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Description  |
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FIELD OF THE INVENTION
The invention relates generally to a method and apparatus for detecting
analyte bands and more particularly to a method and apparatus for
time-delayed integration with a solid-state detector such as a
charge-coupled device in capillary electrophoresis and capillary
chromatography, or other sample separation techniques.
BACKGROUND OF THE INVENTION
Numerous research areas in biochemistry depend on the ability to analyze
minute quantities of nucleic acids, amino acids, and peptides, yet many
present detection schemes have inadequate sensitivity. Examples are the
analyses of single cells and subcellular compartments. One of the few
techniques to permit successful assays of the contents of a single cell is
capillary zone electrophoresis (CZE), a powerful separation technique for
the analysis of small sample volumes. Olefirowicz, T. M.; Ewing, A. G.,
Anal. Chem., 1990, 62, 1872-1876; Wallingford, R. A.; Ewing, A. G., Anal.
Chem., 1988, 60, 1972-1975; Chien J. B.; Wallingford, R. A.; Ewing, A. G.,
J. Neurochem., 1990, 54, 633-638; and Kennedy, R. T.; Oates, M. D.;
Cooper, B. R.; Nickerson, B.; Jorgenson, J. W., Science, 1989, 246, 57-63.
Separation efficiencies routinely exceed several hundred thousand
theoretical plates, and typical injection volumes are 10 nL or less.
For CZE, channel diameters usually range from 25 to 100 microns; therefore,
designing methods to detect low concentrations in these small capillaries
is a challenge. Laser-induced fluorescence (LIF) is currently the most
sensitive detection method for CZE; detection limits are in the low
attomole range. Nickerson, B.; Jorgenson, J. W., , J. High Resolut.
Chromatogr. Commun., 1988, 11, 533-534; Drossman, H.; Luckey, J. A.;
Kastichka, A. J.; D'Cunhan, J.; Smith, L. M., Anal. Chem., 1990, 62,
900-903; and Kuhr, W. G.; and Yeung, E. S., Anal. Chem., 1988, 60,
2642-2646. In these systems, the capillary is used as the flow cell, the
laser illumination is perpendicular to the capillary, and a
photomultiplier tube (PMT) monitors the fluorescence. Dovichi and
coworkers developed a more sensitive method that has the same excitation
geometry but uses a sheath flow cuvette as the sample cell and thereby
eliminates much of the scattered light and luminescence from the fused
silica capillary. With this technique, detection limits in the low
zeptomole range (10.sup.-21 moles) for fluorescently tagged amino acids
have been reported. Cheng, Y.-F.; Dovichi, N. J.; Science, 1988, 242,
562-564; Wu, S.; Dovichi, N. J.; J. Chromatogr., 1989, 480, 141-155;
Dovichi, N. J.; and Cheng, Y.-F., Am. Biotechnol. Lab., 1989, 7, 10-14.
However, despite the above advances in detection techniques, there remains
a need for detection methods and devices with greater sensitivity.
SUMMARY OF THE INVENTION
Higher detection sensitivity and other objects are accomplished by the
present invention where components separated from a sample as migrating
analyte bands are detected by use of an array of solid state detectors.
Each detector is capable of collecting radiation signals to produce
charges. The sample is separated by conventional means, including
capillary electrophoresis and capillary chromatography, in which the
separated analyte bands migrate in a detector zone. Thereafter, means are
employed to generate radiation signals that are indicative of said bands
in the zone. Conventional means including ultraviolet absorption,
fluorescence, phosphorescence, or chemiluminescence can be employed to
generate the signals. The array of solid state detectors are correlated
with the detection zone along the migrating direction of the bands so that
as each analyte band migrates through the detector zone the radiation
signals indicative of said band are directed to each detector in sequence.
The solid state detector array is also designed so that the charges from
each detector is shifted to the respective adjacent detector in the
direction of the migration at a rate that is synchronized to the movement
of the analyte band thus permitting the charges corresponding to the
radiation signals of an analyte band to be accumulated at an array output.
The array could be one or two dimensional. In a two-dimensional array where
each row of detectors consists of multiple detector elements, the
intensities at a range of wavelengths of the radiation signals can be
measured. In a one dimensional array where each row consists of a single
element, the intensity measured is the sum of the intensities at different
wavelengths in the range.
In a preferred embodiment, a charge-coupled device (CCD) is employed in a
fluorescence detection system. Analyte bands separated by capillary zone
electrophoresis are detected in a small section of an axially illuminated
capillary column. The CCD is operated in the time-delayed integration
(TDI) mode that allows long exposure times of the analyte zone. In the TDI
mode, the transfer of the photogenerated charge in each row of the CCD
proceeds approximately at the same rate as the migration of the solute
bands in the capillary. Hence, as each analyte band enters the laser
excitation zone, the fluorescence is collected and focused onto the first
row of the CCD. After an appropriate time period, the charge in the CCD is
shifted toward the readout register by one row. This time interval
corresponds approximately to the time required by the band to move in the
capillary by a distance so that the same analyte fluorescence signal
causes an additional charge to be produced in the second row of the CCD,
during the next time period, where this additional charge is accumulated
and added to the charges shifted from the first row. The charge shifting
is then performed for subsequent time intervals in the same manner at a
rate synchronized with the movement of the band until the band exits the
detection zone. At the same time, the total charge thus accumulated will
be shifted to the array output. The effective integration time for a given
analyte band is thus the entire time the band is within a window in the
illuminated area; however, multiple bands can be within the illumination
window simultaneously and still be resolved because of their different
spatial positions. The time-delayed integration method in effect increases
the effective sampling volume of the flow cell without introducing band
broadening.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram of the CCD/LIF system illustrating the
time-delayed integration mode.
FIGS. 2(a) through 2(e) are another illustration of the CCD/LIF system.
FIG. 3 is a schematic diagram showing the Ar ion laser, capillary
arrangement, optics, and CCD detector.
FIG. 4 is schematic of the optical system from FIG. 3, showing the axially
illuminated detector.
FIGS. 5(a), 5(b), and 5(c) are snapshots taken in succession (0.5 s
exposures) of the focal plan output for a 4 nL injection of
8.times.10.sup.-10 M FITC.
FIGS. 6(a), 6(b), and 6(c) are electropherograms illustrating the effect of
changing the TDI shift rate on electrophoretic efficiency for a series of
3 amol fluorescein isothiocyanate FITC injections.
FIG. 7 is a TDI electropherogram of sulforhodamine and fluorescein.
FIGS. 8(a), 8(b), 8(c), and 8(d) are TDI electropherograms of FITC at
different concentrations.
FIG. 9 is a TDI electropherogram of four FITC-amino acids, with the
concentration of injected FITC-arginine, FITC-valine, and FITC-glycine at
7.times.10.sup.-11 M, and FITC-glutamate at 8.times.10.sup.-11 M.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
General Description. The present invention encompasses a method and
apparatus employing time-delayed integration for detecting components
separated from a sample. The invention employs an array of solid state
detectors to respond to radiation signals indicative of said components.
Conventional means including ultraviolet absorption, fluorescence,
phosphorescence, or chemiluminescence can be employed to generate the
signals. Separation techniques that can be employed are any that can
separate sample components into discrete units moving within a confined
region, said region to be referred as the observation or detection zone.
For instance, capillary electrophoresis, capillary chromatography, or
micellar electrokinetic chromatography could be employed. Moreover, a
portion of the capillary where the separated analyte bands traverse that
is transparent to the radiation of interest could be used as the detection
zone where the radiation signals are collected. With CZE, for example,
once the sample is separated into migrating analyte bands they are
detected by the array of solid state detectors, where each detector is
capable of collecting radiation signals to produce charges. The array of
solid state detectors are aligned with the detection zone along the
migrating direction of the bands so that as each analyte band migrates
through the detector zone the radiation signals indicative of said band
are directed to each detector in sequence. The solid state detector array
is also designed so that the charges from each detector is shifted to the
respective adjacent detector in the direction of the migration at a rate
that is synchronized to the movement of the analyte band such that the
charges corresponding to the radiation signals of a particular analyte
band can be accumulated at an array output.
The array could be one or two dimensional. In a two-dimensional array where
each row of detector consists of multiple detector elements, both the
intensity and wavelength characteristics of the radiation signals can be
measured. In a one dimensional array where each row consists of a single
element the measured intensity is normally the sum of the intensities
measured at different wavelengths.
Charge-Coupled Device. A solid state device comprising an array of
detectors which can be readily adapted for use in the present invention is
the charge-coupled device (CCD). A CCD is a monolithic large-format
silicon array detector. Characteristics that make it ideally suited for
detection are extremely high quantum efficiency (.ltoreq.80%), virtually
no dark current, and up to 10.sup.6 individual detector elements in the
array. The CCD is conceptually similar to an electronic photographic film
in that both integrate signal information. The integrating ability of the
CCD and lack of dark current allow the CCD to perform exceptionally well
in situations where several seconds are allowed for integration of the
signal. In microcolumn separations, detection zones can be constructed
such that analyte bands are viewed for many seconds--an ideal measurement
task for a CCD.
As is described below, the two-dimensional nature of the CCD allows a
detection scheme known as time-delayed integration to be used to increase
even further the sensitivity, selectivity, and utility of CZE. After a
normal exposure of the CCD, the charge information in every row of the CCD
is shifted toward one end of the detector and the charge/signal
information in the last row is quantified. Thus, the photogenerated charge
originally created in a particular row of the CCD is transferred in a
step-wise fashion toward the readout register.
Normally, the transfer of charge is started after an exposure and continues
until the entire CCD has been read. In the time-delayed integration mode,
the transfer of each row of the CCD proceeds approximately at the same
rate as the migration of the solute bands in the capillary. Hence, for
instance, in LIF, as the analyte band enters the laser excitation zone,
the fluorescence is collected and illuminates the first row of the CCD.
After the appropriate delay, the charge in the CCD is shifted toward the
readout register by one row at approximately the same time as the band has
moved down the capillary so that the analyte fluorescence signal is still
contributing to the same charge information. The effective integration
time for a given analyte band is the entire time the band is within the
illuminated area; however, multiple bands can be within the illumination
window simultaneously and still be resolved because of their different
spatial positions aligned to different rows of detectors. The TDI method
in effect increases the effective sampling volume of the flow cell without
introducing band broadening. The effective increase in either sampling
volume or integration time (two aspects of very closely related
parameters) is a function of the number of rows of the CCD. With the
availability of 512 by 512 element and larger CCDs, the TDI mode results
in large increases in sensitivity.
The synchronization of the shift rate to the analyte velocity needs to be
fairly accurate or a loss in separation efficiency is observed.
Fortunately, the shift rate synchronization is not difficult. Because the
start of the CZE run is known exactly, as is the distance from the
injector to the center of the observation zone, the shift rate is
determined by several geometrical parameters.
The invention will be described herein using a LIF/CZE system but the
invention is applicable to capillary chromatography as well. Moreover, the
invention is applicable to radiation detection techniques in general
including, for instance, absorption, fluorescence, phosphorescence, and
chemiluminescence, or the emission of high energy radiation (e.g.,
radioactive decay).
The LIF/CZE system uses a two-dimensional CCD containing an array of 516 by
516 detector elements. CCDs are available in arrays ranging from 64 by 64
to more than 2048 by 2048 elements. The system employs an unique axial
illumination arrangement for CZE that has several advantages when used
with multichannel detectors. The output of a laser is focused into the end
of the capillary, the fluorescence emission from the analyte is collected
over a 2 cm section of the channel, and the entire fluorescence spectrum
is measured using the CCD array. In this way, the fluorescence cell is
on-column, and the complete fluorescence spectrum is acquired
simultaneously. Residence times for analytes in the 2 cm detection or
observation zone range from 2 to 45 s. The axial illumination method
allows the CCD to be operated in the snapshot and TDI modes and provides
significant advantages over conventional illumination and CCD detection.
Time-Delayed Integration Mode. The TDI readout mode is ideally suited to
LIF/CZE with axial illumination, in which the analyte band passes through
an extended detection zone. Using this technique, the CCD becomes a highly
sensitive and flexible multichannel fluorescence detection system. TDI,
which does not use a shutter as in the snapshot mode, can acquire entire
spectra at a 50 ms rate, and can spatially resolve multiple bands that are
in the observation zone. Furthermore, far less data are generated than in
the snapshot mode.
Description of CCD Operation. In a CCD, all the photogenerated charge in
the photoactive elements is transferred toward the serial register one row
at a time, and the charge information in the serial row is read using the
single on-chip amplifier. Sweedler, J. V.; Billhorn, R. B.; Epperson, P.
M.; Sims, G. R.; Denton, M. B.; Anal Chem., 1988, 60, 282A-291A. For a 516
by 516 element CCD, each time a single imaging area is transferred to the
serial register, 516 readouts are performed; each readout corresponds to a
different spectral element. This process continues until all 516 rows have
been read 516 times.
Normally these transfers are done with a closed shutter to prevent exposure
of the CCD to the illumination source; if such exposure occurs, the image
becomes blurred. With the present invention the shutter is eliminated and
in the CCD the shifting of rows is synchronized to the migration rate of
the analyte band in the capillary. FIG. 1 illustrates the TDI mode using a
simplified 3 by 6 element CCD and two separated analyte bands 101 and 102
that are migrating in detection or observation zone 120 of a capillary.
The detection zone (or illumination window) is that portion of the
capillary where the radiation signals (which is light in the embodiment of
FIG. 1) indicative of the analtye bands are collected and directed to the
CCD. As is apparent from FIG. 1, while the length of the detector zone may
be the same as the length of the CCD array, it will be understood (as
shown in FIG. 3) that they may be different, since light passing through
the band or originating therefrom may be directed by optical means to the
rows of the CCD. CCD 100 has a serial readout register 110 and six rows of
photoactive elements denoted 1, 2, 3, 4, 5, and 6, respectively. Each row
contains three photoactive elements. In FIG. 1, each row is perpendicular
to the band motion; however, the rows need only be transverse to band
motion direction 104 to practice the invention. The CCD is oriented so
that the parallel shift direction 105 is the same as the direction of
analyte band motion 104. As an analyte band enters the laser excitation
zone, the fluorescence through the proper optics and spectrograph
(collectively represented as element 103) is collected and illuminated
onto row 1 of the CCD. The image that is projected onto each row of the
two-dimensional CCD is a spectrum that is representative of the particular
analyte band. As shown in FIG. 1, each row has three detector elements
that are designated A, B, and C, respectively. Thus in this
two-dimensional array, the image focused onto elements A, B, and C,
respectively, would be from different portions of the spectrum of the
light passing through or originating from the analyte band. As is
apparent, by increasing the number of detector elements per row in a CCD
one can measure radiation over a larger band of wavelengths. When an image
is focused onto a detector element, the element generates a charge.
Meanwhile, the band takes a period of time to migrate to the point on the
capillary from which its image is projected onto CCD row 2. This is shown
in FIG. 1 by the image from analyte band 101 that is focused onto row 2.
Once the image of the band reaches row 2, each detector element therein
will generate a charge. As the image of the analyte band moves from row 1
to row 2, the charge from row 1 is also shifted to row 2. Specifically,
any charge generated in element A of row 1 is shifted to element A of row
2, any charge generated in element B of row 2 is shifted to element B of
row 2, and so on. The shifting permits the charges to be accumulated so
that when, for instance, the image is focused onto row 2, the charge
generated by row 2 will be added to the charge shifted from row 1. This
synchronized accumulation and shifting of charge continues step-wise over
each pair of adjacent rows of the detector array over six consecutive time
intervals until all the charges produced by all six rows 1-6 have been
accumulated and shifted in the manner described in row 6, where upon the
charges are read by means of read out register 110. In this fashion, the
light signals from a particular band contribute to the same charge
information which is read at the serial readout register of the CCD. An
electropherogram illustrating the measured intensities over a wavelength
range for different analytes is shown in FIG. 7 which is described below.
The effective integration time for a given analyte band is the entire time
the band is in the detection zone; however, multiple bands can be within
this zone simultaneously and still be resolved because of their different
spatial positions.
FIG. 2(a)-(e) is another illustration of the TDI method using a 3 by 4
element CCD. Here the four rows are designated 11, 12, 13, and 14,
respectively. As each of the two analyte bands 201 and 202 migrates across
the detection zone 205 of the capillary (not shown) in direction 210, its
spectrum is focused onto the CCD integration area beginning at row 11. In
FIG. 2(a), neither ban has entered the detection zone, but in FIG. 2(b),
band 201 is in the zone. As shown on FIG. 2(b), the spectrum 206 of the
analyte band 201 illuminates all three detector elements of row 11. Thus,
with a two-dimensional solid state array like a CCD, the invention also
permits intensity measurements at different wavelengths. If a
one-dimensional array is used, the intensity over a band of wavelengths
would be detected. As shown in FIG. 2(d), the photogenerated charges 208
generated by spectrum 206 are accumulated as the spectrum traverses the
CCD integration area, and the charges are transferred to the serial
readout register where they are read and digitized. Analyte band 202 forms
spectrum 207.
The light signals in FIG. 1 may be generated by fluorescence of the analyte
bands by laser induced fluorescence. The TDI mode has several important
advantages. Only one row of the CCD is read at a time, reducing the data
produced and the readout time up to 516-fold (depending on the spatial
binning factor, which is the binning factor in the dimension that is
imaging the capillary). Also, a row contains the fluorescence from a
single analyte and not from a point on the capillary; the fluorescence is
integrated over the entire time the band is in the observation zone. A
third advantage is that the fluorescence information from the analyte band
can be obtained from a single CCD readout instead of the approximately 4
to 20 readouts required for the snapshot mode. The single readout produces
a two- to five-fold reduction in read noise.
Synchronization of Shift Rate. In CZE, the TDI mode is complicated by the
fact that the analyte bands move at different rates because of their
different electrophoretic mobilities. Hence, the shift rate must be
continually decreased during the run to follow different bands. The
synchronization of the shift rate to the analyte velocity needs to be
accurate to avoid a loss in separation efficiency. Fortunately, such
synchronization is not difficult to obtain. The CCD shift rate can be
programmed to account for analyte band velocity. For instance, if
detection is in a capillary region in which analtye separation is
occurring, the photogenerated charge is shifted step-wise from row to row
at periodic intervals wherein each interval is longer than the preceding
interval by a fixed percentage to account for the slower rate of later
bands. Moreover, because both the start time of the CZE run and the
distance from the injector to the center of the detection zone are known,
the shift time can be determined by the following Formula I:
T.sub.shift
=[(T.sub.elapsed)(X.sub.obs)(N.sub.bin)]/[(N.sub.ccd)(L.sub.cap)]
where T.sub.shift is the time until the next CCD shift, T.sub.elapsed is
the time since the start of the CZE run, X.sub.obs is the length of the
observation zone, N.sub.bin is the binning factor in the observation
(imaging) dimension, N.sub.ccd is the number of CCD elements the capillary
image illuminates, and L.sub.cap is the length of the capillary to the
center of the observation zone. The integration times range from 2 to 45 s
for a 80 cm capillary, 2 cm observation zone and analyte elution times
from 1 to 30 min.
A CCD must shift charge from all rows in concert (i.e., the shift rate is
the same for all rows). Because each row receives a signal that originates
from a different point in the capillary, the shift rate must be incorrect
for all but one specified row. To visualize this concept, consider two
analyte bands that are resolved and are in the detection region
simultaneously. They must be moving at slightly different velocities
because they are resolved; the CCD, which shifts at a single velocity,
cannot independently track both. In the experiments described herein, the
shift rate was matched to the center of the 2 cm observation zone at the
end of an 80 cm capillary, a .+-.1% difference between the zone velocity
at the observation boundaries and TDI shift rate is expected. Thus, in the
configuration employed, almost no increase in analyte zone width is
expected.
Immediately after injection, the required time between shifts is less than
50 ms. Because the system cannot shift this fast, the tracking of this
extremely fast moving (hypothetical) band is not exact. Currently, the
system shifts for the maximum 50 ms per spectrum until the calculated
shift rate is longer than 50 ms (approximately 2 min.), at which point it
shifts at the appropriate rate. Because the shift rate is always
decreasing, data are obtained with a nonuniform time resolution, although
the spatial resolution remains constant (the system images every band with
the same 80 .mu.m resolution). Thus, the early fast bands are sampled
faster than later bands. This type of sampling scheme fits well with
constant resolution and data acquisition per band. Huang, X.; Coleman, W.
F.; Zare, R. N., J. Chromatogr., 1989, 480, 95-110.
A simple method exists to preclude the need for a changing shift rate and
thus avoid the potential blurring caused by the TDI method. If the
capillary is grounded just prior to the observation zone, the analyte
bands would not undergo electrophoresis while being detected, and instead
would move at the solution flow velocity. In addition, when separation is
accomplished by capillary chromatography instead of electrophoresis, the
analyte bands will move at the same velocity once the bands pass the
stationary phase of the capillary.
Using the TDI mode, CZE electropherograms with over 700,000 theoretical
plates can be obtained. Theoretical plate calculation is based on the full
width of a peak at one half of the maximum intensity. With the present
invention it is possible to obtain sensitivities for fluorescent tags in
the low zeptomole range and differentiate between multiple fluorophores
based on different migration times and spectral characteristics.
Description of Axial Illumination. To achieve high sensitivity with the CCD
system, longer exposure of the CCD to the analyte fluorescence is
advantageous. In most LIF/CZE systems, the laser beam diameter is focused
to less than 50 .mu.m and strikes the capillary perpendicular to the
separation channel; thus, the analyte is illuminated by the laser for only
several milliseconds. In the present system, the capillary is illuminated
end-on, and the resultant fluorescence from a 2 cm section is imaged onto
the CCD. Fluorescence is collected during the entire residence time of the
analyte band in this section.
Problems could occur when axially illuminating a capillary that contains a
leading band at high concentration closely followed by other bands. The
leading band could absorb a significant fraction of the channeled
excitation light, thus reducing the fluorescent signal from later bands
also resident in the observation zone. However, such a shadowing effect
was not observed at the fluorophore concentrations used in these
experiments.
A buffer-filled capillary does not fulfill the requirements of a
light-pipe; the index of refraction of the aqueous center is lower than
that of the fused silica "cladding." Thus, laser illumination tends to
travel in the fused silica wall rather than propagate through the center
of the capillary, unless care is taken in the choice of laser-focusing
lens and the alignment of the laser beam with respect to the capillary.
The focusing lens is chosen to provide a beam waist just smaller than the
capillary i.d. This lens provides a large depth of field and hence an
extended illumination zone. To facilitate alignment of the laser beam with
the capillary, they must be almost exactly parallel to each other. Even
with these precautions, significant illumination travels only several
centimeters in the capillary channel.
An axially illuminated open tubular liquid chromatography (OTLC) system has
been described using capillaries of similar dimensions, but in that case,
the solvent was chosen to provide a higher index than the quartz capillary
material. Xi, X.; Yeung, E., Anal. Chem., 1990, 62, 1580-1585. For axial
illumination of the OTLC system, the laser beam must be propagated through
the length of the capillary. In the present case, however, the loss of
light after several centimeters is desirable. Many fluorophores, including
fluorescein, photodegrade rapidly under moderate-intensity illumination;
thus illumination of the analyte band prior to the detection zone is
deleterious. For a water-filled fused silica capillary, light radiates
from the aqueous channel into the walls as the capillary bends. Thus, just
before the 2 cm detection zone, the channel is bent sharply (90 bend over
1 cm). This bending prevents laser light from propagating much past the
observation zone and thus greatly reduces photodegradation of the
fluorophore before detection. Additionally, the bend in the capillary
prevents ambient room light from propagating through the capillary to the
detection region.
In conventional systems, when the laser power is high, photodegradation
occurs but the fluorescence of the fluorophores is still monitored. In the
present system, a 1 cm distance separates the bend in the capillary and
the beginning of the observation zone. Thus for 1 cm, or up to 20 s, the
fluorescently tagged analyte is subject to laser illumination without the
fluorescence being collected. To limit premature photodegradation and a
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