Optics: measuring and testing – By dispersed light spectroscopy – With sample excitation
Reexamination Certificate
2001-01-19
2002-10-15
Evans, F. L. (Department: 2877)
Optics: measuring and testing
By dispersed light spectroscopy
With sample excitation
C250S458100, C356S073000, C356S417000
Reexamination Certificate
active
06466316
ABSTRACT:
CROSS-REFERENCES TO RELATED MATERIALS
This application incorporates by reference the following U.S. patent applications: Ser. No. 09/156,318, filed Sep. 18, 1998; and Ser. No. 09/349,733, filed Jul. 8, 1999.
This application also incorporates by reference the following PCT patent applications: Ser. No. PCT/US98/23095, filed Oct. 30, 1998; Ser. No. PCT/US99/01656, filed Jan. 25, 1999; Ser. No. PCT/US99/03678, filed Feb. 19, 1999; Ser. No. PCT/US99/08410, filed Apr. 16, 1999; Ser. No. PCT/US99/16057, filed Jul. 15, 1999; and Ser. No. PCT/US99/16453, filed Jul. 21, 1999.
This application also incorporates by reference the following U.S. provisional patent applications: Ser. No. 60/094,275, filed Jul. 27, 1998; Ser. No. 60/094,276, filed Jul. 27, 1998; Ser. No. 60/094,306, filed Jul. 27, 1998; Ser. No. 60/1,00,817, filed Sep. 18, 1998; Ser. No. 60/100,951, filed Sep. 18, 1998; Ser. No. 60/104,964, filed Oct. 20, 1998; Ser. No. 60/114,209, filed Dec. 29, 1998; Ser. No. 60/116,113, filed Jan. 15, 1999; Ser. No. 60/117,278, filed Jan. 26, 1999; Ser. No. 60/119,884, filed Feb. 12, 1999; Ser. No. 60/121,229, filed Feb. 23, 1999; Ser. No. 60/124,686, filed Mar. 16, 1999; Ser. No. 60/125,346, filed Mar. 19, 1999; Ser. No. 60/126,661, filed Mar. 29, 1999; Ser. No. 60/130,149, filed Apr. 20, 1999; Ser. No. 60/132,262, filed May 3, 1999; Ser. No. 60/132,263, filed May 3, 1999; Ser. No. 60/135,284, filed May 21, 1999; Ser. No. 60/136,566, filed May 28, 1999; Ser. No. 60/138,311, filed Jun. 9, 1999; Ser. No. 60/138,438, filed Jun. 10, 1999; Ser. No. 60/138,737, filed June 11, 1999; Ser. No. 60/138,893, filed Jun. 11, 1999; and Ser. No. 60/142,721, filed Jul. 7, 1999.
This application also incorporates by reference the following publications: Max Born and Emil Wolf,
Principles of Optics
(6
th
ed. 1980); Richard P. Haugland,
Handbook of Fluorescent Probes and Research Chemicals
(6
th
ed. 1996); and Joseph R. Lakowicz,
Principles of Fluorescence Spectroscopy
(1983).
FIELD OF THE INVENTION
The invention relates to spectroscopic assays. More particularly, the invention relates to apparatus and methods for conducting spectroscopic measurements, including absorbance, scattering, reflectance, and luminescence.
BACKGROUND OVERVIEW OF SPECTROSCOPIC ASSAYS
Generally speaking, spectroscopy involves the study of matter using electromagnetic radiation. Spectroscopic measurements can be separated into three broad categories: absorbance, scattering/reflectance, and emission. Absorbance assays involve relating the amount of incident light that is absorbed by a sample to the type and number of molecules in the sample. Absorbance assays are a powerful method for determining the presence and concentration of an analyte in a sample. Most commonly, absorbance is measured indirectly by studying the portion of incident light that emerges from the sample. Scattering assays are similar to absorbance in that the measurement is based on the amount of incident light which emerges or is transmitted from the sample. However, in the case of scattering, the signal increases with the number of interactions, whereas, in the case of absorbance, the signal is inversely proportional to the interactions. Emission assays look at electromagnetic emissions from a sample other than the incident light. In each case, the measurements may be broad spectrum or frequency specific depending on the particular assay.
1. Absorbance Assays
FIG. 1
shows a schematic view of a typical absorbance experiment. Generally, absorbance measurements are made by directing incident light from a light source through a sample and through two walls of a sample container, and measuring the transmitted light using a detector. Unfortunately, this approach has a number of shortcomings. In particular, the sample container may absorb part or all of the incident and transmitted light, decreasing or eliminating the sample signal and increasing the background signal. Moreover, correcting for absorbance by the sample container requires the performance of two experiments, one involving the sample and sample container, and the other involving only the sample container.
The amount of light absorbed by a sample in an absorbance experiment generally may be described by the Beer-Lambert law:
Absorbance
=
-
log
(
I
(
λ
)
I
0
(
λ
)
)
=
ϵ
(
λ
)
c1
(
1
)
The Beer-Lambert law states that when light of wavelength &lgr; passes through an absorbing sample, its intensity, I, decreases exponentially. Here, I
0
(&lgr;) is the intensity of the incident light at wavelength &lgr;, I(&lgr;) is the intensity of the transmitted light, &egr;(&lgr;) is the decadic molar extinction coefficient, c is the concentration of absorbing molecules, and 1 is the path length. The quantity −log(I/I
0
) is termed the absorbance and is the logarithm of the reciprocal of the fraction of transmitted light.
Generally, absorbance measurements are most accurate when the absorbance is in the range 0.1-2.0, corresponding to absorption of about 20-99% of the incident light. Yet, in many biological and pharmaceutical applications, such “high” absorbances may be difficult to obtain, because the absorbing molecules may be expensive and/or available in small quantities. Moreover, in many biological and pharmaceutical applications, small samples are desirable, because experimental procedures may involve studying hundreds of thousands of samples, such that small samples decrease reagent costs and the overall space required.
As seen from Equation 1, absorbance may be increased by increasing the concentration of absorbing molecules. Unfortunately, this approach has a number of shortcomings. In particular, because concentration is the number of molecules per unit volume, increasing the concentration involves increasing the number of molecules and/or decreasing the volume. Yet, increasing the number of molecules is undesirable if the molecules are expensive and/or rare. Similarly, decreasing the volume is undesirable because it may decrease the path length and so decrease absorbance.
Also as seen from Equation 1, absorbance may be increased by increasing the path length. Unfortunately, this approach also has a number of shortcomings. In particular, increasing the path length may involve increasing the volume of sample, and hence increasing the number of molecules and the overall space required. Alternatively, increasing the path length may involve decreasing the cross section of the sample, decreasing signal.
2. Scattering Assays
Scattering assays can be used to detect the motion, size, concentration, aggregation state, and other properties of molecules in a sample. For example, by looking at the spectral spread of scattered light, it is possible to determine the average velocity of scattering particles in a sample. By observing the intensity of scattered light, the concentration of scattering objects can be measured. By observing the angular distribution of scattered light, various physical characteristics of scattering molecules can be deduced.
3. Luminescence Assays
Luminescence is the emission of light from excited electronic states of atoms or molecules. Luminescence generally refers to all kinds of light emission, except incandescence, and may include photoluminescence, chemiluminescence, and electrochemiluminescence, among others. In photoluminescence, including fluorescence and phosphorescence, the excited electronic state is created by the absorption of electromagnetic radiation. In chemiluminescence, which includes bioluminescence, the excited electronic state is created by a transfer of chemical energy. In electrochemiluminescence, the excited electronic state is created by an electrochemical process.
Luminescence assays are assays that use luminescence emissions from luminescent analytes to study the properties and environment of the analyte, as well as binding reactions and enzymatic activities involving the analyte, among others. In this sense, the analyte may act as a reporter to provide information about another material or target substa
Modlin Douglas N.
Owicki John C
Petersen Jon F.
Evans F. L.
Kolisch Hartwell PC
Lauchman Layla
LJL BioSystems, Inc.
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