Electrophoresis method, electrophoresis device, and marker...

Details Electrophoresis method, electrophoresis device, and marker... Electrophoresis method, electrophoresis device, and marker...

1999-12-02

2003-03-18

Warden, Jill (Department: 1743)

Chemistry: electrical and wave energy

Processes and products

Electrophoresis or electro-osmosis processes and electrolyte...

Reexamination Certificate

active

06533913

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to an electrophoresis method, an electrophoresis device, and a marker sample used for the same. More particularly, the present invention relates to an electrophoresis method and an electrophoresis device for separating and detecting a sample labeled with two or more luminescent reagents having different luminescent wavelength ranges, and to a marker sample used for such method and device.
BACKGROUND OF THE INVENTION
Various devices for detecting a sample by utilizing luminous phenomenon (e.g., fluorescence, chemiluminescence and fluorescent chemiluminescence) are known, such as a fluorescence measuring device, a chemiluminescence measuring device, an electrophoresis device and a biochip reading device. When such devices are used to determine a sample labeled with two or more luminescent reagents with different luminescent wavelength ranges, optical interference filters having high wavelength range selectivities are used for color separation. However, if the wavelength ranges of the luminescent reagents, even partially, overlap each other, a light component of an untargeted luminescent reagent passes through the optical filter, causing leak that needs to be excluded for the subsequent evaluation.
FIG. 1
is a schematic view showing an exemplary device for detecting a sample
2
labeled with two or more luminescent reagents with different luminescent wavelength ranges by using optical interference filters with high wavelength range selectivity. The sample
2
labeled with luminescent reagents is carried on a substrate
1
. The light emanating from the luminescent reagents labeling the sample
2
is collected by a condenser lens
3
, then transmitted through an optical filter
4
, and focused by a convergence lens
5
to a photomultiplier
6
to be detected. The detected signal from the photomultiplier
6
is amplified by an amplifier
7
, converted into a digital signal by an A/D converter
8
, and processed with a data processor
9
.
The optical filter
4
of the device shown in
FIG. 1
transmits only a light component within a wavelength range of a luminescent reagent labeling a targeted molecule, and eliminates light components in other wavelength ranges derived from the other luminescent reagents. For sample
2
containing molecules labeled with two or more luminescent reagents with different wavelengths, each molecule may be separately detected by changing the optical filter
4
that allows transmission of light emanated from the luminescent reagent to be detected.
However, the wavelength ranges of luminescent reagents that are actually used often overlap each other. Particularly, when a plurality of (three or more) luminescent reagents are used, it is difficult to select a combination of luminescent reagents such that it does not cause overlapping of their wavelength ranges.
FIG. 2
is a diagram showing wavelength patterns of luminescent light obtained when three types of fluorescent dyes (i.e., fluorescein
14
, TMR (carboxy-tetramethyl-rhodamine)
15
and CXR (carboxy-X-rhodamine)
16
) are excited with excitation light
13
of 532 nm. The horizontal axis
11
represents wavelength which becomes longer towards right while the vertical axis
12
represents luminous intensity. Generally, when a plurality of luminescent reagents are used as labels, their wavelength ranges overlap as shown in FIG.
2
. Thus, even if an optical filter is used for the purpose of obtaining only the light component from the luminescent reagent of interest, light components from other luminescent reagents may leak and pass through the optical filter.
FIG. 3
is the same diagram as that shown in
FIG. 2
showing wavelength patterns of luminescent light obtained when the above-mentioned three types of fluorescent dyes are excited with excitation light
13
(532 nm). For example, when an optical filter that transmits light in a wavelength range
17
shown in
FIG. 3
is used in a detection system to obtain a light component
16
emanated from the fluorescent dye CXR (hereinafter, referred to as “CXR light component”), a light component
15
emanated from the fluorescent dye TMR (hereinafter, referred to as “TMR light component”) partially overlaps the wavelength range
17
as leakage
18
of the light component
15
through the optical filter for detecting the light component
16
. The leakage of a light component of a luminescent reagent other than the luminescent reagent of interest causes detection of a band that is absent in one-dimensional electrophoresis, or detection of a band intensity greater than the band intensity originally obtained in one-dimensional electrophoresis.
FIG. 4
is a diagram illustrating that a measured waveform is deformed due to a leak of an irrelevant light component. Due to the leak of the TMR light component, as shown in
FIG. 4
, the waveform (electrophoresis pattern)
43
of the CXR-labeled molecule obtained by using the optical filter for extracting CXR light component is deformed from a waveform
41
obtained by one-dimensional electrophoresis of the CXR-labeled molecule. Suppose that the waveform pattern
41
of the CXR-labeled molecule obtained by one-dimensional electrophoresis has two peaks
44
and
45
, and the waveform pattern
42
of the TMR-labeled molecule has two peaks
46
and
47
. If the TMR light component leaks through the optical filter for extracting CXR light component, the detected electrophoresis pattern is influenced as shown in FIG.
4
. Where the molecular weight of the CXR-labeled molecule approximates the molecular weight of the TMR-labeled molecule, the peaks obtained by electrophoresis of both molecules by using the optical filter for detecting CXR overlap each other (peaks
45
and
46
) and the electrophoresis pattern
43
gives a peak
48
which is greater than its actual peak
45
. Where the TMR-labeled molecule is present and the CXR-labeled molecule is absent, a peak
49
appears on the electrophoresis pattern
43
as influenced by the leakage of light component at peak
47
where there should be no peak.
Such misdetection caused by the leakage of light component emanated from a luminescent reagent other than the luminescent reagent of interest is conventionally corrected by software means. Such software calculates the leakage value, and subtracts that value from the actually measured value. First, positions where or time when a molecule labeled with a luminescent reagent A is solely present are empirically predetermined. Then, values at these positions or time as measured with an optical filter a that transmits light emanated from luminescent reagent A and values at the same positions or time as measured with an optical filter b that is not intended to transmit light emanated from the luminescent reagent A are determined. Based on these values, a leakage rate R
ab
of the light component emanated from the luminescent reagent A leaking through the optical filter b is calculated. The leakage values at the predetermined points are determined based on the value measured with the optical filter a and the leakage rate R
ab
. Each leakage value is then subtracted from the value measured at the same point with the optical filter b, thereby eliminating the influence of the leak of the light component from the luminescent reagent A through the optical filter b.
Hereinafter, the process will be described in more detail with reference to FIGS.
5
and
6
A-
6
C. Molecules
53
and
54
are labeled with luminescent reagents A and B, respectively, the luminescent reagents emitting light having different but partially overlapped wavelength ranges. Then, the molecules
53
and
54
are simultaneously but separately subjected to one-dimensional electrophoreses.
FIG. 5
is a diagram showing waveforms
51
and
52
measured with the optical filters a and b for detecting light components from luminescent reagents A and B, respectively. The optical filters a and b have selectivity towards the wavelength ranges of the luminescent reagents A and B, respectively. Provided that the molecule
53
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