Abstract:
An apparatus adapted to obtain a profile of a density gradient sample independently of fractionation is provided. The apparatus includes a light source, a probe comprising a first probe needle actuatable to extend into a tube containing a sample, a first light-transmitting means to receive light from the light source and transmit light through the sample as the probe needle extends into the sample, a second light-transmitting means to receive light transmitted by the first light-transmitting means and transmit the received light to a signal-producing means capable of translating the received light into a recordable signal to produce a profile of the sample. The apparatus may additionally be adapted to fractionate the sample following generation of the gradient profile.

Description:
This application claims the benefit of the U.S. Provisional Patent Application No. 60/860,217, filed on Nov. 21, 2006. 

   FIELD OF THE INVENTION  
   The present invention relates to an apparatus useful to probe a solution gradient. In particular, the present invention relates to an apparatus comprising a probe adapted to generate a gradient profile of a density gradient independent of fractionation. 
   BACKGROUND OF THE INVENTION  
   Solution gradients or density gradients are utilized in biochemical research to separate macromolecules such as proteins, DNA and RNA, and larger aggregates such as viruses and cells. More recently, density gradient centrifugation has found application in the field of nanotechnology. Researchers at Northwestern University have used gradients to separate and purify different classes of carbon nanotubes. 
   Solution gradients usually utilize a solute of varying concentrations to aid in the separation of particles. Examples of appropriate solutes are: sucrose, glycerol, CsCl, Optiprep™, Percoll™, ficoll, metrizamide, Nycodenz™ and/or sodium acetate. Particles are separated during centrifugation either by their velocity of sedimentation, or by their density if there is an isopycnic point within the solution column in the tube. Faster, or denser particles, respectively, will appear lower in the tube. 
   After the sample has been subjected into the appropriate density gradient in the centrifuge, the particles are recovered from the gradient for analysis. Fractionation methods and apparatus used to recover the sample in the gradient involve the transfer of the entire gradient or certain layers or bands of the solution gradient to other vessels. It is often desired to extract only desired bands from the solution gradient for electron microscopy, liquid scintillation or gel electrophoresis. 
   One of the earliest and simplest methods of fractionation is to pierce the bottom of the centrifuge tube with a fine bevelled needle and collect the drops of the solution gradient as it flows through the needle into a second vessel. The flow of the solution into the opening of the needle becomes conical. In other words, the particles directly in front of the needle opening and within a zone best described as an inverted cone above the needle are drawn into the needle opening before particles outside the cone. The resulting fractionation of different layers of the solution gradient significantly degrades the resolution achieved in the gradient. 
   Bottom puncture with side hole needles have also been used for fractionation. Side hole needles have a hole on each side of the needle tip. Side hole needles are more effective than the bevelled needle, but side hole needles also draw the solution into the needle in a conical fashion preventing high resolution of the fractionation. 
   One of the most common methods for fractionating solution gradients introduces a dense solution at the bottom of the centrifuge tube, which floats the gradient up to an inverted collection funnel placed on the top of the gradient. Some loss of resolution results from the retardation of particles near the tube wall during this upward movement, and at any but the slowest flow rates, the shallow collection cone fails to prevent the shallow collection cone fails to prevent the same cone-shaped extraction of liquid directly below the cone&#39;s central orifice experienced by the bevelled needle described above. The result is mixing of different layers in the gradient and the resultant loss of resolution. 
   These problems were addressed in U.S. Pat. No. 4,003,834 to Coombs, issued Jan. 18, 1977, an apparatus is disclosed for the fractionation of a solution gradient by displacement with a piston, and in U.S. Pat. No. 5,645,715 to Coombs issued 1995 which discloses a piston collection tip with a unique trumpet shape collection face. The use of a piston to displace the gradient from the top down solves the problem of particles adhering to the wall during the upward movement of the entire gradient since the gradient remains stationary until it is displaced by the downward movement of the piston. The trumpet tip prevents the cone-shaped mixing by gradually compressing horizontal bands into thin vertical columns prior to collection. Tubing carrying both air and rinse is disposed within the piston to allow for cleaning of the collection tubing, further improving resolution by preventing cross contamination between fractions. Pumping air into the piston tip transfers any solution gradient left in the tubing to a second vessel. 
   U.S. Pat. No. 4,003,834 also provides a means for visualizing bands of particles large enough to scatter visible light. However, many particles of interest are too small to scatter visible light or are present at too low a concentration to be detected. Since the nucleic acids and proteins found in these particles absorb UV light in the 260-280 nm range, it is the current practice to detect bands of these particles by passing the gradient outflow through a UV flow cell as is frequently done in HPLC and FPLC. The UV gradient profile obtained by the flow cell can be used as a diagnostic tool in its own right; however, in this application, the profile is generated as the gradient is being removed from the centrifuge tube. 
   There are two potential problems with this type of UV-based fractionation. Firstly, it is difficult to accurately and reproducibly identify the beginning and end of UV absorbance peaks (bands) in the profile as it is being generated. Secondly, unless the user manually interrupts the flow at the start and end of each peak, the fraction collector typically used to separate the gradient outflow into discrete fractions is doing so at a constant time interval or rate of flow. Thus, there is no relationship between the peaks of absorbance and the fractions and this requires the user to scan a range of fractions to identify those containing the particles of interest. Some UV-based collection systems have “peak-picking” algorithms built into their software so that rapid changes in UV absorbance in the outflow trigger sample collection into a new vessel. While providing adequate separation of discrete peaks of particles, these devices have difficulty detecting and separating overlapping peaks or shoulders. Volume- or time-based fractionation of the UV-flow cell output is disrupted by peak-picking, so the overall sampling profile is then lost. Thus, one must choose between obtaining uniform size samples for analysis or isolating peaks, as they are mutually exclusive. 
   Certain inventions (i.e. U.S. Pat. Nos. 4,873,875; 6,479,239) have attempted to obtain a UV or fluorescent profile of the contents of a centrifuged gradient by vertically scanning the gradient with a beam of light from the outside of the tube. These have not seen widespread use because the only centrifuge tubes that can withstand the severe stress of ultracentrifugation (100,000-1,000,000×g) are made of a UV-absorbing plastic, effectively preventing the beam from penetrating the tube. Consequently, the only devices currently capable of producing a UV profile of a gradient are those which pass the gradient through a detached UV flow cell. 
   It would be desirable, thus, to develop a means of generating a gradient profile independent of fractionation that may be used as a guide to fractionation. 
   SUMMARY OF THE INVENTION 
   An apparatus has now been developed which is useful to obtain a profile of a density gradient. The apparatus comprises a probe adapted to obtain a gradient profile optically. The probe advantageously causes minimal disturbance of the gradient and thereby provides a gradient profile of high resolution. 
   Thus, in one embodiment, an apparatus is provided adapted to obtain a gradient profile. The apparatus includes a light source, a probe comprising a first probe needle actuatable to extend into a tube containing the gradient, said probe being in communication with the light source and comprising a first light-transmitting means to receive light from the light source and transmit light through the gradient as the probe needle extends into the gradient, and a second light-transmitting means to receive light transmitted by said first light-transmitting means and transmit the received light to a signal-producing means. 
   In another aspect of the present invention, there is provided a fractionation apparatus adapted to obtain a gradient profile of a density gradient independently of fractionation. The fractionation apparatus comprises:
         a light source;   a probe comprising a first probe needle actuatable to extend into a tube containing the gradient, said probe being in communication with the light source and comprising a first light-transmitting means to receive light from the light source and transmit light through the gradient as the probe needle extends into the gradient, and a second light-transmitting means to receive light transmitted by said first light-transmitted means and transmit the received light;   a signal-producing means positioned to receive light from the second light-transmitting means, wherein said signal-producing means translates the received light into a recordable signal to produce a profile of the gradient; and   a piston actuatable to extend into the gradient-containing tube and to fractionate the gradient according to the sample profile;
 
wherein the probe and piston are moveable between a resting position wherein the probe and piston are clear of the gradient tube, a probing position wherein the probe is in a gradient access position and a fractionating position wherein the piston is in a gradient access position, said probe and piston being independently actuatable.
       

   In another aspect of the present invention, a method of fractionating a gradient is provided comprising:
         i) determining the profile of a gradient;   ii) selecting a fractionation plan according to the profile; and   ii) executing fractionation of the gradient according to the plan.       

   
     These and other aspects of the present invention will become apparent by reference to the detailed description, and the drawings in which: 
     BRIEF DESCRIPTION OF THE DRAWINGS  
       FIG. 1  is a perspective view of an apparatus according to an aspect of the invention; 
       FIG. 2  is a side view of the fractionation portion of an apparatus of  FIG. 1  and an exploded view (A) of the valves therein; 
       FIG. 3  is a perspective view of the probe portion of an apparatus of  FIG. 1  and exploded views of the tip of the probe needle (A) and light source/photodetector (B); 
       FIG. 4  is a top view illustrating the arc of movement of a probe, piston and actuator of an apparatus of  FIG. 1 ; 
       FIG. 5  illustrates the first probing position (A/B) and second fractionating position (C/D) of the apparatus of  FIG. 1 ; 
       FIG. 6  illustrates an embodiment of the invention; 
       FIG. 7  illustrates an embodiment of the invention; 
       FIG. 8  illustrates an embodiment of the invention; 
       FIG. 9  illustrates an embodiment of the invention 
       FIG. 10  illustrates different geometries of the probe needle tip; 
       FIG. 11  illustrates the output signal including zone marking of an apparatus of  FIG. 1 ; and 
       FIG. 12  illustrates the probe (B) and piston (C) of an embodiment of the invention extended within a sample tube. 
   

   DETAILED DESCRIPTION OF THE INVENTION  
   A fractionation apparatus  100  is provided as shown in  FIG. 1  comprising a fractionation or collection portion  110  and a probe portion  120  useful to generate a profile of a density gradient that can be converted into a fractionation run. The fractionation portion  110  of the apparatus  100  generally corresponds with that described in U.S. Pat. No. 5,645,715, the relevant portion of which (found in columns 3-7) is incorporated herein by reference. As described, and as generally shown in  FIG. 2 , the fractionation portion  110  comprises a piston 10 having an internal passageway or valve  12 , and inserted within the valve  12  are a collection tube  14 , an air tube  16  and a rinse tube  18 . Mounted below the valve  12  on the end of the piston  10  is a collection tip  19 , which may optionally be an interchangeable collection tip as described in U.S. Pat. No. 5,645,715. 
   As one of skill in the art will appreciate, the configuration described in U.S. Pat. No. 5,645,715 may be modified as shown in  FIG. 2(A)  to increase efficiency. For example, the valve  12  may incorporate two one-way valves to permit the rinsing and drying of the sample tubing without disturbing the gradient. The first one-way valve is a ball valve  3  which prevents backflow of air or rinse into the gradient, while the second one-way valve is a rubber duckbill  4  valve which prevents backflow of rinse and gradient into the air tubing. Backflow of air and gradient into the rinse line is prevented by a rubber duckbill one-way valve mounted in the tubing between the rinse pump and the piston. 
   Referring to  FIGS. 1 and 3 , the fractionation apparatus  100  of an embodiment of the invention comprises a probe portion  120  situated adjacent to the piston  10 . The probe portion  120  comprises a probe  20 . The probe  20  consists of at least one hollow probe needle  22  which is open at both ends. The probe needle  22  has an upper end  24 , which is secured to a mounting block  27 , and a lower end  26 . The probe  20  and piston  10  are both mounted on a platform or swing arm  17 . The swing arm  17  is mounted onto an actuator  15 . As shown in  FIG. 4 , the center of the piston  10  and the center of the probe needle(s)  22  are positioned on the same radial arc  21 . The arc is centered on the actuator  15  such that rotation of the actuator  15  positions either the probe  20  or the piston  10  over the gradient tube holder  50  ( FIG. 1 ) of the apparatus  100 . Thus, as shown in  FIG. 5 , the swing arm  17  and actuator  15  are moveable between a fully retracted resting position in which both the piston  10  and probe  20  are clear of a tube holder  50  which holds the gradient, a first or probe position in which the probe  20  is in position centered above tube holder  50  ( FIG. 5A )) and a second or fractionating position in which the piston  10  is positioned centered above the tube holder  50  ( FIG. 5(C) ). 
   The actuator  15  also functions to lower each of the probe  20  and piston  10 , respectively, when in position above the tube holder  50 , into a sample gradient tube  13  as illustrated in  FIG. 9 . The actuator  15  may be manual, as illustrated and described in U.S. Pat. No. 4,003,834, the relevant disclosure of which (e.g. columns 3-7) is incorporated herein by reference. Alternatively, the apparatus may be fully automated by incorporating a computer to drive a stepper motor to rotate an acme screw which raises and lowers the actuator  15 , providing means for precisely determining the position and velocity of both the piston  10  and the probe  20 . This ensures that the extension of the probe  20  into the sample is conducted at a constant velocity such that each data point in the UV profile is coupled with its precise position in the gradient. 
   The probe needle  22  contains fiber optic bundles  28 . In one embodiment, as illustrated in  FIG. 6 , the fibre optic bundles  28  within the probe needle  22  include a mixed and randomized fibre optic transmitting bundle  29  and a fibre optic receiving bundle  30  ( FIG. 6B ), each of which extend the length of the probe needle  22 . The transmitting bundle  29  is connected to a suitable light source  32 , such as an LED emitting in a selected wavelength range. For detection of UV-absorbing particles, the transmitting bundle  29  connects to a suitable UV source such as an ultra-violet light emitting diode (UV LED). In alternative embodiments, the light source may be an LED emitting in the visible range. The light source  32  is mounted onto the mounting block  27 . A photodetector  42  is also mounted on the mounted block  27  approximately adjacent to the light source  32  ( FIG. 6A ). 
   As one of skill in the art will appreciate, each fibre optic bundle will incorporate fibres manufactured of material appropriate for the transmission of the wavelength of the light emitted from the light source  32 . For example, if the light source  32  emits in the UV range from 250 to 350 nm, quartz (fused silica) fibres may be used. The number and diameter of the fibres in the fibre optic bundle is optimized empirically to provide the highest signal to noise ratio and the highest resolution in a given application. For example, in certain embodiments, such as those illustrated in  FIGS. 6-9 , 80 fibres with 0.1 m diameter are utilized. In these embodiments, the choice of 80 fibres was based on the fact that fewer fibres produce a weaker signal while more fibres require a larger diameter needle and result in greater disturbance during the probing of the gradient. In certain embodiments, such as those illustrated in  FIGS. 6 ,  8  and  9 , the fibres are split into two independent bundles, a transmitting bundle  29  and a receiving bundle  30 . 
   A reflection means  34  is permanently attached to the bottom end  26  of the probe needle  22  which functions to reflect a beam of light received from the light source  32 , conducted the length of the probe needle  22  by the transmitting bundle  29 , into the gradient. The reflecting means  34  reflects the light beam into the gradient towards a second reflection means  36 , for example, at a 90° angle to the probe needle  22 . The reflected beam exits the needle  22 , travels a gap  38  through the gradient and is then deflected by the second reflection means  36  back along the same plane towards the first reflection means  34 , e.g. at an angle of 180°. The gap  38  between the first and second reflection means  34 ,  36  is sufficient to render a suitably accurate reading of the gradient. The first and second reflection means  34 ,  36  may be any means capable of reflecting the light beam at the required angle. In this embodiment, for example, a prism is appropriate for use as the first reflection means  34  having a 45° reflecting angle. While many sizes of prism will be suitable, the prism exemplified in one embodiment has cross section dimensions of: 1.0 mm.×1.0 mm on both 90° faces and 1.4 mm along the hypotenuse reflector surface. The length of the prism matches the length of the end of the needle, e.g. 2.9 mm. 
   The second reflection means  36  is affixed to a support  45  sufficient to position it appropriately from the probe needle  22 . The support  45  may be a support needle (as shown in  FIG. 3 ) generally aligned parallel to the probe needle  22  such that it is appropriately spaced from the needle  22  to provide gap  38 . Generally, the gap  38  between the first and second reflection means  34 ,  36  is in the range of 1-10 mm. Since an increase in gap size will result in increased absorbance, and a decrease in gap size will result in a stronger signal, the gap  38  between the first and second reflection means  34 ,  36  may be adjusted in order to maximize resolution in view of the variability among gradients. The support needle  45  containing the reflecting means  36  is shown mounted onto mounting block  27  such that it will co-extend into the gradient simultaneously with the probe needle  22  on actuation of actuator  15 . 
   The second reflecting means  36  comprises a 180° reflecting angle and may be, for example, a planar mirror. Its minimum dimensions are the dimensions of the beam it is to reflect, for example, 0.3×2.6 mm, but may, of course be larger to reduce the stringency of its positioning on the support needle  45 . As indicated above, the second reflection means  36  is positioned to reflect the incident beam from the probe needle  22  back and into the receiving fibres  30  within the end of the probe needle  22 . 
   As shown in  FIG. 10 , the geometry of the probe needle end  26  has a significant impact on the resolving power of the probe needle  22 . In one example, the transmitting/receiving fibre bundle  29 ,  30  may be circular  48  ( FIG. 10A ) at the bottom end  26  of the probe needle  22 , producing a cylindrical beam of light  49  that is reflected by the first reflecting means  34  across the gap  38  to the second reflecting means  36 . The band of particles in the gradient  51  will first be detected when the bottom edge of the cylindrical beam  49  first encounters the band  61 . The band will be detected until the top edge of the beam  52  leaves the band, giving a total distance of detection  53 . If the probe needle  22  is flattened at its lower end  26  to a rectangular shape  54  ( FIG. 10B ), the light beam  55  crossing the gap  38  is much thinner, resulting in a smaller total distance of detection  56  and increased resolution. The dimensions of the rectangle are constrained by the number, diameter and arrangement of the fibers in the bundle. For example, using a bundle of 80 fibers generates a beam having 0.3×2.9 mm rectangular shape. If a single row of fibers is used in a flattened needle  57  ( FIG. 10C ), the thinnest beam of light  58  is produced and (theoretically) the smallest total distance of detection  59 . However, the amount of light available for detection is also much reduced (30% of the two other versions shown since the number of fibres is decreased), so the signal to noise ratio suffers. If the number of fibres remains the same as in the circular and rectangular examples (FIG.  10 A/B) ( 48  and  54 ), the end of the needle shown in  FIG. 10C  would be 8 mm across, giving a greater wetted surface area and producing more disturbance of the gradient during insertion and withdrawal of the probe. Thus, the dimensions of the end of the probe needle impact on both resolution and gradient disturbance, and require selection in order to provide an appropriate balance. Dimensions of 0.3 mm×2.9 mm represent an example of a suitable compromise between resolution and disturbance. While bands of particles in a gradient are occasionally 1 mm thick, most bands lie in the 2-5 mm range of thickness, so the 0.3 mm thickness produces a tolerable loss of resolution. 
   The required electronic circuitry  60  to send and receive the light signal is attached directly to the mounting block  27  (as shown in  FIG. 3A ) to minimize the sensitivity of the photoreceptor to spurious electronic interference and vibration. 
   The light beam received and conducted by the receiving fibres  30  is transmitted for detection by a photodetector  42  as shown in  FIG. 3 . One suitable photodetector for UV light is a 1 mm 2  SiC chip contained in a small can with a quartz window (JEC 1S, Boston Electronics, Boston, Mass.). Since the end of the receiving bundle  30  cannot be physically coupled to this chip, and since the light beam diverges at an angle of, for e.g., 17° after it leaves the upper end of the receiving bundle  30  the light beam is transmitted onto a set of condensor lenses  40  which collect and refocus the beam onto the SiC chip inside the can. An example of a suitable set of lenses consists of two plano convex lenses (01LQF005, f=10.0 mm, dia=5 mm, Melles Griot, Ontario, Canada) arranged as shown in the exploded view  FIG. 3B . 
   The photodetector  42  translates the light beam into a recordable output such as current or voltage which is then digitized by a microprocessor  61  such as a Burr Brown microprocessor (DDC-112) and displayed on a display unit  44 , such as a monitor, which is connected to the control panel  43  ( FIG. 1 ). The absorbance values collected at regular user-determined intervals, for example, 10 data points/mm, are stored as a spreadsheet associated with the depth in the gradient from which they are taken. The display unit  44  functions in real-time to display the gradient UV absorbance profile throughout the depth of the gradient. 
   Using the real-time profile, the fractionation is planned by dividing the profile into fractionation zones and by further dividing each zone into a desired number of fractions. This can be accomplished using, for example, a rotary encoder with push switch  63  to move a vertical line cursor across the displayed profile, pressing it down to set the position of the various zones. To synchronize the probing and fractionation functions, both stages of analysis begin with the actuator  15  in its full up resting position where it contacts a limit switch. For each different size tube, for example Beckman&#39;s SW28, SW28.1, SW40, SW41, SW55, SW60 and TLS55, the precise vertical offset between the tip of the probe and the point of gradient capture inside the piston tip is determined empirically and stored in memory. Thus, the position of the cursor on the display can be precisely translated into a corresponding piston position during fractionation. Likewise, the position of each zone and the sub-fractions within each zone can readily be calculated. Once the plan has been set on the display, the computer converts the fractionation plan into a series of downward movements of the piston interrupted by a user-selected rinse protocol at the end of each fraction. The speed of the piston&#39;s movements is set automatically, but is user-adjustable, with 0.3 mm/sec being a typical speed. 
   In practice, a gradient profile is obtained using an apparatus according to an aspect of the invention by placing a gradient-containing tube  13  into the tube holder  50 . As shown in  FIG. 5 , the probe  20  is swung into position above the tube and actuated to probe the sample. The probe and support needles ( 22 ,  45 ) are simultaneously lowered into the gradient at a constant speed within the range of 0.1-6.0 mm/sec. As the needles extend into the gradient, light transmitted from the light source  32  is captured by the transmitting fibre optic bundle  29  and is conducted the length of the probe needle  22  to the bottom end  26  thereof where it is reflected at a 90° angle off of the first reflecting means  34 . The reflected light travels the gap  38  through the gradient and is reflected at a 180° angle off of the second reflecting means  36 . The reflected light re-travels the gap  38 , is reflected 90° by the first reflecting means  34 , is captured by the receiving fibre optic bundle  30  and is conducted the length of the probe needle  22  to the top end  24  thereof. The returning beam leaves the end of the receiving bundle and is captured by a set of condensor lenses  40  which refocus it for detection by the photodetector  42 . The photodetector  42  translates the light received into recordable output that may be displayed graphically, numerically or otherwise on a display unit  44 . Upon reaching the bottom of the sample tube, the needles are withdrawn automatically. 
   The planning stage involves using a combined rotary encoder+push switch  63  connected to the display unit  44 , for example, as is a standard feature of any oscilloscope, to move a vertical line cursor along the X-axis of the displayed gradient profile to set the various zones for fractionation. As the user sets each new zone along the profile, the user is prompted to set the number of fractions within that zone as illustrated in  FIG. 11 . For example, the gradient may be a single zone divided into a number of equal fractions. Alternatively, the gradient may be divided into several zones with a variable number of fractions per zone. This latter mode permits the isolation of individual bands of particles in the gradient for further analysis. 
   Once the fractionation zones and fractions are selected, the piston  10  is swung into position over the gradient tube and the gradient is then fractionated automatically. The fractionation plan developed by the user in the previous stage is converted by the computer into a series of downward movements of the actuator  15  and piston  10 , interrupted by the insertion of brief bursts of rinses and air to expel sample left in the tubing and remove any cross contamination between fractions. 
   This profiling function of the probe  20  of the present apparatus  100  advantageously provides a means to view the sample gradient and particles of interest therein, and thus, a means to plan the fractionation of the gradient prior to the actual fractionation. 
   To this point, an embodiment is described in which the apparatus  100  comprises a dual needle probe ( 22 ,  45 ) with one needle ( 22 ) containing both the sending and receiving light bundles ( 29 ,  30 ) and the other support needle ( 45 ) having mounted thereon a second reflecting means ( 36 ) to deflect the beam reflected by the first reflecting means ( 34 ) from the transmitting bundle ( 29 ) back to the receiving bundle ( 30 ), thus effectively doubling the path length of the probe. 
   In another embodiment of the present invention, as shown in  FIG. 7 , the probe  20  includes dual probe needles, a first transmitting probe needle  22  containing a transmitting fibre optic bundle  29  and a second receiving probe needle  23  containing a receiving fibre optic bundle  30 . Both bundles will contain an appropriate number of fibers to permit a maximum beam strength and sensitivity, for example, 80 fibres each. As set out above, the first and second probe needles are spaced by an adjustable gap  38  as shown in  FIG. 7B . In this case, the first reflecting means  34  is attached to the bottom of the first probe needle  22  such that the light beam passing through the first probe needle  22  is reflected at a 90° angle through the gradient across gap  38 . The reflected light beam is received by the second reflecting means  36  which is attached to the bottom of the receiving probe needle  23  such that it reflects the incoming light beam at an angle of 90° to be received by the receiving fibre optic bundle  30  in the receiving probe needle  23 . As described, the receiving fibre optic bundle  30  carries the light beam to the top end of the second probe needle  23  where it is transmitted onto a set of lenses  40  for refocusing and then translation by a photodetector  42  to an output signal that forms the profile of the gradient ( FIG. 7A ). The fractionation plan and fractionation are then executed as described. 
     FIG. 8  illustrates another embodiment of the present invention, in which the apparatus  100  incorporates a dual wavelength probe. In this embodiment, the first transmitting probe needle  22  contains two fiber bundles of 40 fibers each. A first wavelength fibre bundle  29   a  is in communication with a first LED light source  32   a  that generates light at a first wavelength and a second wavelength fibre bundle  29   b  is in communication with a second LED light source  32   b  that generates light at a second wavelength ( FIG. 8A ). For example, in the case of UV light, the first light source may generate UV light at a wavelength of 260 nm for detection of nucleic acids, while the second light source may generate UV light at a wavelength of 280 nm for detection of proteins. The second receiving probe needle  23  contains a single receiving fibre optic bundle  30  of 80 fibers. In this embodiment, the first and second light sources  32   a,    32   b  transmit light in alternating pulses, which are absorbed by the appropriate transmitting bundle, reflected by the first reflecting means  34  to the second reflecting means  36 , reflected by the second reflecting means  36 , received and transmitted by the receiving fibre optic bundle  30  in the second receiving probe needle  23 . The light received by the photodetector  42  at the top of the receiving needle  23  is sorted by wavelength into two data streams by the microprocessor  61  (e.g. Burr-Brown processor DDC-112), one for each wavelength, generating a dual wavelength profile at the output stage. The fractionation plan and fractionation are then executed as described. 
   A further embodiment of the present invention is illustrated in  FIG. 9 . This embodiment comprises a single needle, dual bundle probe; however, rather than measuring the amount of light absorbed by particles in the gradient, this probe is designed to detect particles within the gradient or sample by fluorescence. In this embodiment, the single probe needle  22  houses both transmitting and receiving fibre optic bundles  29 ,  30  consisting of, for example, 40 fibers each ( FIG. 9A ). The light source  32  transmits an excitation beam which is carried by the transmitting fibre optic bundle  29  and reflected into the gradient by the first reflecting means  34  attached at the bottom of the probe needle  22  as previously described. Particles of interest  39  within the solution are detected when they absorb light from the emitting bundle at an absorption wavelength and emit it back to the receiving bundle  30  at an emission wavelength ( FIG. 9B ). The returning light beam strikes the reflecting means  34 , is received by the receiving fibre optic bundle  30  and transmitted to the photodetector  42  for translation as an output signal. A narrow bandpass filter  47  placed between the two condensor lenses  40  in front of the photodetector  42  will prevent the emitted beam from reaching the photodetector  42 , as is standard practice in any commercial fluorometer. This configuration can detect particles by their natural fluorescence, by the enhanced fluorescence of a wide variety of commercial dyes that bind specifically to biological molecules of interest or by fluorescent dye-tagged antibodies. For example, viruses can be detected using a DNA-binding dye called PicoGreen (Molecular Probes, Invitrogen, USA) (for example, see “Quantitation of Adenovirus DNA and Virus Particles with the PicoGreen Fluorescent Dye, Murakami P.; McCaman M. T. Analytical Biochemistry, Volume 274, Number 2, October 1999, pp. 283-288”). The excitation light source for PicoGreen delivered by the transmitting bundle 29 is at 485 nm and the emission wavelength received by the receiving bundle  30  in the same probe needle  22  is 538 or 518 nm. These dyes offer sensitivity that is reportedly 10,000 times more sensitive than UV absorbance. Using these dyes converts this absorbance device into a fluorometer probe. 
   As will be appreciated by one of skill in the art, the present apparatus  100  may be modified to solely incorporate a probe portion  120  without a fractionation portion  110  as partially shown in  FIG. 12B . Such a probing apparatus comprises the features detailed in the foregoing for the probing function of the apparatus and lacks those features necessary to conduct fractionation. Thus, in instances where the desired end product of a gradient-based analyses is the absorbance profile, an apparatus incorporating only a probe portion as described herein may be used. 
   Although the disclosure describes and illustrates the preferred embodiments of the invention, it is understood that the invention is not limited to these particular embodiments. Many variations and modifications will occur to those skilled in the art. For definition of the invention, reference is made to the appended claims. 
   References referred to herein are incorporated by reference.