof; and an anion selected from the group consisting Cl
−
, ClO
4
−
, NO
3
−
and SO
4
2−
, and a water soluble second salt having a catio
Matijevic Egon
Siiman Olavi
Sondi Ivan
Alter Mitchell E.
Chin Christopher L.
Coulter International Corp.
Kodroff Cathy A.
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