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Description  |
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BACKGROUND OF THE INVENTION
1. Field
The invention is in the field of capillary electrophoresis, detection
methods for capillary electrophoresis and apparatus based on such methods,
and on Schlieren optics.
2. State of the Art
It has been known for some time that a refractive index gradient such as
produced by a concentration gradient in a fluid such as a gas, liquid, or
supercritical fluid, will cause deflection of light passed through the
gradient. The optical method of observing and measuring the deflection of
light caused by refractive index gradient fields is generally referred to
as Schlieren optics. In the past, Schlieren images resulting from light
deflections have been recorded on photographic plates and the plates then
analyzed for light intensity distribution using densitometers. Recently,
evaluation of the photographic images has been done by computer. These
methods are useful in studying plasmas where very complicated toroidal and
parabolic shapes are generated.
U.S. Pat. No. 4,547,071 discloses a sensor for measuring density gradients
in a nonhomogeneous fluid sample using Schlieren optics. In such sensor, a
laser light beam is directed through a sample chamber and is moved along
said chamber. A quadrant light position sensor located on the opposite
side of the chamber detects the deflection of the laser light beam as it
is moved through the sample. The amount of deflection indicates the
density gradient at any point in the sample. Rather than moving the laser
beam along the sample chamber, the beam can be held constant and the
sample chamber with sample therein moved. However, moving a laser and
detector together in relation to a sample chamber and keeping the laser
beam focused on the sample chamber, even a relatively large chamber, is
difficult, as is moving a sample chamber through the laser beam so as to
keep the laser beam properly focused. Trying to do either with a small
capillary sample chamber is very difficult and impractical.
My U.S. Pat. Nos. 4,784,494, 4,940,333 and 4,993,832 show detectors that
can be used to detect concentration and thermal gradients in very small
samples. The detectors utilize a light source to generate one or two probe
beams of light that pass through the sample having the gradient to be
detected and the deflection of the probe beam or beams is measured on a
beam position detector. Various light sources may be used to generate the
probe beam or beams, such as a laser or light emitting diode (LED). These
detectors, however, are designed generally to be used where the gradients
to be detected move through the probe beam or beams of light.
Capillary electrophoresis has become an important separation method in
bioanalytical chemistry. Separation and detection of very small amounts of
biological samples, about pL-nL volumes, can be achieved with capillary
electrophoresis. This is generally not possible with more conventional
methods of separation, even with high performance liquid chromatography.
There are several capillary electrophoresis separation methods in use for
different kinds of samples. They include capillary zone electrophoresis,
moving boundary capillary electrophoresis, capillary isotachophoresis, and
capillary isoelectric focusing. Capillary zone electrophoresis, moving
boundary capillary electrophoresis, and isotachophoresis all have the
advantage that the sample moves through a capillary sample separation
chamber during the separation so can be used with the detectors of my
cited prior patents. Capillary zone electrophoresis and moving boundary
capillary electrophoresis are dynamic processes where separation occurs at
an instant in time and then the zones immediately begin to diffuse and
disperse. The diffusion takes place as the samples move through the sample
chamber to the detector. This makes detection of the various zones more
difficult and less accurate than may be desired. Isotachophoresis has the
advantage that the zones stay relatively sharp as the sample moves through
the capillary, but isotachophoresis is a difficult process to work with.
Isoelectric focusing has been employed for separation of sample components
based on differences in their isoelectric points. Recently, the
development of capillary electrophoresis techniques has generated interest
in preforming the isoelectric focusing in capillaries, since efficient
dissipation of Joule heat from a 10-100 .mu.m diameter capillary
eliminates convection effects which occur in larger sample chambers and
enables highly efficient separations. Capillaries with microbores, i.e.,
with very small inner diameter, also require only small amounts of sample,
which is desirable for analysis of biological materials, such as
monoclinal antibodies and other proteins. The capillary isoelectric
focusing process involves establishing an electrical field between the
ends of the capillary and establishing a stable pH gradient inside the
capillary using a mixture of amopholytes. At the same time, an ampholytic
analyte, such as a protein, moves along this pH gradient and is focused at
the point where the pH is equivalent to its isoelectric point. The
migration then ceases. Thus, a stationary condition is reached and
maintained in the capillary. During this separation process, narrow
Gaussian bands are generated with high peak concentrations which results
in high separation resolution of the analytes. In order to detect the now
focused analytes with available detectors, the focused zones must be moved
through a stationary detector which is usually located at one end of the
capillary. Thus, the focusing of the sample in the capillary is followed
by a mobilization process. The commonly used mobilization process requires
addition of salt to the electrolyte at one end of the capillary. The salt
causes changes in pH at that end of the capillary. Because of this pH
shift, the analytes focused in the capillary are no longer at their
isoelectric points and will consequently move or migrate toward the end of
the capillary and will pass through the detector. During the mobilization
process, distortion of the focused zones and loss in resolution are
unavoidable. Further, the mobilization process also takes at least about
15 minutes compared to the about five minutes required for the focusing
itself using commonly available isoelectric focusing systems. Since the
detection is necessary, the required mobilization makes the isoelectric
focusing a relatively slow separation method thought to have little
advantage compared to other capillary electrophoretic techniques.
Therefore, it is necessary to develop an on-line detection method to
eliminate the mobilization step and thereby improve the speed and
performance of detection using the isoelectric focusing separation
technique.
Several on-line scanning spectroscopic and radiometric detection methods
have been developed for electrophoresis performed on slabs. However, such
methods cannot be satisfactorily used with electrophoresis carried out in
microbore capillaries because of their small size. Recently, there have
been attempts made to continuously monitor capillary isoelectric focusing
separation. In one instance, photographs were taken of the focusing of
blue dye stained proteins inside a 0.4-0.6 mm i.d. capillary, and the
photographs used to study the zones of proteins. However, this technique
requires labeling of the proteins and can not give good quantitative
information. In another instance, the separation in the capillary was
monitored using chemical electrodes spaced along the length of the sample
chamber. Although a complicated 100 chemical electrode array was used, the
resolution obtained in these experiments was very poor.
With currently available capillary electrophoresis equipment, the capillary
tube is generally about a meter in length and each end must be manually
positioned in a container holding solute or sample to be separated. The
longer the capillary tube, the longer the time necessary for a sample to
move through the tube. When a new sample is to be separated, one end of
the capillary tube has to be moved to another container which contains the
new sample. Also, as the ends of the capillary are moved from container to
container, the electrodes necessary for operation of the system must also
be moved. Since voltages up to about 10 KV are required to operate the
system, the person moving the capillary tube and electrodes from container
to container may easily come into dangerous contact with the electrodes.
One of the most promising applications for capillary electrophoresis is for
routine analysis in research laboratories, pharmaceutical manufacturing
facilities, and hospitals. However, in many cases, relatively rapid
separation and accurate detection of samples is required, because the
feedback of the analyzed data is essential for observing effectiveness of
a therapy, adjusting drug doses in treatment of patients in hospitals, or
controlling process conditions in industrial manufacturing. Also, since
different methods of capillary electrophoresis apply to different types of
samples and situations, it would be convenient to be able to run different
methods on the same instrument. It is impossible for current commercial
capillary electrophoresis instruments to change from one separation method
to another. Each instrument and capillary cartridge is designed for a
particular type of separation, e.g., for capillary isotachophoresis, or
for moving boundary capillary electrophoresis. A further problem is that
current commercially available capillary electrophoresis instruments lack
sensitive, universal, and inexpensive detectors. Although conventional
absorption spectrophotometric detectors can be universal, they are not
sensitive enough for capillary electrophoresis using narrow capillaries,
and an expensive monochromator is required. The fluorometric detectors in
use not only need expensive lasers and photomultipliers but also require
fluorescent derivatization for most analytes. The commercial capillary
electrophoresis instruments with such detectors are usually expensive and
large devices.
Also, it is sometimes desirable to separate and identify sample components
or determine if such components are present in a sample, for components
which do not lend themselves to separation by electrophoretic techniques.
Thus, other more complicated detectors and detection methods are required
to detect these components.
SUMMARY OF THE INVENTION
According to the invention, it has been found that concentration gradient
detection is uniquely suited for use as a detection means and method for
detecting the various bands of components as separated by capillary
isoelectric focusing techniques. It has also been found that isoelectric
focusing can be effectively accomplished in relatively short capillary
tubes, such as capillary tubes with about ten centimeters overall length,
and that the actual separations take place within an even shorter portion
of such tubes. Further, it has been found that a light beam can be
generated as a sheet or line of light, i.e., a beam of predetermined width
with very small height dimension, that can be focused on the capillary
passage in the capillary tube and the separated sample therein so as to
extend along the capillary passage through the portion containing the
separated sample. The light within the light beam is deflected by the
concentration gradients in the sample along the width of the light beam to
produce variations in the intensity of the light beam along its width
which are representative of the concentration gradients established in the
capillary passage by the isoelectric focusing. By monitoring the light
beam after passing through the capillary tube and sample therein, the
separation of the sample can be easily and quickly determined.
In a preferred embodiment of the invention, the intensity of the light beam
after passing through the sample is monitored by a light detector in the
form of a photodiode array extending along the portion of the capillary
tube where the expected separations take place. The photodiode array is
made up of individual sensing elements of a size small enough to be able
to resolve and detect the differences in light intensity caused by the
refraction of the light passing through the concentration gradients in the
sample. With such an arrangement, the light source, detector, and sample
chamber, i.e., capillary passage, are all fixed relative to one another to
maintain accurate light beam focusing and detection, yet the measurements
can be made with a stationary sample separation in the capillary. It is
not necessary to move the sample through the light beam to obtain a
measurement. The entire sample of interest is comprehended by the light
beam passing through the sample. Further, in the preferred embodiment, the
light beam is generated by an inexpensive He-Ne laser with a cylindrical
lens to convert the beam from the laser into a sheet of light and to focus
the sheet onto the capillary passage. Various other means of producing the
sheet of light could be used, however.
In an alternate preferred embodiment of the invention, the intensity of the
light beam after passing through the sample is monitored by a light
detector in the form of a single photodiode having a narrow sensing area
compared to the width of the light beam to be detected so that the
detector senses only a small portion of the beam at any one time. The
photodiode is mounted for movement along the sample chamber where the
expected separations take place. By moving the photodiode along the sample
chamber through the beam, the diode measures the light intensity at the
particular location it moves through and thereby provides an output
proportional to the intensity of the beam at each point the photodiode
moves through. Since the light beam is not moved with the detector, i.e.,
the sample chamber and the light beam remain fixed in relation to one
another, the problem with maintaining alignment of the light beam and
sample chamber is not present.
A simple and easy to use capillary electrophoresis apparatus can be
constructed with a first reservoir and a second reservoir connected and
held together with a sheet of glass, such as a microscope slide. A
capillary tube secured to the slide extends between the reservoirs so that
the capillary passage through the capillary tube extends between and
connects the two reservoirs. Electrodes are inserted into the reservoirs
and the reservoirs are configured to easily accept a tube extending from a
syringe to enable liquid to be easily added to or withdrawn from a
reservoir. Thus, the solute or sample can be easily changed in a reservoir
by withdrawing it from the reservoir with the syringe and adding new
solute or sample, or mixture, with a syringe. In such apparatus, the
capillary passage will usually be about ten centimeters long and have a
capillary diameter of between ten and one-hundred microns. The reservoirs
have small capacity so that only small sample volumes are needed. The
volume of the reservoir, however, will be larger than the volume of the
capillary so that enough sample will be present to fill the capillary.
With such an arrangement, any method of electrophoresis may be used. It is
not necessary to use a different apparatus for each different method of
electrophoresis.
A method of the invention allows electrophoretic separation, particularly
using the isoelectric focusing technique, for various sample components
not otherwise subject to such separation. The method involves selecting a
reagent capable of separation and having a high degree of specificity for
the component to be detected. The reagent is added to the sample,
preferably in an amount sufficient to interact with all of the sample
component to be detected that might be in the sample. The reagent has its
own isoelectric point and the product of reagent and component has a
different isoelectric point. Thus, with isoelectric focusing, the reagent
in the sample, and the product of reagent and component in the sample,
will each be separated to form different bands in the sample which can be
detected by the detector of the invention.
THE DRAWINGS
The best mode presently contemplated for carrying out the invention is
shown in the accompanying drawings in which:
FIG. 1 is schematic representation of a light beam passing through a
gradient;
FIG. 2, a schematic representation of a light beam passing through a sample
chamber, showing the deflection angle produced by the presence of a
refractive index gradient in the chamber;
FIG. 3, a second schematic representation of a light beam passing through a
sample chamber with a concentration gradient;
FIG. 4, a schematic representation of a light beam passing through a sample
chamber similar to FIG. 2, but showing a wide beam with a gradient in part
of the beam;
FIG. 5, a further schematic representation of a wide light beam passing
through a sample chamber with a concentration gradient in a portion of the
beam;
FIG. 6, a vertical section through the capillary passage of an
electrophroesis apparatus of the invention;
FIG. 7, a fragmentary horizontal section taken on the line 7--7 of FIG. 6,
but showing the light source, lenses, and detector in top plan view;
FIG. 8, a fragmentary vertical section taken through a capillary tube
showing sample component separation resulting from isoelectric focusing;
FIG. 9, a vertical section through a light beam formed by the apparatus of
the invention;
FIG. 10, a fragmentary vertical section taken on the line 10--10 of FIG. 7;
FIG. 11, a fragmentary top plan view of an alternate detector of the
invention;
FIG. 12, a block diagram of a detector of the invention;
FIG. 13, a curve representing the light intensity profile of a probe light
beam passing through a sample separated by isoelectric focusing in the
apparatus of the invention and produced as the output signal by a moving
detector;
FIG. 14, a set of three curves labeled a, b, and c with curve a
representing a peak of the curve of FIG. 13, curve b representing the
integral of curve a, and curve c representing the integral of curve b and
second integral of curve a;
FIG. 15, a set of three curves labeled a, b, and c representing the light
intensity profiles of a probe light beam passing through a sample during
separation by isoelectric focusing in the apparatus of the invention and
produced as the output signals by a moving detector;
FIG. 16, a set of three curves labeled a, b, and c representing the light
intensity profiles of a probe light beam passing through a sample during
separation by isoelectric focusing in the apparatus of the invention and
produced as the output signal by a diode array detector;
FIG. 17, a fragmentary vertical section of an alternate sample receiving
reservoir usable with the apparatus of FIGS. 6 and 7;
FIG. 18, a set of three curves labeled a, b, and c being schematic
representations of light intensity profiles showing separations obtained
through a method of the invention;
FIG. 19, a curve representing the concentration profile of a sample
separated in the apparatus of the invention and mobilized to move through
a detector and produced as the output signal by the detector;
FIG. 20, a set of two curves labeled a and b showing the concentration
gradient profiles of the same sample separated using capillary zone
electrophoresis and moving boundary capillary electrophoresis; and
FIG. 21, a curve showing the concentration gradient profile for the same
sample as used for FIG. 20, showing the sample separated by isoelectric
focusing.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
It is well known that light passing through a refractive index gradient in
a solution is deflected. The physical reason for light deflection when
passing through this gradient lies in the relationship between the
refractive index and light propagation velocity. Different parts of the
light advance to a different degree with time, which generates the phase
shift. Thus, as shown in FIG. 1, during a given time period t+dt, light at
the top of a light beam indicated by arrow 10 which is passing through a
solution with a refractive index of n+dn will travel a distance of D1. The
light at the bottom of the light beam indicated by arrow 11 which is
passing through a solution with a refractive index of n will travel a
distance D2. This results in a tilt of the light wavefront and since light
travels perpendicular to the wavefront, the light beam is tilted as
illustrated. In FIG. 1, D2 is greater than D1 resulting in an upward tilt,
but depending upon the values of n and n+dn, the tilt could be downward.
The light path through the refractive index gradient can be calculated by
using the Fermat principle that the light path through the medium is such
that the time necessary for its traversal is minimum. The relationship
between the angle of deflection, .theta., and the refractive index
gradient normal to the light propagation dn/dx and path length through
this gradient, D, can be written as
tan .theta.=sinh (D/n)(dn/dx)=(D/n)(dn/dx)+(dn/dx).sup.3 (D.sup.3 /n.sup.3
3!)+(dn/dx).sup.5 (D.sup.5 /n.sup.5 5!)+. . .
where n is the refractive index of the medium. In situations where the
sensor of the invention will be used, D and .theta. are small. We can then
approximate:
.theta.=(D/n)(dn/dx)
FIG. 2 illustrates the detection principle behind this method. With a
nonuniform distribution of a solute in the sample chamber shown
schematically between sample chamber walls 12 and 13 giving a sample
chamber distance D, a concentration gradient is established. This gradient
forms the corresponding refractive index gradient dn/dx=(dn/dc)(dc/dx),
which then tilts or deflects the propagating light beam by angle
.theta.=(D/n) (dn/dc)(dc/dx). This deflection can be measured by measuring
the position of the light beam on the position detector 14. The
information produced during the measurement of the concentration gradient
relates to the universal property of the solute--refractive index n.
Consequently, the concentration gradient produced by any solute that has a
different n than the solvent will be detected by noting a deflection or
tilt in the light beam.
FIG. 3 shows the same principal as FIG. 2, but illustrates it somewhat
differently. Thus, if a concentration gradient represented by line 16
exists in a sample in a sample chamber defined by walls 12 and 13, a probe
beam of light 17 directed through the sample will be deflected as
indicated above by an angle .theta.. This causes the position of the beam
to move on the surface of the position sensor 14 as indicated above.
FIG. 4 is similar to FIG. 2, but shows a wide light beam with a
concentration gradient 18 within the width of the light beam. Thus, rather
than the light beam being uniformly deflected as shown in FIGS. 1, 2, and
3, a portion of the light beam 19, on one side of the gradient 18, which
does not pass through a gradient, passes straight through the sample to
detector 14. Portion 20 of the light beam passes through the gradient 18
and is deflected by angle .theta. as shown by the broken arrows, and falls
onto detector 14 partially overlapping portion 19. Where the overlap
occurs on the detector, indicated at 22, the light striking detector 14 is
brighter than in non overlapping areas. Portion 21 of the light beam on
the other side of the gradient 18, again passes straight through the
sample and onto detector 14. As indicated in FIG. 4, there will be an area
23 where little or no light will fall. Thus, the single light beam after
passing through a sample with one or more gradients therein will have
varying intensity indicating the gradients present in the sample. A
gradient in the sample will generally result in a bright spot followed by
a spot of very little intensity, or, if just measuring intensity, a level
representing the light passing straight through the sample, an increased
or positive signal (the increased intensity), followed by decreased or
negative signal (the decreased intensity), followed again by the level
representing the light passing straight through the sample.
If the detector 14 is broken down into many small detectors, such as an
array of detectors, each detector detecting the light from a small portion
of the beam, a comparison or scanning of the individual detectors will
produce an output signal representative of the intensity of the light beam
falling on the array along its length. Alternatively, a single, small
detector which detects only a portion of the light beam could be
positioned to be moved through the beam along its width and measure the
light intensity as it is moved.
FIG. 5 shows the same principal as FIG. 4, but relates it more directly to
a system of the invention. Thus, again, the light beam may be considered
as many parallel light rays 25. A gaussian refractive index gradient
produced by a similar gaussian concentration gradient as would appear in a
sample in a sample chamber is represented schematically by curve 26. Line
27 represents both the plane of the sample chamber where the light beam
passes through the sample and an axis indicating length along the sample
chamber for the refractive index gradient curve 26. Axis n represents the
refractive index of the sample within the sample chamber. Line 28
represents the detector plane and also an axis representing the intensity
of light passing straight through the sample chamber when no refractive
index gradient is present. Curve 29 represents the intensity I of the
light striking the detector plane. When the light beam passes through the
sample chamber, the individual light rays 25 are refracted and bent out of
their original path upon encountering the refractive index gradient 26
produced by a concentration gradient inside the sample chamber. If the
intensity of each light ray is constant, equalling I.sub.o, the relative
changes of the light intensity on the detector plane can be given by:
##EQU1##
Here, x is the direction along the sample chamber, z is the direction
along the light beam, n is the refractive index inside the sample chamber,
d is the diameter of the sample chamber, and L is the distance between
sample chamber and the detector plane. In this equation, [1/n(x)] dn/dx
corresponds to the light beam deflection angle, and is small. Its high
power in the second term of the equation can be neglected, compared with
the first term, then:
##EQU2##
which shows that the relative changes of the probe beam intensity on the
detector plane are proportional to the second derivative of the refractive
index inside the capillary. The relationship between the magnitude of the
refractive index change and the sample's concentration is approximately
linear. Hence, the relative changes of probe beam intensity on the
detector plane are also expected to be proportional to the second
derivative of the sample's concentration inside the capillary.
FIGS. 6 and 7 show a capillary electrophoresis apparatus of the invention.
As shown, a capillary tube 35, preferably of glass, has a capillary
passage 36 therein. Open containers 37 and 38, forming reservoirs 39 and
40, respectively, are secured to the ends of capillary tube 35 so that
capillary passage 36 communicates with reservoirs 39 and 40. The assembly
is held together as a unit by a member 41 to which the capillary tube 35
and containers 37 and 38 are secured. If the capillary tube is glass, it
is preferred that member 41 also be glass and that the capillary be
secured thereto by epoxy so that no refractive interface is formed between
the two. The containers 37 and 38 may be of any suitable material, such as
polyethylene, and secured to member 41 by any suitable adhesive, such as
epoxy. Containers 37 and 38 may be provided with tops 42 and 43,
respectively, which hold electrodes 44 and 45 in position in reservoirs 39
and 40, respectively. Tubes 46 and 47 extend through tops 42 and 43,
respectively, to open near the bottom of reservoirs 39 and 40,
respectively. Fittings 48 and 49 on the tops of tubes 46 and 47 where they
pass through the respective tops are adapted to receive tubes from a
source of, or receptacle for, liquid to be added to, or withdrawn from,
the reservoir. Such means may conveniently take the form of a syringe such
as shown schematically as 50 with a tube 51 to connect it to the
appropriate fitting 48 or 49. The syringe may be manually operated or
motor driven, or various other types of pumps or delivery systems could be
used. Of course, electrodes 44 and 45 and tubes 46 and 47 could be
positioned in the reservoirs by various other means and containers 37 and
38 could remain with open tops. An advantage of providing containers 37
and 38 with tops, and providing that the tops seal such containers, is
that the pressure in the containers can then be controlled. Thus, a
syringe or other delivery system can be used to pressurize a reservoir to
force liquid into the capillary passage, or can be used to draw a partial
vacuum in a reservoir to draw liquid from the opposite reservoir into the
capillary passage. The unit described may be mounted on a base 52, if
desired.
For the unit described, the capillary tube may vary in length as desired,
but tube lengths between three centimeters and fifteen centimeters have
been found satisfactory for various types of capillary electrophesis.
Further, the diameter of the capillary passage may also vary as desired,
with diameters of between 10 .mu.m and 100 .mu.m having been found
satisfactory. Either round or square capillary passages may be used, but
square passages have been found particularly suitable for use with the
detector and detection method of the invention. When a square capillary is
used, diameter of the capillary refers to the length of a side of the
square. The size of the reservoir is not critical, although the volume of
a reservoir must be large enough so that the capillary passage can be
filled by the particular method being used to fill the capillary. In
addition, the electrodes must be in contact with liquid in the reservoir.
Thus, the volume of the reservoirs will generally be larger than the
volume of the capillary passage extending between them. For example, in
the configuration shown in FIGS. 6 and 7, reservoirs each having a volume
of about 0.2 milliliters has been found satisfactory for various size
capillary passages, such as a fifteen centimeter long passage of 20 .mu.m
diameter.
The electrodes 44 and 45 are connected to a source of high voltage (not
shown) by wires 53 and 54, respectively. The voltage will preferably be in
the range of between 5 KV and 10 KV, depending upon the type of separation
being used. Generally, higher voltages will result in faster separation
times, but the voltage is limited by the current flow through the sample
in the capillary passage which generates heat in the sample. The heat
generation must be kept below the level of heat that is readily dissipated
through the capillary tube or the capillary tube may explode. The current
flow is generally monitored by monitoring current f | | |
