Optics: measuring and testing – By electrophoresis
Reexamination Certificate
2002-06-06
2004-09-28
Nguyen, Tu T. (Department: 2877)
Optics: measuring and testing
By electrophoresis
Reexamination Certificate
active
06798509
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods and instruments for fluorescence detection for use in electrophoretic instruments and electrophoresis that are applied to analyzing biopolymers such as nucleotides and proteins.
BACKGROUND OF THE INVENTION
Electrophoresis in which to detect fluorescence by laser induction is widely used as one fundamental technique for analyzing biopolymers such as nucleotides and proteins because of its high sensitivity and convenience. In the biopolymer analysis field, capillary electrophoresis has lately been used commonly, superceding slab gel electrophoresis that was a mainstream analysis method. For the capillary electrophoresis, less Joule heating is generated when analytes electophoretically migrate and therefore high voltage can be used. As a result, analysis can be performed in a shorter time. The length of a migration (separation) channel is generally 50 cm to 20 cm. Aiming at reducing analysis time and downsizing the analysis system, diverse techniques have been developed to shorten the migration (separation) channel.
Such a method is described in Science, 261, 895-897 (1993) (prior art-1) that, by forming capillary channels on a substrate by application of photolithography technique and making analytes electrophoretically migrate through the channels, separating a plurality of fluorescence labeled amine acid is achieved with migration (separation) channel length of 0.75 cm to 2.2 cm.
Under conditions that unimolecular detection is performed, several species of deoxyribonucleic acid (DNA) can be separated in a migration (separation) channel with effective length of 0.25 mm, which is described in Anal. Chem., 67, 3181-3186 (1995) (prior art-2).
A method in which fluorescence emitted from analytes migrating through channels is sequentially detected through 55 300 &mgr;m-wide detection slits arranged at intervals of 700 &mgr;m and the velocity of the analytes is measured by Fourier transform of fluorescence intensity is described in Anal. Chem., 71, 2130-2138 (1999) (prior art-3).
A method in which interference fringes of excitation light are generated across analytes including fluorescent material and fluctuation of fluorescence radiated from the fluorescent material is measured in a correlation function, thereby measuring the fluid velocity of the analytes (one type of the method called Fluorescence Correlations (FCS)) is described in Kokai (Japanese Unexamined Patent Publication) No. Sho-53-40586 (No. 40586 of 1978) (prior art-4).
Using the analysis apparatus configured in the same way as for prior art 1, when the migration channel length is made shorter by 5 mm, the number of theoretical plates will be 5800, which is described in Anal. Chem., 65, 2637-2642 (1993) (prior art-5).
Under general conditions, electrophoresis of prior art posed the following problem. Band broadening of analytes injected into the sample inlet end of a channel in which electrophoresis takes place restricts separation of the analytes. When analytes with close mobilities are used, it is difficult to achieve good separation in a migration (separation) channel with length of the order of millimeters. Actually, in prior art-1, a 2.2-cm long migration (separation) channel separates analytes with mobility difference of 10% or less, whereas a 0.75-cm long channel can separate only analytes with mobility difference of 20% or more.
Prior art-3 also presents problems. Detection slit pitch is restricted by the band broadening of injected analytes and cannot be reduced unlimitedly. The number of detection slits cannot be reduced arbitrarily because it influences separation performance. In prior art-3, the pitch (clearance) between detection slits is 0.7 mm, the number of detection slits is 55, and the effective migration (separation) channel length is about 4 cm which is longer than the channel length in prior art-1.
In prior art-1 and -3, because analytes in narrow band broadening are injected, it is necessary to form two or more crossing channels on the substrate.
Application of the FCS technique described in prior art-4 to the electrophoresis field has not been reviewed heretofore. The present invention is made through consideration of improving the FCS technique described in prior art-4 and applying it to electrophoresis.
SUMMARY OF THE INVENTION
The object of the present invention is to provide methods and instruments for florescence detection, enabling better separation and detection of a plurality of species of analytes with different mobility in a migration (separation) channel with length of the order of millimeters without being restricted by band broadening of the analytes injected into the sample injection end of the channel, thereby solving the above-described problems.
Methodology for fluorescence detection of the present invention is as follows. Analytes are caused to electophoretically migrate in a migration channel such that the analytes disperse in succession across a detection region where they are detected. Excitation light is applied to the detection region. The excitation light is controlled to have an intensity profile that periodically changes in a cycle equaling a pitch greater than the size of a analyte molecule in the direction that the analytes move (in the direction of electric field application). Instead, a slit is located between the detection region and a detector for detecting fluorescence. The slit is designed to have a transmittance profile that periodically changes in a cycle equaling a pitch greater than the size of a analyte molecule. For detected fluorescence emission from the analytes in the detection region, its power spectrum is obtained. Alternatively, an array sensor is used as the detector for detecting fluorescence and fluorescence emission from the detection region is measured. Distribution in the migration direction appears in the fluorescence measurements. Calculation is executed for the sum of the products of fluorescence intensity detected by the photoelectric elements of the array sensor and a function of predetermined pitch and the power spectrum as the sum of the products is obtained.
Description of the Principle Underlying the Invention
First, the principle underlying the invention is now described hereinafter. On the assumption that fluorescence labeled analytes of one species are irradiated by monochromatic excitation light, we consider that fluorescence emitted from the analytes exposed to the excitation light is detected by a photomultiplier (PM). Output current i (t) of the PM is expressed by mathematical expression 1 using molarity C(r, t) of analyte at time t and position r=(x, y, z), and the product I® of multiplying the following excitation light intensity at position r, and efficiency of collection of fluorescent light emitted from an analyte on the photoelectric plane of the PM.
Mathematical Expression 1
i
(
t
)={
ge&eegr;&egr; Qln
10/(
hc
/&lgr;)}∫
I
(
r
)
C
(
r, t
)
dr
[Mathematical expression 1]
where dr=dxdydz, &egr; is a molar excitation coefficient of analyte, Q is a fluorescence quantum yield of analyte, h is a plank constant, c is the velocity of light, &lgr; is wavelength of the excitation light, &eegr; is quantum efficiency of the PM, e is elementary electric charge, g is current gain of the PM. By setting g=1, mathematical expression 1 can be applied to photodiodes. FCS (Fluorescence Correlations) is based on analysis of fluctuation &dgr;i (t)=i (t)−<i (t)> of i (t) when the concentration distribution of the analytes stays in a thermal equilibrium state. In this relation, <X (t)> is an average in the ensemble of X (t). A normalized auto-correlation function G (t) that represents fundamental quantity of time dependency of fluctuation &dgr;i (t) is defined by mathematical expression 2.
Mathematical Expression 2
G
(
t
)=<&dgr;
i
(0)&dgr;
i
(
t
)>/<(&dgr;
i
(
t
))
2
> [Mathematical expression 2]
Furthermore, to analyze fluctuation &dgr;i (t) in the frequenc
Irie Takashi
Kojima Kyoko
Sonehara Tsuyoshi
A. Marquez, Esq. Juan Carlos
Fisher Esq. Stanley P.
Hitachi , Ltd.
Nguyen Tu T.
Reed Smith LLP
LandOfFree
Methods and instruments for fluorescence detection does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods and instruments for fluorescence detection, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods and instruments for fluorescence detection will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3228572