Sample collection preparation methods for time-of flight miniature mass spectrometer

Information

  • Patent Grant
  • 6806465
  • Patent Number
    6,806,465
  • Date Filed
    Tuesday, January 15, 2002
    22 years ago
  • Date Issued
    Tuesday, October 19, 2004
    19 years ago
Abstract
A field portable mass spectrometer system comprising a sample collector and a sample transporter. The sample transporter interfaces with the sample collector to receive sample deposits thereon. The system further comprises a time of flight (TOF) mass spectrometer. The time of flight mass spectrometer has a sealable opening that receives the sample transported via the sample transporter in an extraction region of the mass spectrometer. The system further comprises a control unit that processes a time series output by the mass spectrometer for a received sample and identifies one or more agents contained in the sample.
Description




FIELD OF THE INVENTION




The invention relates to a time-of-flight (TOF) miniature mass spectrometer (MMS), and more particularly to an automated TOF MMS collection, measurement and analysis system for acquisition of mass spectra.




DESCRIPTION OF THE RELATED ART




One of the most powerful laboratory tools for analyzing a broad spectrum of chemical and biological material is the mass spectrometer. Mass spectrometry is a proven technique for analyzing many types of environmental samples. Mass spectrometry is used to determine the masses of molecules formed following their vaporization and ionization. Detailed analysis of the mass distribution of the molecule and its fragments leads to molecular identification. Mass spectrometry is especially suited for aerosol analysis because micrometer-sized heterogeneous particles contain only about 10


−12


moles of material and thus requires a sensitive technique such as mass spectrometry for proper analysis. Liquid samples can be introduced into a mass spectrometer by electrospray ionization (1), a process that creates multiple charged ions. However, multiple ions can result in complex spectra and reduced sensitivity.




A preferred technique, matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS), has become popular in the analysis of biological polymers for its excellent characteristics, such as ease of sample preparation, predominance of singly charged ions in mass spectra, sensitivity and high speed. Time-of-flight MALDI-TOF-MS is established as a method for mass determination of biopolymers and substances such as peptides, proteins, and DNA fragments. The analytical sensitivity of TOF MS is such that under the right conditions only a few microliters of analyte solution at concentrations down to the attomolor (10


−12


moles) range are required to obtain a mass spectrum. The MALDI-MS technique is based on the discovery in the late 1980s that desorption/ionization of large, nonvolatile molecules such as proteins can be effected when a sample of such molecules is irradiated after being co-deposited with a large molar excess of an energy-absorbing “matrix” material, even though the molecule does not strongly absorb at the wavelength of the laser radiation. The abrupt energy absorption initiates a phase change in a microvolume of the absorbing sample from a solid to a gas while also inducing ionization of the sample molecules. Detailed descriptions of the MALDI-TOF-MS technique and its applications may be found in review articles by E. J. Zaluzed et al. (Protein Expression and Purifications, Vol. 6, pp. 109-123 (1995)) and D. J. Harvey (Journal of Chromatography A, Vol. 720, pp. 429-4446 (1996)), each of which is incorporated herein by reference.




In brief the matrix and analyte are mixed to produce a solution with a matrix:analyte molar ratio of approximately 10,000:1. A small volume of this solution, typically 0.5-2. microliters, is applied to a stainless steel probe tip and allowed to dry. During the drying process the matrix codeposits from solution with the analyte. Matrix molecules, which absorb most of the laser energy, transfer that energy to analyte molecules to vaporize and ionize them. Once created, the analyte ions the ions formed at the probe tip are accelerated by the electric field toward a detector through a flight tube, which is a long (on the order of 0.15 to 1 m) electric field-free drift region. Since all ions receive the same amount of energy, the time required for ions to travel the length of the flight tube is dependent on their mass to charge ratio. Thus, low-mass ions have a shorter time of flight (TOF) than heavier ions. All the ions that reach the detector as the result of a single laser pulse produce a transient TOF signal. Typically, ten to several hundred transient TOF mass spectra are averaged to improve ion counting statistics. The mass of an unknown analyte is determined by comparing its experimentally determined TOF to TOF signals obtained with ions of known mass. The MALDI-TOF-MS technique is capable of determining the mass of proteins of between 1 and 40 kDa with a typical accuracy of +−0.1%, and a somewhat lower accuracy for proteins of molecular mass above 40 kDa. The ability to generate UV-MALDI mass spectra is critically dependent upon the co-crystallization or very close special proximity of the analyte and a molar excess of the matrix compound. In routine practice, a small volume of matrix solution that delivers a one thousand-fold molar excess of matrix is manually mixed with a small volume of the analyte solution which then dries on a sample stage. A spatially heterogeneous distribution of analyte and matrix typically develops as the droplet dries to form a sample spot. Under laboratory conditions, the incident laser is rastered across the sample to identify so called “sweet spots” that preferentially yield for an abundance of analyte ions. Although a motorized x-y stage may be incorporated for automated searching for the spot providing the best spectrum, this procedure can be a time consuming step.




MALDI is typically operated as an offline ionization technique, where the sample, mixed with a suitable matrix, is deposited on the MALDI target to form dry mixed crystals and, subsequently, placed in the source chamber of the mass spectrometer. Although solid samples provide excellent results, the sample preparation and introduction into the vacuum chamber requires a significant amount of time. Even simultaneous introduction of several solid samples into a mass spectrometer or off-line coupling of liquid-phase separation techniques with a mass spectrometer do not use TOF mass spectrometer time efficiently.




To improve on these procedures, microfabricated targets have recently been developed for automated high throughput MALDI analysis. In these designs, pL-nL sample volumes can be deposited into a microfabricated well with dimensions similar to the spot size of the desorbing laser beam about 100 micrometers to 1,000 micrometers diameter). Thus, the whole sample spot can be irradiated and the search for the “sweet spot” eliminated. Analysis of short oligonucleotides has been demonstrated with about 3.3 s required to obtain a good signal to noise ratio for each sample spot. Although the total analysis time, including the data storage, takes nearly an hour, theoretically all 96 samples could be recorded in about five minutes.




While the miniaturization of the sample target simplifies the static MALDI analysis, on-line coupling would allow continuous analysis of liquid samples including direct sample infusion and the monitoring of chromatographic and electrophoretic separations. Compared to ESI, MALDI provides less complex spectra and, potentially, higher sensitivity. There have been numerous reports in the literature about the MALDI analysis of flowing liquid samples. In one arrangement, the sample components exiting a CE separation capillary were continuously deposited on a membrane presoaked with the matrix and analyzed after drying. In other cases, the liquid samples were analyzed directly inside the mass spectrometer using a variety of matrices and interfaces. MALDI was then performed directly off rapidly dried droplets. In another design, a continuous probe, similar to a fast atom bombardment (FAB) interface, was used for the analysis of a flowing sample stream with liquid matrix. Glycerol was used to prevent freezing of the sample. Other attempts for liquid sample desorption were also made using fine dispersions of graphite particles and liquid matrices instead of a more conventional matrices. More recently, an outlet of the capillary electrophoresis column was placed directly in the vacuum region of the TOF mass spectrometer. The sample ions, eluting in a solution of CuCl.sub.2, were desorbed by a laser irradiating the capillary end. On line spectra of short peptides separated by CE were recorded. Attempts to use ESI to introduce liquid sample directly to the evacuated source of a mass spectrometer have also been reported.




Standard MALDI sample preparation techniques as just discussed are not applicable to a real-time TOF-MS systems, the constraints of which do not permit either the analyte and matrix to be mixed in solution or the laser to be rastered across the sample. An additional major design goal of a real-time system is increased throughput speed by avoiding or minimizing the extent to which samples must be processed prior to acquisition of mass spectra. Since MALDI-MS is being used, ideally it is preferred to intimately mix the concentrated sample with a large molar excess of MALDI matrix to produce a uniform analyte-matrix lattice across the sample spot. An alternate technique of depositing an analyte sample in aerosol form directly on a bare collection substrate, or pre-coated surface with a MALDI matrix might not provide the degree of intimate mixing and co-crystallization of the analyte with the matrix that for generation of high quality UV-MALDI mass spectra. Thus, with this second method, additional post-collection steps, e.g., over-spraying with MALDI matrix, may be required.




Another shortcoming of current TOF MS designs are the long pump-down times associated with the introduction of the samples into the vacuum chamber. In the operation of a conventional mass spectrometer a test sample must be introduced through a valve into a vacuum chamber to a location less than a millimeter from an ion extraction source. The introduction of a sample into the MMS vacuum chamber in a real-time system requires rapid sample exchange while maintaining a high vacuum. Current mass spectrometer models require about 5 minutes to pump-down to high vacuum after the introduction of a new sample. A pump-down time of seconds would better meet the requirements of a real-time device.




Although the above-listed examples show efforts to address various different problems related to sample preparation and extraction for a real-time spectrometer, currently there is no-real time device that would permit continuous on-line processing of multiple samples. A device for continuous introduction of individual samples into a time-of-flight mass spectrometer so that on-line MALDI-MS analysis can be carried out would be highly desirable.




SUMMARY




In view of the above described state of the art, the present invention seeks to realize the following objects and advantages.




It is a primary object of the present invention to provide a mass spectroscopic analysis system and method which is fully automated requiring no operator interaction.




It is also an object of the present invention to provide a mass spectroscopic analysis system which is portable and reliable enough to survive transport on a range of vehicles, allows handling by two persons, and operates from a portable power source.




It is also an object of the present invention to provide a mass spectroscopic analysis system and method which can carry out spectrographic analysis results faster than previously possible.




It is also an object of the present invention to provide a mass spectroscopic analysis system and method that is suitable for field applications.




It is another object of the present invention to provide a mass spectroscopic analysis system and method which includes provisions for thoroughly mixing an analyte with a matrix composition, thus facilitating real-time spectral analysis.




It is a further object of the present invention to provide a mass spectroscopic analysis system and method which may use, but does not necessarily, require post-collection fluid matrix processing prior to performing a mass spectral analysis.




It is also a further object of the present invention to provide a mass spectroscopic analysis system and method which reduces contamination of the procedure.




It is also an object of the present invention to provide a mass spectroscopic analysis system and method provides a permanent storage medium that has the ability to record pertinent data associated with the collection and measurement of the sample.




It is also a further object of the present invention to provide a mass spectroscopic analysis system and method which includes an external ionization source and electrostatic lens, thus removing the necessity of inserting the sample into the mass spectrometer's vacuum chamber, thus keeping vacuum pump-down times to a minimum and allowing real-time spectral analysis.




It is a further object of the present invention to provide a mass spectroscopic analysis system and method which promotes rapid throughput and utility of MALDI-TOF MS.




It is also an object of the present invention to capture infectious and toxic agents on a substrate in small spots that allow maximum coverage by an irradiating laser beam. The beam may cover less than about 0.1 mm diameter to greater than 1.0 mm in diameter.




It is another object of the present invention to provide a mass spectroscopic analysis system and method which provides for a variety of techniques for applying and mixing matrix with analyte, thus facilitating real-time spectral analysis.




These and other objects and advantages of the invention will become more fully apparent from the description and claims which follow, or may be learned by the practice of the invention.




As will be appreciated, the present invention provides an automated mass spectroscopic analysis system that may be characterized as an “end-to-end” process of sample collection, preparation, measurement and analysis. The present invention is distinguishable from prior art approaches in that conventional approaches are neither integrated nor automated. That is, in the prior art each process is manually performed under operator control and guidance. In accordance with the present invention, a mass spectroscopic analysis systems is provided which performs the following method steps: (1) collect, concentrate, and separate aerosols from breathable ambient air at concentrations on the order of 15 ACPs per liter of air and of 0.5 to 10.0 um aerodynamic diameter. It should be noted that while concentrations on the order of 15 ACPs per liter and of 0.5 to 10.0 um aerodynamic diameter are described, other particle concentrations and densities are also within the contemplation of the present invention; (2) capture infectious and toxic agents from the collected, concentrated and separated aerosols on a continuous substrate (e.g., flexible tape) in small spots that allow coverage by an irradiating laser beam on the order of 1.0 mm in diameter. It should be noted that using a laser with a spot size greater than or less than 1.0 mm in diameter is also within the contemplation of the present invention; (3) prepare the collected samples for the MALDI process by adding a matrix, (4) introduce the collected samples directly into the analysis system in real-time on the continuous substrate. That is, after collection is completed for each sample, the tape transports the sample into a time-of-flight (TOF) mass spectrometer analyzer. The apparatus of the present invention provides a novel vacuum interface which advantageously reduces the vacuum pump loading by isolating the main vacuum chamber from the sample port around the tape sample when samples are being changed. The vacuum interface is formed in part by utilizing the tape as a temporary boundary to form a vacuum chamber seal at or below micro-Torr pressure levels and (5) once inside the high vacuum chamber, a laser than ionizes the sample, and the resulting mass spectrum is analyzed for specific biomarkers that indicate the presence and identity of a biological agent.




The automated system of the present invention provides a number of advantages over prior art approaches including, a minute volume of fluid required for sample processing, eliminating the need for large storage reservoirs, stationary and level mounting configurations, or large power-hungry heating and cooling systems. Further advantages include the concurrent collection of multiple samples, allowing both the application of different analysis protocols and the archiving of samples for later confirmatory analysis.




In practice of the method of the invention, a sample is placed on a permanent storage medium (e.g., a VCR tape) that limits cross sample contamination and undergoes a variation of a matrix-assisted laser desorption/ionization (MALDI) preparation. Each sample is then advanced on the tape to the mass spectrometer analyzer for acquisition of mass spectra. A movable platen forces the tape against a sealing surface, thus creating a vacuum seal with an external vacuum chamber. A triggered laser and an external electric field ion extraction source provides the necessary ionization to initiate mass spectra analysis using a time-of-flight mass spectrometer. When the analysis is complete, the tape advances and a new sample can be analyzed.




Although the analyzer of the invention is achievable in a number of configurations, an acceptable configuration includes: (1) An aerosol interface including a particle collector/impactor stations for collecting, concentrating, and separating analyte from the sample aerosol. A nebulizer for injecting MALDI matrix particles into a sample aerosol upstream of one or more tape particle collector/impactor stations. Continuous tape substrate to collect, hold, and store the analyte and matrix mixture. The nebulizer is preferably automatically controlled to inject metered amounts of MALDI matrix aerosol from the one or more MALDI dispensers into an incoming air stream bearing the analyte to provide thorough mixing prior to collection on a VCR tape. Typically, the aerosol of interest have concentrations of 15 agent containing particles (ACPs) per liter of air and an aerodynamic diameter 0.5 to 10.0 um, (2) a tape transport system for advancing the concentrated samples into a mass spectrum analyzer instrument one at a time for acquisition of mass spectra while continuously and simultaneously collecting new aerosols (samples). The tape transport system includes one or more closed-loop control motors to independently position the tape both inline with the one or more aerosol collectors and with the inlet to the mass spectrometer, (3) a micro applicator may optionally be included to apply MALDI matrix to the samples after collection or to supplement co-deposited matrix to increase sensitivity; (4) a time-of-flight mass spectrometer including an ionization/desorption cell located outside the walls of the vacuum chamber, and (5) a data acquisition system for collecting data, preferably digitized, to be stored in a computing device.




It is noted that it is within the contemplation of the present invention to perform sample preparation by means other than co-deposition, such as, for example, interspersed collection deposition and a post-collection deposition. Other means not explicitly recited herein are also within the scope of the present invention.




Advantages of the apparatus of the present invention include short analysis times (e.g., less than 5 minutes), high sensitivity, wide agent bandwidth, portability, low power consumption, minimal use of fluids required for sample processing thereby eliminating the need for large storage reservoirs, stationary and level mounting configurations, or large power-hungry heating and cooling systems, extending unattended operation, automated detection and classification, and the concurrent collection of that multiple samples allowing both the application or different analysis protocols and the archiving of samples for later confirmatory analysis.











BRIEF DESCRIPTION OF THE DRAWINGS





FIG. 1

is a pictorial illustration of a portable analyzer of the invention;





FIG. 2

is a schematic diagram of an embodiment of the system of the present invention;





FIG. 3

depicts details of the aerosol interface of the system of

FIG. 2

; and





FIG. 4

is a partial perspective view of the external ionization source and vacuum interface portion of the system of FIG.


2


.











DETAILED DESRIPTION OF THE PREFERRED EMBODIMENTS




As will be appreciated shortly, the present invention provides an automated spectrographic analysis system which collects biological samples on a permanent storage medium, such as a VCR tape, advances the prepared samples on the tape to a mass spectrum analyzer for acquisition of mass spectra, as well as performing other required steps. The present invention includes an aerosol interface for collecting, concentrating and separating aerosols from breathable ambient air. The aerosol interface uses a modified MALDI sample preparation technique that may co-deposit MALDI matrix as an aerosol with the sample analyte, or include post-collection sample matrix processing before analysis in a mass spectrometer. As will become evident below, the system is designed to run automatically. That is, it may be placed where detection of chemical or biological agents is desired, and it will sample the environment and analyze and identify such agents on an ongoing basis. The present invention solves the problem of carrying out tasks associated with the acquisition of mass spectra quickly and efficiently which has prevented mass spectra analysis from achieving rates which have been long desired in the art.




System Overview




With reference now to the drawings, and particularly to

FIG. 1

, there is shown a perspective view of a presently preferred embodiment of an automated spectrographic analysis system


100


in accordance with the invention. The system


100


is transportable and sufficiently small and rugged to allow its dependable use in a field environment. Importantly, the system


100


is configured to remain in alignment, even with rough handling. The system


100


is configured to be suitably reliable to survive transportation on a range of vehicles, allow handling by two persons, and to be operable from a portable power source.




The principal parts of the system


100


are illustrated in FIG.


2


. The system


100


includes an aerosol interface


10


which provides means for preparing a sample which is to undergo mass spectrum analysis. In particular, a sample is prepared in accordance with a modified MALDI sample preparation technique in which a MALDI matrix is either co-deposited as an aerosol with the sample analyte, or applied with post-collection processing


252


before analysis in a mass spectrometer


22


. The sample analyte is derived by collecting, concentrating and separating aerosols from a sample collector airflow


45


at concentrations of typically 15 ACPs per liter of air and of 0.5 to 10.0 um aerodynamic diameter onto a permanent storage medium such as a movable tape


120


′ (to be described).




As shown in

FIG. 2

, the mixing method of the present invention includes a matrix nebulizer


12


dispensing metered amounts of matrix into the sample collector airflow, thus avoiding the use of post-collection fluids. This process allows for intimate mixing of matrix and analyte throughout the deposited sample and negates the need for additional post-collection processing prior to introduction of the MALDI-analyte combination into the spectrometer.




As is appreciated in the art, the ability to generate UV-MALDI mass spectra is critically dependent upon the co-crystallization or very close spatial proximity of the analyte and a molar excess of the matrix compound. As currently practiced in conventional non-field deployable TOF-MS analyzers, UV-MALDI mass spectra is generated in accordance with a procedure in which a small volume of matrix solution that delivers a one thousand-fold molar excess of matrix is manually mixed with a small volume of the analyte solution which then dries on a sample stage. A spatially heterogeneous distribution of analyte and matrix typically develops as the droplet dries to form a sample spot. Under laboratory conditions, the incident laser is rastered across the sample to identify so called “sweet spots” that preferably yield an abundance of analyte ions. This technique is not applicable to a field deployable TOF MS, such as the one described herein, because constraints do not permit either the analyte and matrix to be mixed in solution and to raster the laser across the sample makes the system unnecessarily complex.




An alternate matrix application approach for a field-deployable automated TOF MMS system consists of depositing an analyte sample in aerosol from directly on tape pre-coated with a MALDI matrix. This does not provide the intimate mixing and co-crystallization of the analyte with the matrix that is essential for the generation of high quality UV-MALDI mass spectra. Thus, additional post-collection steps, e.g., using a dispenser


252


to apply MALDI matrix over the sample prior to introduction of the MALDI-analyte combination into a spectrometer, maybe required.




Referring now to

FIG. 3

, a more detailed illustration of system


100


is shown. In one embodiment, the aerosol interface


10


includes one or more impactor/concentrator stations (


104


/


106


, one station is shown) which is made up of a concentrator


104


and a set of second stage impactors


106


. The impactors


106


serve to separate the particles from the airflow and provide sample deposits


108


on a transport tape


120


through a number of impaction nozzles


106


′. Interposed between the impactor/concentrator stations are one or more matrix-assisted laser desorption/ionization (MALDI) dispensers


110


. The MALDI dispensers


110


re-wet the sample areas on the tape


120


to provide for additional concentration of aerosol at each impactor/concentrator station. This technique intersperses MALDI matrix as an aerosol with the sample analyte, thus requiring no post-collection processing before analysis in a mass spectrometer. Alternately, the dispensers,


110


may be located after the aerosol collection stage and before the spectrometer,


170


, as shown in

FIGS. 1 and 2

,


252


, to provide post-collection matrix application or over-spraying.




While impactors were chosen for this embodiment, other sample separator and collection systems may be used depending on the MMS application, e.g., collection from a solid surface may require a different approach from an application where the sample is collected from air.




The present invention solves the problems discussed above for an automated TOF MMS system suitable for field deployment by co-depositing the matrix with the analyte as an aerosol on video recorder tape.




The inventive mixing method, according to one embodiment, for co-depositing the matrix with the analyte as an aerosol on video recorder tape is now described in greater detail with reference to

FIGS. 2 and 3

. A nebulizer


12


is used to inject metered amounts of MALDI matrix particles into a sample collector airstream


45


. The airstream


45


is drawn (via a vacuum) into a collector


102


via an inlet


104


. Upon entering the collector


102


, the airstream


45


passes through a concentrator/impactor station


104


/


106


. The impactor


106


serves to separate the desired particles from the airstream and provide sample deposits


108


on a transport tape


120


(described further below) through a number of impaction nozzles


106


′. The air collection portion so configured has a high throughput and high collection efficiency. Thus, a high concentration of dry particles are withdrawn from the environment and deposited on a small area of the tape


108


as shown. The collector


102


therefore collects particulate agents from the environment, such as biological agents and chemical agents that are attached to particles (such as residue of explosive material in the earth left by mine placement). Thus, the sample is not collected or transported in a liquid state, thus avoiding freezing, spoiling, etc. In addition, samples


108


deposited on the tape


120


are extremely thin, which is advantageous when introduced into the extraction region of the mass analyzer, as described further below.




After collection, the samples


108


are transported by the tape


120


for treatment and analysis. The tape


120


may be a standard VHS tape, which is withdrawn from a tape supply end


120




a


of a video cassette


120


′ and collected at the tape collection end


120




b


. The video tape


120


from the tape supply side


120




a


runs between the impaction nozzles


106


′ (from which the samples


108


are deposited, as described above) and a backing platen


113


. The tape


120


is wound in a loop pattern between the drive shaft


140




a


, a take up idler wheel


142


and a rubber tape roller


140




b


of a first stepper motor


140


, around a tensioning shaft and roller arrangement


142


, and between a drive shaft


144




a


and a rubber tape roller


144




b


of a second stepper motor


144


.




The tape


120


then passes through an input portion to the mass analyzer


170


, and is then collected by the cassette


120


′ at the tape collection end


120




b


. Referring to

FIG. 3

, the take up tensioning shaft


142


provides for a variable length tape loop prior to the sample introduction into the mass analyzer


170


. A similar function can also be provided with a vacuum column. The idler wheel


141


serves to allow incremental motion of the tape


120


under the impactors


106


independent of incremental motion of the tape


120


into the mass analyzer


170


.




The tape


120


provides for permanent storage of samples which may be ‘replayed’ into the analyzer


170


at a later time. Separation of the sample collection areas on the tape so that they are not cross contaminated by winding on to a take up reel and contacting the backside of the tape is provided by limiting the contact to areas where other samples never touch, if the tape is rewound. This consistency of tape wrapping is controlled by the tensioning wheel and the consistency of the drive on the take up reel of the tape cartridge or reel so that each time the tape is played and re-wrapped on the take up reel the samples will contact the back side of the tape nearly in the same spot and never as far away as areas touched by adjacent samples.




A groove or notch in the drive wheel capstan and tape guide provides for tape motion without touching the sample area on the tape thus eliminating a possible source of cross contamination between the individual samples on the tape. Referring to

FIG. 2



a


which illustrates a cross-section of the drive shafts


140




a


,


144




a


and the rubber tape roller


140




b


,


144




b


is shown, with the tape


120


there between. As shown, both the drive shafts


140




a


and


144




a


have a reduced diameter at a mid region M than at end regions E. The end regions E between the drive shafts


140




a


,


144




a


and the tape rollers


140




b


,


144




b


serve to pinch the edges of the tape


120


, while the middle region M allows the sample


108


to pass through untouched. The friction the tape


120


and the drive shafts


140




a


,


144




a


created by the pinching between the drive shafts


140




a


,


144




a


and the tape rollers


140




b


,


144




b


allows the drive shafts


140




a


,


144




a


to advance the tape


120


. Rollers of like grooved design placed along the tape path guide the tape lateral alignment.




Driving of the tape uses commercially available closed-loop motor control drivers for the positioning of the tape. The embodiment of

FIG. 2

includes a three axis stepper motor driver


150


that receives control signals from control unit


160


. The stepper motor driver


150


independently controls first stepper motor


140


, second stepper motor


144


and a third stepper motor (not shown) that serves to load the video cassette


120


′. By sending the appropriate control signals to the first stepper motor


140


, a portion of the tape is positioned in the collector


102


. By sending appropriate control signals to the second stepper motor


144


and coordinating simultaneous collection of the tape into the cassette by the third stepper motor, samples are positioned in the mass spectrometer vacuum interface


180


. Thus, the tape segment associated with the collection of the samples moves independently of the segment associated with the analysis of the samples. Thus, additional samples may be collected by the collector


102


while a particular sample continues to be analyzed by the mass spectrometer


170


. Controllable motors other that stepping motors may work as well for this application.




When the analysis is completed, the second stepper motor


144


is stepped by the control unit


160


to move the next sample into the mass analyzer


102


. Likewise, samples may continue to be collected within unit


10


while independently moving previously collected sample into the analyzer. Upon completing the sample collection, the first stepper motor


140


, controlled by unit


160


, advances fresh tape into the collector


102


for collection of a subsequent sample. Tension is maintained in the tape


120


during independent movement of stepper motors


140


,


144


because shaft


142


moves against spring tension as required in the directions of the arrows shown in

FIG. 2

associated with roller


142


.




The stepper motors


140


,


144


(as well as the cassette stepper motor) may, of course, also be stepped together to position a collected sample


108


from the collector


102


to the mass analyzer


22


. This may occur, for example, if the sampling is initiated manually (for example, by a security office at an airport gate), or during automatic collection and processing where a remote command provides instructions to bypass the analysis of the last sample and proceed with analysis of the actively collected samples. In any case, the control unit


160


keeps track of the movement of each sample


108


leaving the concentrator


102


by using magnetic write head


132


to write a reference marking on the tape


120


adjacent the exiting sample


108


, and by tracking control motor rotation angles.




As described below, a read head prior to the mass analyzer is used to identify and provide a position of the sample


108


to the control unit


160


. Thus, the control unit


160


uses stepping motor counts and magnetic tape markings to keep track of the position of the sample


108


while being transported between the collector


102


and the mass analyzer


170


. For ease of description, the ensuing description will focus on the collection of a single sample


108


by the collector


102


and its treatment, transport and analysis by the mass analyzer.




Following collection of sample


108


by collector


102


, association of a reference marking by write head


132


and movement of the sample


108


through the tape loop of the stepper motors (described above), a magnetic read head


134


reads the reference marking on the tape


120


associated with sample


108


provided by write head


132


. This identifies the sample


108


to the control unit


160


and also provides a reference position for subsequent movement by the control unit


160


. Using the reference position, the control unit


160


steps stepper motor


144


by a known amount to position sample


108


adjacent the nozzle of a MALDI micro dispenser


150


. The MALDI micro dispenser


150


adds a small amount of MALDI matrix to the sample to facilitate ionization in the mass spectrometer (described below), especially for desorption of large macromolecules previously described. The MALDI treatment provides a small amount of matrix, thus the sample


108


remains relatively flat. In addition, the post-collection MALDI treatment occurs just prior to introduction into the mass analyzer, thus minimizing exposure to the elements.




The control unit


160


then steps stepper motor


144


by a known amount to move treated sample


108


into the mass analyzer


170


. The software run by the control unit


160


and the stepper motors position the sample


108


within {fraction (1/10)}


th


the diameter the sample target region of the mass analyzer


170


, thus ensuring that the sample


108


is illuminated with the laser, as described further below.




Referring now to

FIG. 4

, in accordance with another aspect of the present invention, an improved design is provided whereby an extraction ionization source


190


and


194


is located outside the vacuum chamber


260


to a location between the sample surface and an isolation valve. In a conventional design, the ionization cell normally resides within the walls of the vacuum chamber


260


and is reachable only by a long probe. The improved design of the present invention removes the requirement of using a long probe and associated multiple vacuum seals.




The inventive external ionization source reduces the complexity of repeatedly breaking and restoring a high-vacuum seal as each tape sample is repositioned over the sample port. Eliminating the need for a probe allows this invention to use a sample collection substrate consisting of continuous tape [or disk, or other medium]. This adds the capability of rapidly advancing a continuous series of samples through the MS analyzer stage. In a conventional design where the extraction source is located inside the vacuum chamber


260


, typically many tens of minutes are required to restore the mass analyzer chamber to a high vacuum if the whole chamber were exposed to the atmosphere. The vacuum interface of the present invention reduces the vacuum pump loading by isolating the main vacuum chamber


260


from the sample port around the tape sample when samples are being changed, while simultaneously providing a clear passage for the ions during a measurement (described further below).




In

FIG. 4

, the external extraction source-valve design for an MMS is shown which retains certain desired features of the prior art, e.g., providing space for an electrostatic lens and allowing a laser beam


232


to impact a sample surface


108


directly, but is different in that it locates the extraction source outside the vacuum chamber


260


to a location between the sample surface


108


and the valve. The novel configuration eliminates the need to introduce the sample


108


into the vacuum chamber via a long probe by overcoming the dimensional separation (i.e., between the sample surface and extraction source) caused by the valve mechanism. That is, the correct sample-surface and extraction source electric field geometry needed for the proper voltage potential gradient and sample ion acceleration is achieved with the placement of the extraction source outside the chamber.




The external placement of the extraction source advantageously provides sufficient room for an isolation valve which facilitates the collection and sample preparation techniques of the present invention. Without the external source, an isolation valve could not fit in the space between the source and the sample collection substrate. The sample collection tape


120


serves to form the vacuum seal. This function was performed by an extended probe in the conventional design. The tape


120


must be made of a nonporous material that holds a vacuum seal at or below micro-Torr pressure levels such as, for example, a polyester film as used for magnetic recording tape. Candidate materials also include a wide variety of polyester, polyamide, and polytetra fluoroethylenes. In general, any tape material sufficient to hold an adequate vacuum is a candidate material.




With continued reference to

FIG. 4

, additional details of the ionization source


190


,


194


and vacuum interface


180


of the mass analyzer portion


170


, is shown. The interface


180


comprises housing


182


having a roughing vacuum chamber portion


184


therein, and a pressure platen


196


. A sample


108


is introduced into the vacuum system of the mass analyzer by moving tape


120


so that sample


108


is positioned in upper opening


186


of roughing vacuum chamber portion


184


. An insulating disc


188


surrounds the upper opening


186


and is supported by an electrode assembly


190


that projects axially from the roughing vacuum chamber portion


184


. The upper surface of the insulating disc


188


is flush with the upper surface of the housing


182


, thus providing an even surface across which the tape


120


extends. An O-ring


192


is positioned in circumferential groove


194


in the surface of the insulating disc


188


.




When the sample


108


is positioned, the stepper motor


204


is stepped by control unit


160


to position the source ionization platen


196


over the sample


108


and the upper opening


186


. Platen assembly


196


is an insulating material with a set of electrodes


197




a


, surrounding the opening


186


, which create an electric field with the electrodes


190


, and form an electrostatic lens to focus the ions on the MS detector. The platen


196


has a circumferential groove


194




a


and O-ring


192




a


in its bottom surface opposite the circumferential groove


194


and O-ring


192


of the insulating disc


188


. When the platen


196


is positioned as shown, and


196


is drawn downwards, the compression of


192


,


192




a


creates a vacuum seal in the roughing vacuum chamber portion


184


.




While the sample


108


is being positioned, the roughing vacuum chamber portion


184


is exposed to atmospheric pressure. A ball valve


251


remains closed during the positioning process to isolate the high vacuum (micro-Torr) in the mass spectrometer vacuum chamber


260


. This is done via a motor (not shown) associated with the ball valve


251


that receives commands from the control unit


160


when a new sample


108


is to be positioned. The roughing pump


198


is switched off by the control unit


160


and the vacuum in roughing vacuum chamber portion


184


rises to atmospheric pressure. Control unit


160


moves platen


196


away from upper opening


186


in the Z direction by sending the appropriate stepping signals to stepper motor


204


, which removes platen


196


via cantilever arms


202


. Stepper motor


144


is then stepped by control unit


160


so that tape


120


positions the next sample


108


in line with the upper opening


186


. Guides keep the sample from contacting the top surface of housing


182


and insulating disc


188


during positioning. Once the sample


108


is in position, motor


204


is activated to close platen


196


. This compresses the tape between O-rings


192




a


and


194




a


to from a vacuum seal. Control unit


160


initiates a vacuum roughing pump


198


, which evacuates the roughing vacuum chamber portion


184


through port


200


. It has been experimentally determined that approximately 10 seconds is required to rough the vacuum chamber portion


184


. After the roughing operation is complete (removal of the air), the roughing pump ball valve


250


closes and the isolation valve


251


opens. This creates a direct straight-line path from the sample surface


108


to the spectrometer detectors (not shown). At this point, approximately 20 additional seconds is required to pump the cavity


235


to a micro-Torr pressure. Once at high vacuum, a potential of at least 4,600 V is applied between an electrode on the contact surface inside the sealing ring of the platen


196


and the extraction source electrodes


190


. A laser


232


then ionizes the sample by firing a beam


226


through an optically clear vacuum window to a spot focused on the tape surface. The vacuum isolation valve


251


closes upon completion of the spectrometer measurement, the roughing port valve


250


opens, and the platen


196


releases, allowing the tape to advance for the next measurement. In practice, valves


250


and


251


may be combined in a single three-port-two position valve. Tests thus far have demonstrated the capability to handle extraction voltages exceeding 6,000 V, with feasible designs up to 12,000 V. The seal between the platen


196


and the O-ring


192


has a Helium leak rate of less than 10


−7


cc/s, which is well within the capability of the vacuum pump to maintain the required micro-Torr vacuum.




To prevent deformation of the tape


120


caused by a pressure differential between the two sides of the tape


120


, the platen


196


contains a port opening on the backside of the tape. The port connects to a compensating vacuum formed by the main vacuum chamber. This compensating vacuum eliminates the differential pressure forces, thereby preventing unacceptable tape deflection. Alternatively, the tape may be perforated with pins during closure to the aerosol platen


113


during the aerosol collection step. The perforations allow excavation of the volume between the tape and the source ionization platen


196


, which equalizes the pressure across the tape and minimizes tape deformation.




In summary, numerous benefits have been described which result from employing the concepts of the present invention. Advantageously, the apparatus of the present invention provides for real-time mass spectra analysis. As used herein, the term “real-time” refers to the apparatus and accompanying methods which provides for the collection, concentration and separation of aerosols onto a permanent storage medium (the tape) and for advancing the concentrated samples into an analyzer instrument one at a time for analysis while continuously sampling new aerosols. It will be further appreciated that the apparatus


100


may run automatically and be readily used by unskilled personnel for field analysis of biological samples.




It will be understood that various modifications may be made to the embodiments disclosed herein, and that the above descriptions should not be construed as limiting, but merely as exemplifications of preferred embodiments. Those skilled in the art will envision other modifications within the scope and spirit of the claims appended hereto.



Claims
  • 1. A field portable mass spectrometer system comprising:a) an aerosol interface comprising: an inlet having a vacuum therein, the inlet collecting an environmental specimen containing one or more analytes; and a nebulizer for injecting metered amounts of MALDI matrix particles into the environmental specimen prior to the inlet collecting the environmental specimen; b) a sample transporter, the sample transporter interfacing with a sample collector to receive sample deposits thereon; c) a time of flight (TOF) mass spectrometer, the time of flight mass spectrometer having a sealable opening that receives the sample transported via the sample transporter in an extraction region of the mass spectrometer; and d) a control unit that processes a time series output by the mass spectrometer for a received sample and identifies one or more agents contained in the sample.
  • 2. The field portable mass spectrometer system of claim 1, wherein the metered amounts of MALDI matrix particles mixed with the one or more analytes contained in the environmental specimen form a spatially heterogeneous distribution of analyte and matrix.
  • 3. The field portable mass spectrometer system of claim 1, wherein the metered amount of matrix solution injected into the environmental specimen is adjusted in accordance with differing amounts of environmental background.
  • 4. A field portable mass spectrometer system comprising:a) an aerosol interface comprising: an inlet having a vacuum therein, the inlet collecting an environmental specimen containing one or more analytes; and one or more tape particle collector/impactor stations for collecting, concentrating and separating said one or more analytes contained in said environmental sample; b) a sample transporter, the sample transporter interfacing with a sample collector to receive sample deposits thereon: c) a time of flight (TOF) mass spectrometer, the time of flight mass spectrometer having a sealable opening that receives the sample transported via the sample transporter in an extraction region of the mass spectrometer; and d) a control unit that processes a time series output by the mass spectrometer for a received sample and identifies one or more agents contained in the sample.
  • 5. A field potable mass spectrometer system comprising:a) an aerosol interface; b) a sample transporter, the sample transporter interfacing with a sample collector to receive sample deposits thereon, the sample transporter comprising a tape that receives the sample deposits from the sample collector, the tape being received at the scalable opening of the mass spectrometer, thereby allowing a sample thereon to be received in the extraction region of the mass spectrometer, the movement of each small being tracked between the sample collector and the mass spectrometer by using a magnetic write head to write a reference marking on the tape adjacent the sample upon exiting the sample collector; c) a time of flight (TOF) mass spectrometer the time of flight mass spectrometer having a scalable opening that receives the sample transported via the sample transporter in an extraction region of the mass spectrometer and d) a control unit that processes a time series outwit by the mass spectrometer for a received sample and identifies one or more agents contained in the sample.
  • 6. The field portable mass spectrometer system of claim 5, wherein the tape is perforated during the time that it receives sample deposits thereby permitting equalization of pressure across the tape and resultant minimization of tape deformation when the tape with sample thereon is received at the sealable opening of the mass spectrometer.
  • 7. A field portable mass spectrometer system comprising:a) an aerosol interface; b) a sample transporter, the sample transporter interfacing with a sample collector to receive sample deposits thereon; c) a time of flight (TOF) mass spectrometer, the time of flight mass spectrometer having a scalable opening that receives the sample transported via the sample transporter in an extraction region of the mass spectrometer, wherein the sealable opening and the extraction region of the TOF mass spectrometer are provided in a housing attached to or part of the TOF mass spectrometer and the housing further comprises a roughing vacuum chamber portion that connects between the sealable opening of the housing to a vacuum valve; and d) a control unit that processes a time series outwit by the mass spectrometer for a received sample and identifies one or more agents contained in the sample.
  • 8. The field portable mass spectrometer system of claims 1, 4, 5, or 7, wherein movement of the tape when interfacing with the sample collector is independent of movement of the tape when being received in the mass spectrometer.
  • 9. The field portable mass spectrometer system of claims 1, 4, 5, or 7, wherein the sample transporter further comprises a first controllable motor that receives control signals from the control unit and enables independent movement of the tape when interfacing with the sample collector and a second controllable motor that receives control signals from the control unit and enables independent movement of the tape when being received in the mass spectrometer.
  • 10. The field portable mass spectrometer system of claims 1, 4, 5 or 7, wherein the TOF mass spectrometer comprises a linear TOF mass spectrometer.
  • 11. The field portable mass spectrometer system of claims 1, 4, 5 or 7, wherein the TOF mass spectrometer comprises a linear and/or reflectron TOF mass spectrometer.
  • 12. The field portable mass spectrometer system of claim 8, wherein the independent movement of the tape is provided at least in part by a movable tensioner that interfaces with the tape, the movable tensioner being interposed between the sample collector and the mass spectrometer.
  • 13. The field portable mass spectrometer system of claim 12, wherein the tensioner is a spring-loaded shaft and roller arrangement, the tape being wound around at least a part of the shaft and roller components.
  • 14. The field portable mass spectrometer system of claim 12, wherein the consistency of tape wrapping in a tape cartridge is controlled by the tensioner and the consistency of a drive on a take up reel of the tape cartridge such that the contact point on the backside of the tape for a sample is limited to areas where other samples never touch thereby allowing samples deposited on the tape to be permanently stored for later analysis without being cross contaminated by other samples deposited on the tape.
  • 15. The field portable mass spectrometer system of claim 14, wherein the hint and second controllable motors each comprise a drive shaft and tape roller, the drive shaft and tape roller each having a groove formed therein such that the end regions of the drive shaft and tape roller contact and drive the tape while the groove prevents the sample from contacting the drive shaft and tape roller and thereby contaminating other samples.
  • 16. The field portable mass spectrometer system of claim 7, wherein the housing further comprises a removable cover that is engageable with the sealable opening, the removable cover and the sealable opening forming a vacuum seal when engaged.
  • 17. The field portable mass spectrometer system of claim 16, wherein a roughing pump interfaces with the roughing vacuum chamber portion and serves to evacuate the roughing vacuum chamber portion when (a) the vacuum seal is formed between the removable cover and the sealable opening and (b) the vacuum valve is closed.
  • 18. The field portable mass spectrometer system of claim 16, wherein the vacuum seal is provided by at least one o-ring in each of the removable cover and the sealable opening the o-rings engaging to form a vacuum seal when the removable cover engages the sealable opening.
  • 19. The field portable mass spectrometer system of claim 16, wherein the cover is a platen.
  • 20. The field portable mass spectrometer system of claim 16, wherein a surface of the cover that covers the sealable opening comprises an electrode and defines one end of an extraction region of the TOF mass spectrometer in the roughing vacuum chamber portion.
  • 21. The field portable mass spectrometer system of claim 20, wherein one or more additional electrodes surrounding the roughing vacuum chamber portion and lying between the sealable opening and the vacuum valve defines an another end of the extraction region.
  • 22. The field portable mass spectrometer system of claim 21, wherein a vacuum pump that interfaces with a main mass spectrometer vacuum chamber serves to evacuate the main mass spectrometer vacuum chamber.
  • 23. The field portable mass spectrometer system of claim 22, wherein an open valve between the main mass spectrometer vacuum chamber and the extraction region forms part of the time of flight path of the spectrometer.
  • 24. The field portable mass spectrometer system of claim 23, wherein the vacuum pump that interfaces with the main mass spectrometer vacuum chamber serves to evacuate the main mass spectrometer vacuum chamber and the roughing vacuum chamber when the valve is opened, thereby providing a connected vacuum between the main mass spectrometer vacuum chamber and the roughing vacuum chamber when the valve is opened.
  • 25. The field portable mass spectrometer system of claim 24, wherein the sample transporter comprises a tape and the removable cover contains a port opening on the backside of the tape, the port opening being connected to a compensating vacuum formed by the main mass spectrometer vacuum chamber, the compensating vacuum eliminating differential pressure forces thereby preventing unacceptable tape deflection.
Parent Case Info

This application claims the benefit of Provisional Application No. 60/207,825 filed May 30, 2000.

PCT Information
Filing Document Filing Date Country Kind
PCT/US01/16697 WO 00
Publishing Document Publishing Date Country Kind
WO01/93307 12/6/2001 WO A
US Referenced Citations (6)
Number Name Date Kind
3922546 Livesay Nov 1975 A
4296322 Wechsung Oct 1981 A
4757396 Ishiguro et al. Jul 1988 A
4819477 Fisher et al. Apr 1989 A
5376788 Standing et al. Dec 1994 A
20030020011 Anderson et al. Jan 2003 A1
Provisional Applications (1)
Number Date Country
60/207825 May 2000 US