Chemistry: electrical and wave energy – Apparatus – Electrophoretic or electro-osmotic apparatus
Reexamination Certificate
1999-07-09
2002-03-26
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Apparatus
Electrophoretic or electro-osmotic apparatus
C204S452000, C356S344000
Reexamination Certificate
active
06361672
ABSTRACT:
TECHNICAL AREA
The present invention relates primarily to induced fluorescence, or multiple angle light scattering, mediated identification of sample analytes in solution, and more particularly to a multiple channel electrophoresis system which comprises a multiple laser diode array electromagnetic radiation source, individual laser diodes of which multiple laser diode array electromagnetic radiation source are situated so as to each independently provide a beam of electromagnetic radiation to a specific electrophoresis channel, in said multiple channel electrophoresis system.
BACKGROUND
It is well known to separate different sample analytes present in a solution by electrophoresis, whereby different sample analytes present in a micro-channel, capillary or flow-cell or the like, are caused to migrate at different rates under the influence of an applied electric field. It is also well known to, for instance, analyze fluorescence which is induced by application of electromagnetic excitation energy to sample analytes in a solution present in a micro-channel, capillary or flow-cell, to identify said sample analytes.
Additionally, combination electrophoresis sample separation systems and sample analyte identifying fluorescence inducing systems are known. In fact, a search of Patents has provided a number of relevant references. For instance, two Patents to Yeung et al, U.S. Pat. Nos. 5,498,324 and 5,324,401 describe multiplexed fluorescence detector systems for capillary electrophoresis. Multiplexing is achieved by causing simultaneous application of fluorescence inducing energy from a single laser source to multiple capillaries via a multiplicity of light fibers, each of which light fibers can approach a capillary axially or orthogonally. Another Patent, U.S. Pat. No. 5,584,982 to Dovichi et al., also describes the use of a single laser source, which single laser source simultaneously provides excitation energy to a number of sample containing capillaries. Light fibers are positioned so as to detect the results of said excitation in each capillary and each said light fiber carries excitation energy to an individual detector. U.S. Pat. No. 5,582,705 to Yeung et al, describes a similar system in which a single laser source simultaneously provides excitation energy to a multiplicity of sample analyte containing capillaries present in a multi-channel electrophoresis system. U.S. Pat. No. 5,141,609 to Sweedler et al. is also identified as it describes a CCD array detector system in a capillary electrophoresis system. Additional known Patents are U.S. Pat. Nos. 5,567,294 and 5,439,578 to Dovichi et al.; U.S. Pat. Nos. 5,516,409 and 5,366,608 to Kambara; U.S. Pat. No. 5,413,686 to Klein et al.; U.S. Pat. No. 5,312,535 to Waska et al.; U.S. Pat. No. 5,274,240 to Mathies et al.; U.S. Pat. No. 5,540,825 to Yeung et al.; U.S. Pat. No. 5,585,069 to Zanzucchi et al.; U.S. Pat. No. 5,547,849 to Baer et al.; U.S. Pat. No. 5,356,525 to Goodale et al.; U.S. Pat. No. 5,332,480 to Datta et al.; U.S. Pat. No. 5,239,360 to Moring et al.; U.S. Pat. No. 5,215,883 to Chu; U.S. Pat. No. 4,648,715 to Ford, Jr. et al.; U.S. Pat. No. 5,571,680 to Chen; Japanese Patent No. 4-264859 and WO 89/01620. Additional known Patents which focus on the use of light fibers in electrophoresis systems are Patent to Zare et al., U.S. Pat. No. 4,675,300 describes a method of detecting laser excited fluorescence in an electrokinetic separation system; U.S. Pat. No. 5,140,169 to Evens et al.; U.S. Pat. No. 5,096,671 to Kane et al.; Patent, U.S. Pat. No. 4,837,777 to Jones; Patent to Buckles, U.S. Pat. No. 4,399,099; U.S. Pat. No. 4,740,709 to Leighton et al.; U.S. Pat. No. 4,682,895 to Costello; Another Patent, U.S. Pat. No. 5,068,542, to Ando et al. A known Patent to Zhu, No, U.S. Pat. No. 5,432,096 describes a system and method for identifying sample analyte, (eg. DNA, RNA and Protein etc., based upon electromagnetic radiation absorbtion).
Further disclosed is a paper by Yeung et al, titled “Laser Fluorescence Detector For Capillary Electrophoresis”, J. Chromatography, 608(1992), 73-77, describes a laser-based fluorometer for use in detection in capillary electrophoresis. While laser induced fluorescence, in combination with electrophoresis mediated provision of sample analyte into the described system is reported to be a very efficient approach to sample analyte identification, the use of axially oriented optical fibers in a system for detection of sample analyte identifying fluorescence is not described. Said article is incorporated by reference hereinto.
Another known method of identifying analytes in a solution which is caused to be present in a micro-channel, capillary or flow-cell, is that of Multiple-Angle-Light-Scattering (MALS). An article, which describes the technique is titled “Multi-Angle Light Scattering Combined With HPLC”, by Wyatt, LC-GC, Vol. 16, No. 2, (Febuary 1997). Briefly, said technique involves impinging light upon a sample analyte containing solution in a flow cell, and intercepting light scattered therefrom with detectors oriented at a number of angular positions. The amount of light intercepted at any angular position is proportional to the product of “Molar Mass and Concentration”, and variation in light intercepted at the various angular positions is related to “Molecular Size” of the reflecting molecules. While the theoretical basis of inducing and detecting components in a sample solution utilizing fluorescence is fairly straight forward, (one provides energy to a sample and detects the wavelength of fluorescence emitted), the theoretical basis of multiple-angle light scattering (MALS) requires a bit of elaboration. First, as described in cited article by Wyatt, it is to be understood that the “Excess Rayleigh Ratio” R(⊖) is defined as the ratio of light intensity at a (MALS) detector positioned at an scattering angular position (⊖), divided by incident laser intensity I
0
. Next, the relationship between said “Excess Rayleigh Ratio” and the weight average molar mass Mw, and something called the “Second Viral Coefficient) is:
K*c/R
(⊖) (1/(
Mw×P
(⊖))+2(
A
2
c
) 1
where the P(⊖) is the form factor, which describes the angular variation of the molecular scattering given by:
P
(⊖)−1=sin
2
(⊖/2)+sin
4
(⊖/2)− 2
The constant K* is a function of measured quantities, including the refractive index increment (dn/dc), the refractive index of the solvent n
0
, and the wavelength of the incident light. The coefficients &agr;
i
depend on the structure of the molecule and are usually determined by fitting the collected light-scattering data measured at various concentrations and angles ⊖
i
, i=1 to N, where N is the total number of angles at which measurements are taken. It is noted that ⊖
i
=0.0, P(⊖)=1.0. The coefficient &agr;
i
is always proportional to the molecular mean square radius (<r
g
2
>)
α
;
α
<
r
9
2
>
=
∑
i
r
i
2
mi
∑
i
mi
=
1
m
∫
r
2
ⅆ
m
where the summation is taken over each mass element m
i
, and the distances r
i
are measured with respect to the molecule's center of mass. It is noted that after separation by chromatography, the concentrations generally are so small that the term involving the solvent-solute interaction 2(A
2
c) can be ignored. This simplifies Eq. 1. If it is then assumed that the separation is complete and that each chronological “slice” of effluent from a chromatography column contains a unique molar mass, then the polydispersity within the slice and all molar mass moments are equal. Therefore as (⊖) approaches (0.0), the molar mass may be calculated directly using the extrapolated value R(⊖) and equation 1, and the mean square radius can be determined by Eq. 2 by using the initial slope of R(⊖). Again, this description of (MALS) theoretical basis is adapted from the cited Wyatt article, and said Wyatt artic
D'Silva Arthur P.
Fry Robert C.
Zhu Jianzhong
Starsiak Jr. John S.
Transgenomic Inc.
Walker William B.
Warden Jill
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