Patent Publication Number: US-2007110628-A1

Title: Apparatus and method for dispensing fractions of an analyte solution

Description:
FIELD OF THE INVENTION  
      The present invention relates to an apparatus and method for dispensing low volumes of an analyte solution for subsequent testing or analysis.  
     BACKGROUND OF THE INVENTION  
      Apparatus used in analytical chemistry include mass spectrometers. One type of mass spectrometer is a matrix assisted laser desorption ionization (MALDI) mass spectrometer of which there are also several types. MALDI is typically operated as an off-line ionization technique, where a solid or liquid sample, mixed with a suitable matrix, is deposited on a MALDI target or medium to form dry mixed crystals and, subsequently, placed in a source chamber of the mass spectrometer for analysis. An example of a MALDI target is a rectangular plate having a plurality of microfabricated wells on an upper surface thereof for receiving pL-nL sample volumes of analyte solution. The analyte solution may be generated by a variety of separation or processing techniques or apparatus including liquid chromatography apparatus.  
      Liquid chromatography (LC) involves the separation of chemical substances and particles by differential movements through a two phase system. A mixture of materials is typically applied to a column containing a suitable chosen absorbent (e.g. an ion-exchange material) and caused to flow therethrough. Materials in the mixture are absorbed at differential rates, with the least absorbed materials emerging first from the column and the more strongly absorbed materials emerging later.  
      When analyzing liquids eluted from an LC apparatus using a mass spectrometer, it is important to dispense very low volume samples (e.g. pL-nL samples) with no or minimal carry-over. That is, it is desirable to keep the components of a sample as well as the components of subsequent samples from mixing together when being deposited on a sample target or medium. While apparatus and methods exist for dispensing low volumes of liquid, there is a problem with carryover of liquid samples being dispensed. The present invention is intended to address this problem.  
     SUMMARY OF THE INVENTION  
      In accordance with a first aspect of the invention, there is provided an apparatus for dispensing fractions of an analyte solution, the apparatus comprising: 
          (a) a receiver defining an ejection cavity and an outlet downstream of and in fluid communication with the ejection cavity;     (b) first and second fluid conduits coupled to the receiver and defining respective first and second fluid flow paths in fluid communication with the ejection cavity;     (c) an analyte generating apparatus operatively coupled to the first fluid conduit and operable to deliver analyte solution along the first fluid flow path to the ejection cavity;     (d) a dispensing mechanism operatively coupled to the second fluid conduit and operable to deliver a fluid buffer along the second fluid flow path into the ejection cavity to displace analyte solution present therein through the outlet.        

      The ejection cavity may have a volume of between 0.1 nl and 1000 nl, or less than 500 nl, 200 nl, 100 nl, or 50 nl.  
      The dispensing mechanism may comprise an actuator chosen from solenoid, piezoelectric, electro kinetic, mechanical, valve, thermal, magnetic, and pressurized fluid actuators. Furthermore, the analyte generating apparatus may comprise a component chosen from liquid chromatography, capillary electrophoresis, and capillary electro chromatography apparatus.  
      The dispensing mechanism may further include an aspirator operable to selectively reverse the flow of buffer from and out of the ejection cavity and upstream along the second fluid conduit.  
      The outlet of the apparatus may be circular and have a diameter of from 5 to 200 micrometers, or less than 100, 50, or 20 micrometers.  
      In one embodiment, the apparatus is used to dispense fractions of an analyte solution onto a collection medium for subsequent testing by matrix assisted laser desorption ionization, and includes a matrix flow generator, the matrix flow generator having a matrix supply and a third conduit having a first end in communication with the matrix supply and a second end in communication with the ejection cavity, the matrix flow generator being operable to deliver matrix through the third conduit to the ejection cavity, whereby a mixture of the analyte solution and matrix is dispensed when the dispensing mechanism is actuated. The second end of the third conduit may be in communication with the first flow path upstream of the ejection cavity whereby mixing of the matrix and analyte may occur upstream of the ejection cavity.  
      In accordance with another aspect of the invention, there is provided a method for dispensing fractions of an analyte solution comprising the steps of: 
          (a) providing a receiver defining an ejection cavity and an outlet in communication with and downstream of the ejection cavity;     (b) filling the ejection cavity with an analyte solution at a selected rate; and     (c) injecting a fluid buffer into the ejection cavity to displace the analyte solution through the outlet.        

      The analyte solution may be eluted material exiting a liquid chromatography apparatus, and may also be supplied to the ejection cavity at a rate of from 1 nl per minute to 2 ml per minute, from 10 nl per minute to 5000 nl per minute, or from 50 nl per minute to 2000 nl per minute. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
      Embodiments of the invention will now be described with reference to the drawings in which:  
       FIG. 1  is a schematic drawing illustrating an apparatus according to a first preferred embodiment of the invention;  
       FIG. 2  is a schematic drawing illustrating an apparatus according to a second preferred embodiment of the invention; and  
       FIG. 3  is a schematic drawing showing a variety of actuators which may be used in the present apparatus. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       FIG. 1  illustrates an apparatus  10  for dispensing fractions of an analyte solution, according to the first preferred embodiment of the invention. The apparatus comprises a receiver  12  defining an ejection cavity  14  and a circular outlet  16 , which is 50 micrometers in diameter downstream of and in fluid communication with the ejection cavity  14 . The apparatus further comprises first and second conduits  18 ,  20  coupled to the receiver  14  and defining respective first and second fluid flow paths  22 ,  24  in fluid communication with the ejection cavity  14 . The apparatus  10  includes an analyte generating apparatus which, in this embodiment, is a liquid chromatography (LC) apparatus  26 , including an LC column  27  operatively coupled via LC conduit  28  to the first fluid conduit  18 . The LC apparatus is operable to deliver analyte solution in the form of LC fractions along the first fluid flow path  22  to the ejection cavity  14 . A dispensing mechanism designated generally by reference numeral  30  is operatively coupled to the second fluid conduit  20  and functions to deliver a fluid buffer  32  along the second fluid flow path  24  into the ejection cavity  14  to displace LC fractions present therein through the outlet  16 . In this embodiment, the fluid buffer is a solution consisting of a mixture of water and organic solvents. It would be appreciated that the buffer solution is tailored to the particular application and may contain mild acids, matrix material and other suitable components.  
      As shown in  FIG. 1 , the buffer  32  is contained under pressure in a buffer container  34  which is coupled to a solenoid actuator  36 , as is known in the art. The solenoid actuator  36  includes a valve (not shown) which can be selectively opened or closed to cause buffer to flow into the ejection cavity  14  to displace an LC fraction  37  present therein onto a collection medium in the form of a steel plate  38 .  
      In this embodiment, the LC fractions are later analyzed using a matrix assisted laser desorption (MALDI) apparatus. Thus, the apparatus  10  includes a matrix flow generator in the form of a syringe pump  40  containing a matrix solution. Alternatives to the syringe pump include any other suitable pump which is effective to supply a flow of matrix to the ejection cavity. The person skilled in the art would understand which materials would be suitable for use as matrices in MALDI applications. Common matrix components include alpha-cyano hydroxy cynnamic acid, 2,5-dehydroxy benzoic acid, sinappinic acid, succinic acid, glycerol, and picolinic acid. The matrix flow generator includes a conduit  42  having a first end  44  in communication with the matrix supply and a second end  46  in communication with the ejection cavity  14 . The matrix flow generator  40  is operable to deliver matrix through the matrix conduit  42  to the ejection cavity  14  via the first fluid flow path  22 , whereby a mixture of the analyte solution and matrix is formed upstream of the ejection cavity  14  and enters the ejection cavity  14 . The mixture is then dispensed when the dispensing mechanism  30  is actuated.  
      In this embodiment, the dispensing mechanism  30  comprises an aspirator  48  which is selectively operable to reverse the flow of buffer solution  32  from and out of the ejection cavity  14  and upstream along the second fluid conduit  20 . The aspiration is effected by a second solenoid actuator  50 , as is also known in the art. When the second actuator  50  is actuated, the buffer solution  32  flows into a waste conduit  52  from the second fluid conduit  20  and into a waste reservoir in the form of a waste container  5  containing waste buffer solution. In this embodiment, the waste container  54  has an internal pressure of from 0 to 15 psi.  
      In use, the liquid chromatography apparatus  26  delivers LC fractions eluted from the LC column through the LC conduit  28  to the ejection cavity  14  via the first fluid flow path  22 . The solution is delivered at a rate of 100 nl per minute. Matrix solution is dispensed continuously at a similar rate and a mixture of matrix solution and LC fractions arrives at the ejection cavity at a rate of 200 nl per minute. In this embodiment, the ejection cavity has a volume of 20 nl. Since the rate of flow of analyte solution into the ejection cavity  14  is known, and since the volume of the ejection cavity  14  is known, the time within which the ejection cavity  14  is filled with matrix-containing analyte solution can be easily determined. The apparatus  10  includes a timer which is used to actuate the dispensing mechanism  30  to cause buffer solution to flow into the ejection cavity at select intervals to dispense the analyte solution after the ejection cavity  14  is filled. The buffer solution serves to clean the ejection cavity  14  during dispensing so as to prevent cross-contamination of LC fractions being dispensed. Because the outlet  16  does not come in contact with the steel plate  38 , the risk of cross-contamination of samples being dispensed is greatly reduced. To make room for the next sample to be dispensed, the second solenoid actuator  50  is actuated after each dispensing operation to aspirate the buffer and cause it to leave the ejection cavity  14 . Eluted material flowing from the LC column may then flow into the ejection cavity  14  after exit of the buffer.  
      Referring now to  FIG. 2 , an apparatus  60 , according to a second preferred embodiment of the invention, is shown. This embodiment is similar in all respects to the first embodiment described above, and therefore like reference numerals have been used to refer to like parts. One of the differences between this apparatus  60  and the apparatus  10 , is that the matrix flow generator  48  is coupled to the ejection cavity  14  downstream of the first fluid conduit  18 . In this embodiment, the matrix solution is dispensed through a matrix conduit  42   a  directly into the ejection cavity  14  whereupon it mixes with an LC fraction present therein. A second difference is that the fluid buffer is gaseous, namely air. Thus, no aspirator is required. The air is contained under pressure in the buffer container  34  and injected into the ejection cavity  14  using a solenoid actuator  36   a  suitable for use in pneumatic systems, as is known in the art. The solenoid actuator  36   a  works to apply a pulse of air into the ejection cavity  14  thereby dispensing analyte solution present therein onto the steel plate  38  while, at the same time, cleaning the inside surface of the ejection cavity  14  so as to prevent cross-contamination of analyte fractions being dispensed.  
      While specific embodiments have been described, the person skilled in the art will appreciate that many modifications may be made to the present invention. For example, the ejection cavity  14  may have a volume of anywhere between 0.1 and 1000 nl, less than 500 nl, less than 200 nl, or less than 100 nl.  
      Furthermore, the dispensing mechanism may be any one of a number of direct or indirect actuators, some of which are shown in  FIG. 3 . Thus, the actuator may be a piezoelectric, electro kinetic, mechanical, valve, thermal, magnetic, or a pressurized gas actuator.  
      Instead of a liquid chromatography apparatus, any apparatus which operates to generate analyte solution to be dispensed may be used, including capillary electrophoresis and capillary electro chromatography apparatus. It will be appreciated that the present apparatus may be used to dispense low volume samples of any test solution.  
      In the above examples, buffer is injected into the ejection cavity  14  once the ejection cavity is filled with analyte solution. However, buffer may be injected prior to the ejection cavity  14  being filled completely to dispense volumes less than the internal volume of the ejection cavity. It will also be appreciated that an analyte solution may be supplied to the ejection cavity  14  at varying rates. Typically, solution will be supplied at a rate of from 1 nl per minute to 2 ml per minute. The typical rate of flow of eluted material out of the liquid chromatography apparatus is between 50 nl/min and 1 ml/min and often between 50 nl/min and 5000 nl/min for nano/micro LC applications. Furthermore, any non-reactive gas may be used as the gaseous buffer, including nitrogen, argon and helium.  
      The aspiration portion of the dispensing mechanism  30  is optional even in the case in which the buffer is a liquid. For example, the pressure generator may be selected and configured to inject only enough buffer so as to displace an amount of analyte solution equivalent to the volume of the meniscus of analyte solution forming at the outlet. In this case, the ejection cavity  14  would still contain mostly analyte solution after each dispensing operation and there would be no need to aspirate the buffer to make room for analyte solution entering the ejection cavity  14 .  
      Furthermore, the dispensing and aspiration functions of the dispensing mechanism  30  may be combined in a single device. For example, an electrokinetic pump may be used to cause liquid to flow in opposite directions depending on the polarity of the voltage applied. Thus, when dispensing, the voltage of the pump will be of one polarity, and when aspirating, the voltage will be of the opposite polarity. There are other devices which may be used in place of an electrokinetic pump to achieve this combined function. The person skilled in the art will readily understand which alternative devices would be suitable in the present application.  
      The foregoing description is by way of example only and shall not be construed so as to the limit the scope of the following claims.