Abstract:
Systems and methods for analyzing compounds in a sample. In one embodiment, the present technology is directed towards a method of analyzing a sample, comprising: emitting ions from the sample; selectively filtering the emitted ions for at least one designated trigger ion; fragmenting the designated trigger ions; scanning for a designated trigger ion fragment; and upon detecting the designated trigger ion fragment, scanning for at least one confirmatory ion fragment.

Description:
PRIORITY 
       [0001]    The present application claims priority from U.S. provisional patent application No. 61/038,068, filed Mar. 20, 2008, which is incorporated herein by reference in its entirety. 
     
    
     FIELD 
       [0002]    The present application relates generally to the field of mass spectrometry. 
       BACKGROUND 
       [0003]    The analysis of a substance to determine its composition may be necessary for many applications, including toxicology, forensics and environmental testing, as well as food and drug research. Often, samples to be analyzed are analyzed for the presence of numerous different analytes of interest. Such samples may, for example, be in the form of bodily fluids taken from test subjects, which fluids often include both drug metabolites of interest, as well as irrelevant endogenous ions from the test subject. Correctly determining the presence or absence of a large number of analytes of interest from complex substances can be difficult and time-consuming. 
         [0004]    Mass spectrometers are often used for producing a mass spectrum of a sample to find its composition. This is normally achieved by ionizing the sample and separating ions of differing masses and recording their relative abundance by measuring intensities of ion flux. For example, with time-of-flight mass spectrometers, ions are pulsed to travel a predetermined flight path. The ions are then subsequently recorded by a detector. The amount of time that the ions take to reach the detector, the “time-of-flight”, may be used to calculate the ion&#39;s mass to charge ratio, m/z. 
         [0005]    Additional information (in addition to an ion&#39;s precursor mass) can then be obtained by fragmenting the ion via CID (collision induced dissociation) in a collision cell (or other mean) to generate an MSMS spectrum. In most instruments with MSMS capabilities, the process of generating a mass spectrum, selecting a precursor ion and generating an MSMS (mass spectrum/mass spectrum) spectrum can be performed in an automated mode. This mode of acquisition is frequently referred to as Information Dependant Acquisition (IDA) or Data Dependant Experiment (DDE). 
         [0006]    Chromatographic equipment such as a liquid chromatograph may be used to elute or release ions from a sample into the mass spectrometer over a period of time. Multiple reaction monitoring (MRM) or other distributed-analysis and recursive techniques may be used to analyze the ions received by the mass spectrometer. 
         [0007]    Previous MRM techniques involve repeated cycles of scans by the mass spectrometer for predetermined analytes of interest. A “duty cycle” would involve a list of analytes to be “cycled through” and scanned for by the mass spectrometer. During MRM analysis, the mass spectrometer would divide its scans equally amongst the analytes of interest in the duty cycle. As a result, such duty cycles have a practical upper limit in the number of analytes which may be scanned for. Once the number of analytes grows too large (for example, some mass spectrometers require duty cycles to have no more than 50 analytes of interest in order to maintain acceptable data quality), the amount of scan time available for each analyte of interest is insufficient to provide accurate data. 
         [0008]    The applicants have accordingly recognized a need for systems and methods for analyzing and identifying ions from samples. 
       SUMMARY 
       [0009]    In one aspect, the present technology is directed towards a system for analyzing analytes in a sample. The system comprises an ion source for emitting ions from the sample; a mass spectrometer adapted to receive the ions from the ion source; a controller operatively coupled to the mass spectrometer and configured to control the first mass filter to filter for a designated ion of interest and to control the second mass filter to filter for a designated ion fragment of interest; and a trigger data set having at least one trigger entry. It should be understood that “ion fragment(s)” as used herein, are themselves ions and could alternately be referred to as “fragment ion(s)”. 
         [0010]    The mass spectrometer includes: a first mass filter to filter ions received from the ion source, an ion fragmenter configured to fragment ions received from the first mass filter, a second mass filter configured to filter ion fragments received from the ion fragmenter, and at least one detector configured to detect ion fragments received from the second mass filter. As well, each trigger entry includes: a designated trigger ion, a designated trigger ion fragment, a trigger time window, and a confirmatory data set. In turn, each confirmatory data set has at least one confirmatory entry, and each confirmatory entry includes: a designated confirmatory ion, and a designated confirmatory ion fragment. The controller is responsive to the trigger data set, and during the trigger time window for each trigger entry the controller is configured to control the first mass filter to filter for the corresponding designated trigger ion and to control the second mass filter to filter for the corresponding designated trigger ion fragment. Additionally, upon detection of the designated trigger ion fragment by the detector, the controller is configured to control the first mass filter to filter for the designated confirmatory ion and to control the second mass filter to filter for the designated confirmatory ion fragment. 
         [0011]    The system may also include data storage operatively coupled to the controller, wherein the data storage is configured to store data corresponding to the ion fragments detected by the detector. As well, the trigger data set may comprises a plurality of trigger entries. Additionally, at least one confirmatory data set may comprise a plurality of confirmatory entries. Furthermore, the ion source may comprise a liquid chromatograph. 
         [0012]    In another aspect, the technology is directed towards a system for analyzing ions emitted from an ion source. The system comprises: a first mass filter adapted to receive and to filter ions from the ion source, an ion fragmenter configured to fragment ions received from the first mass filter, a second mass filter configured to filter ion fragments received from the ion fragmenter, and a detector configured to detect ion fragments received from the second mass filter. The system also includes: a controller operatively coupled to the first and second mass filters, to the fragmenter and to the detector, wherein the controller is configured to control the first mass filter to filter for a designated ion of interest and to control the second mass filter to filter for a designated ion fragment of interest; a trigger data set having at least one trigger entry; and a confirmatory data set for each trigger entry. Each trigger entry includes: a designated trigger ion, a designated trigger ion fragment, and a trigger time window. Each confirmatory data set has at least one confirmatory entry, and each confirmatory entry includes: a designated confirmatory ion, and a designated confirmatory ion fragment. 
         [0013]    The controller is responsive to the trigger data set and to the confirmatory data set, and during the trigger time window for each trigger entry the controller is configured to control the first mass filter to filter for the corresponding designated trigger ion and to control the second mass filter to filter for the corresponding designated trigger ion fragment. Upon detection of the designated trigger ion fragment by the detector, the controller is configured to control the first mass filter to filter for the designated confirmatory ion and to control the second mass filter to filter for the designated confirmatory ion fragment. 
         [0014]    The system may also comprise data storage operatively coupled to the controller, wherein the data storage is configured to store data corresponding to the ion fragments detected by the detector. As well, the trigger data set may comprise a plurality of trigger entries. Furthermore, in some instances at least one confirmatory data set comprises a plurality of confirmatory entries. 
         [0015]    In yet a further aspect, the present technology is directed towards a method of analyzing a sample, comprising: emitting ions from the sample; selectively filtering the emitted ions for at least one designated trigger ion; fragmenting the designated trigger ions; scanning for a designated trigger ion fragment; and upon detecting the designated trigger ion fragment, scanning for at least one confirmatory ion fragment. 
         [0016]    The method may also include performing the filtering, fragmenting, scanning, and detecting and scanning during a trigger time window corresponding to the designated trigger ion. The trigger time window may be selected to correspond to a time period when the trigger ion is expected to be emitted from the sample. 
         [0017]    The filtering, fragmenting, scanning, and detecting and scanning may be performed for a plurality of designated trigger ions during a plurality of trigger time windows, each trigger time window corresponding to a designated trigger ion. Each trigger time window may be selected to correspond to a time period when the corresponding trigger ion is expected to be emitted from the sample. In some instances, the scanning may be performed substantially simultaneously for at least two different designated trigger ion fragments. 
         [0018]    The method may further comprise generating a report containing data corresponding to the detected designated trigger ion fragments and confirmatory ion fragments. 
         [0019]    In some instances, the method may involve using liquid chromatography for emitting ions. 
         [0020]    In another aspect, the invention may be directed to computer readable media configured to cause a mass spectrometer having a computer controller to perform the method. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0021]    The present invention will now be described, by way of example only, with reference to the following drawings, in which like reference numerals refer to like parts and in which: 
           [0022]      FIG. 1  is a schematic diagram of a mass spectrometer made in accordance with the present disclosure; 
           [0023]      FIG. 2  is a is a representative example of a trigger data set as may be stored in the data storage of the mass spectrometer of  FIG. 1 ; 
           [0024]      FIG. 3A  is a representative example of a first confirmatory data set as may be stored in the data storage of the mass spectrometer of  FIG. 1 ; 
           [0025]      FIG. 3B  is a representative example of a second confirmatory data set as may be stored in the data storage of the mass spectrometer of  FIG. 1 ; 
           [0026]      FIG. 4A  is a representative example of a duty cycle listing as may be stored in the data storage of the mass spectrometer of  FIG. 1  at a first time during an analysis, in this example at or near the beginning of the analysis period; 
           [0027]      FIG. 4B  is a representative example of a duty cycle listing as may be stored in the data storage of the mass spectrometer of  FIG. 1  at a second time during an analysis, after the duty cycles listing of  FIG. 4A  has been updated; and 
           [0028]      FIG. 5  is a flow diagram illustrating the steps of a method of analyzing a compound in accordance with the present disclosure. 
       
    
    
     DETAILED DESCRIPTION 
       [0029]    Referring to  FIG. 1 , illustrated therein is an analysis system referred to generally as  10 , made in accordance with the present disclosure. The system  10  is preferably configured to be capable of performing information dependent acquisition (IDA) in accordance with the present disclosure, as will be understood. 
         [0030]    The analysis system  10  includes a mass spectrometer  11  (which may be an MS/MS system such as a quadrupole hybrid linear ion trap such as the 4000QTRAP LC/MS/MS System sold by Applied Biosystems/MDS SCIEX). The spectrometer  11  comprises a suitably programmed controller or central processing unit (CPU)  12  having a programmed MRM trigger engine  14  stored in RAM or other suitable computer-readable media which may include a clock module  18 . An input/output (I/O) device  16  (typically including an input component  16   A  such as a keyboard or control buttons, and an output component such as a display  16   B ) is also operatively coupled to the CPU  12 . Data storage  17  is also preferably provided. 
         [0031]    The system  10  also includes an ion source  20 , configured to emit ions, generated from the sample  21  to be analyzed. The ion source  20  may be a continuous ion source, for example, such as an electron impact, chemical ionization, or field ionization ion source (which may be used in conjunction with a gas chromatography source), or an electrospray or atmospheric pressure chemical ionization ion source (which may be used in conjunction with a liquid chromatography source), or a desorption electrospray ionization (DESI), or a laser desorption ionization source, as will be understood. A laser desorption ionization source, such as a matrix assisted laser desorption ionization (MALDI) can typically generate a series of pulses in which a pulsed beam of ions is emitted. 
         [0032]    The ion source  20  can also be provided with an ion transmission ion guide, such as a multipole ion guide, ring guide, or an ion mass filter, such as a quadrupole mass filter, or an ion trapping device, as generally known in the art (not shown). For brevity, the term ion source  20  has been used to describe the components which generate ions from the sample  21 , and emit analyte ions of interest for detection. Other types of ion sources  20  may also be used, such as a system having a tandem mass filter and ion trap. Preferred ion sources are those which emit the ions from the sample  21  over a range of times, to enable recursive mass analysis by the mass spectrometer  11  using MRM or other suitable techniques. 
         [0033]    As will be understood, liquid chromatography may be used to separate ions dissolved in solvent from other substances in the sample  21 , and release or emit such ions for MS analysis. As a result of the different timings for the chemical interactions that take place during the LC phase, the reaction products (which include the ions or analytes of interest) are released over time. The release times for specific analytes can be estimated, based on the expected chemical interactions. 
         [0034]    As noted above, the spectrometer  11  may comprise a triple quadrupole mass spectrometer, having triple rod sets Q 1 , Q 2  and Q 3 . The rod sets Q 1  and Q 3  may be controlled by the processor  12  (via the trigger engine  14 ) to select or filter for ions having a particular m/z. In contrast, the Q 2  rod set is provided with a chamber and configured to operate as a collision cell or fragmenter for fragmenting the ions received from Q 1 . The resulting ion fragments may be passed through to, and selectively filtered by, rod set Q 3 , before being detected or recorded by the detector  22 . 
         [0035]    Optics  24  or other focusing elements, such as an electrostatic lens can also be disposed in the path of the emitted ions, typically between the Q 3  rod set and the detector  22 , for focusing the ions onto the detector  22 . 
         [0036]    Referring now to  FIG. 2 , illustrated therein is a representative example of a trigger data set  200  as may be stored in the data storage  17 . The trigger data set  200  includes at least one trigger entry  202 , and each trigger entry  202  includes: at least one m/z value, each such m/z value corresponding to a designated trigger ion  204 , at least one m/z value, each such m/z value corresponding to a designated trigger ion fragment  206 , timing data corresponding to a trigger time window  208 , and linking data such as a unique identifier data  210  providing a link to a confirmatory data set  300 . As will be understood, each confirmatory data set  300  need not be uniquely linked to only one designated trigger ion  204 /fragment  206  couplet. In some instances, such as in the case of background noise or other interference, it may be desirable to have more than one trigger ion  204 /fragment  206  couplet detected before the corresponding confirmatory data set  300  is filtered for, as will be understood. 
         [0037]    As will also be understood, the trigger time window  208  corresponds to a predetermined period of time when the corresponding designated trigger ion  204  is expected to be emitted by the ion source  20  from the sample  21 . It should also be understood that the trigger time or scanning window data  208  is not a requirement, as for certain simplified applications, the “windows” may be treated as running for the entire analysis period. 
         [0038]    Illustrated in  FIG. 3A  is a representative example of a confirmatory data set  300  as may be stored in the data storage  17 . Each confirmatory data set  300  has at least one confirmatory entry  302 , and each confirmatory entry  302  includes: at least one m/z value, each such m/z value corresponding to a designated confirmatory ion  304 , and at least one m/z value, each such m/z value corresponding to a designated confirmatory ion fragment  306 . Each confirmatory entry  302  may also include timing data corresponding to a confirmatory time window  308 . The example confirmatory timing window data  308  corresponds to a duration of scanning time (eg. 5 seconds). As will be understood in the context of the discussion below, in some instances, while the commencement time of an elution period may be uncertain, the duration of an elution period can often be estimated or known with greater accuracy. As a result, once the rising edge of an LC peak corresponding to the analyte of interest has been detected in accordance with the method discussed below, the system  10  may scan for the confirmatory ion(s)  304  for the duration of the expected elution period. 
         [0039]    Each confirmatory data entry  302  also includes a unique confirmatory identifier  310 , corresponding to a confirmatory data identifier  210  in the trigger data  200 . 
         [0040]    In alternate embodiments (not shown), the confirmatory time window data  308  might match the corresponding trigger time window  208  periods. In such instances the confirmatory time window data  308  need not be stored in the confirmatory data set  300 , and the corresponding trigger time window  208  data may be used by the CPU  12  as required. 
         [0041]    Illustrated in  FIG. 3B , is a representative example of an alternate confirmatory data set  300 B as may be stored in the data storage  17 . The alternate confirmatory data set  300 B generally corresponds to the confirmatory data set  300  and has at least one confirmatory entry  302 , with each confirmatory entry  302  including: at least one m/z value, each such m/z value corresponding to a designated confirmatory ion  304 , and at least one m/z value, each such m/z value corresponding to a designated confirmatory ion fragment  306 . Each confirmatory entry  302  also includes timing data corresponding to a confirmatory time window delay  308 B. 
         [0042]    As will be understood, for certain analysis applications, such as proteomics, in which qualification data is desired (ie. a determination as to the presence of a particular analyte of interest), it may be advantageous to conduct a single scan (or limited number of scans) to confirm the presence of the confirmatory ions  304  and confirmatory ion fragments  306 . For example, while a trigger ion  204  will likely be detected at the rising edge of the LC peak corresponding to the analyte of interest, the scanning for the corresponding confirmatory ion(s)  304  and fragment(s)  306  may be delayed by the confirmatory time window delay  308 B to correspond to the expected LC peak apex, as will be understood. 
         [0043]    Turning now to  FIG. 4A , illustrated therein is a representative example of a duty cycle listing  400  as may be stored in the data storage  17  at a first time during an analysis, in this example, at or near the beginning of the analysis period. Each duty cycle entry  402  in the duty cycle listing  400  includes m/z data corresponding to a designated precursor ion  404 , an m/z value corresponding to a designated ion fragment  406 , together with a corresponding scanning window timeframe  408 . Illustrated in  FIG. 4B  is a representative example of the duty cycle listing  400 ′ as may be stored in the data storage  17  at a second time during an analysis, for example at about 8 seconds after the commencement of the analysis period. 
         [0044]    As will be understood, in operation, the CPU  12 /MRM trigger engine  14  is responsive to the trigger data set  200  and to the confirmatory data set  300 . As will be discussed in greater detail below, the trigger engine  14  is configured to manage the duty cycle list  400  during MRM analysis, such that during the analysis period, designated precursor ion  404  and ion fragment  406  couplets  402  are added to and removed from the duty cycle list  400  over time, based on the trigger  200  and confirmatory  300  data sets, as well as the data received from the detector  22 . In turn, the trigger engine  14  utilizes the ion/fragment couplets  404 , 406  in the duty cycle listing  400  to regulate the operation of the mass analyzers Q 1  and Q 3 , to filter for the corresponding precursor ions  404  and confirmatory ion fragments  406 . 
         [0045]      FIG. 5  sets out the steps of the method, referred to generally as  500 , carried out by the spectrometer system  10  during an analysis period. Typically, before the analysis period is commenced, the analytes of interest are determined (for which the sample is being analyzed)(Block  502 ). As noted above, for each analyte of interest, one or more couplets each comprising a designated precursor ion  204  and corresponding designated ion fragment  206  may be stored in a trigger entry  202  in the trigger data set  200 . The corresponding trigger time window  208 , is also determined and stored (Block  504 ). 
         [0046]    The confirmatory data set  300  will also be determined and stored in data storage  17  (Block  506 ). As will be understood, typically an ion will fragment into a plurality of ion fragments. Accordingly, in many instances, the confirmatory data couplets  302  corresponding to a trigger couplet  202 , will share the same precursor ion  204 ,  304 . 
         [0047]    As will be understood, the trigger data  200  (designated precursor ion(s)  204  and designated ion fragment(s)  206 , together with the corresponding trigger time window  208 ) and the related confirmatory data  300  for numerous analytes of interest may be previously calculated and stored as a library of data in the data storage  17 , and simply indexed and retrieved by the user and the CPU  12  utilizing the I/O device  16 . 
         [0048]    The duty cycle list  400  is initiated, being populated with couplets  402  of designated precursor ion  204  and designated ion fragment  206  from the trigger data  200  which have a trigger time window  208  which commences or coincides with the beginning of the analysis period.  FIG. 4A  illustrates an example duty cycle list  400  as one may exist at the commencement of an analysis period. The user will then typically input a command to commence an analysis period (typically via the I/O device  16 ), upon receipt of which the trigger engine  14  is programmed to initiate the analysis period (Block  510 ). 
         [0049]    When the analysis period is commenced, the ion source  20  is activated to commence the emitting of ions from the sample  21  (which may be the commencement of the LC phase as outlined above)(Block  512 ). As will be understood, the sample compound, for example, may include bodily fluid taken from a test subject, which fluid often includes both drug metabolites of interest, as well as irrelevant endogenous ions from the test subject. 
         [0050]    The system  10  is then configured to selectively filter the emitted ions for the designated precursor ions  404  listed on the duty cycle listing  400  (Block  514 ). As will be understood, at least one (if not most) of the precursor ions  404  (and designated ion fragments  406 ) listed on the duty cycle listing  400  corresponds to a trigger ion  204  (and trigger ion fragment  206 ) in the trigger data  200 . As indicated by the dotted line  530 , the CPU  12 /trigger engine  14  is programmed to rapidly and repeatedly cycle through the designated precursor ions  404  on the duty cycle listing, and causes the rod set Q 1  to selectively filter the ions received from the ion source  20  for the designated precursor ions  404 . 
         [0051]    The filtered ions  404  (which as noted, include at least one trigger ion  204 ), are then received by the fragmentation module/rod set Q 2  and fragmented (Block  516 ). The fragments are then received by the Q 3  rod set, which is controlled by the trigger engine  14  to scan or filter for the designated ion fragments  406  on the duty cycle listing  400  (Block  518 ). Such designated ion fragments  406  (if any) are permitted to impact the detector  22 . As will be understood, the filtering, fragmenting and filtering steps of Blocks  514 - 518  are typically all performed for one ion/fragment couplet  402 , prior to the trigger engine  14  cycling to the next couplet  402  on the duty cycle list  400 . 
         [0052]    If the detector  22  detects a designated trigger ion fragment  206  (Block  522 ), the trigger engine  14  may be programmed to cause the system  10  to scan for at least one confirmatory ion fragment. As will be understood, a certain threshold may be predetermined for “detecting” a trigger ion fragment  206 —a certain quantity of trigger ion fragments  206  must be detected in order for the trigger ion fragment  206  to be considered “detected”. Similarly, in the event multiple trigger ion  204 /fragment  206  couplets in a trigger entry  202  must be “detected” the trigger engine  14  may be programmed to determine that such multiple trigger ion  204 /fragment  206  couplets have been detected before the system  10  scans for confirmatory ion fragment(s). 
         [0053]    The trigger engine  14  determines the confirmatory entry identifier  210  corresponding to the designated trigger ion fragment  206  (and designated trigger ion  204 ), and adds to the duty cycle listing  400  the one or more corresponding confirmatory couplets  302  of confirmatory ion  304  and confirmatory ion fragment  306  (linked to by the matching identifiers  210 ,  310 ) and calculates or otherwise determines the scanning window data  408  which is also added to the duty cycle listing  400  (Block  524 ). Referring briefly again to  FIGS. 4A and 4B , the example data provides an illustrative example of the updating of a duty cycle listing  400 ,  400 ′, after the designated ion  204 /designated trigger ion fragment  206  couplet  202 ′, has been detected. The designated confirmatory ion  304  and designated confirmatory ion fragment  306  data in the corresponding confirmatory entry  302 ′ (sharing a matching confirmatory identifier  310 ,  210  with the detected trigger couplet  202 ′) have been added as entries  402 * to the duty cycle listing  400 ′, together with the calculated trigger window data  408 . As can be seen from the example calculated trigger window data  408 * for the added entries  402 *, the trigger engine  14  has detected the couplet  202 ′ at approximately 5 seconds from the commencement of the analysis period and has calculated the trigger window data  408 * as commencing at the time of detection, 5 seconds, for the duration of the corresponding scanning window duration  308  (in this case 3 seconds), to result in an entry  408 * of “5 sec.-8 sec.”, as will be understood. 
         [0054]    Over time, as the time of the analysis period advances as may be tracked by the clock module  18 , the duty cycle listing is updated (Block  526 ). As the analysis period moves into the various trigger time windows  208 , the corresponding couplet  202  of designated trigger ions  204  and ion fragments  206  are added to the duty cycle listing  400 . Similarly, as the time of the analysis period moves beyond the various trigger time windows  408 , the corresponding couplet  402  of designated ions  404  and ion fragments  406  are removed from the duty cycle listing  400 . 
         [0055]    As will be understood, during the updating carried out in Block  526 , when a trigger time window  408  has passed and the corresponding couplet  202 ,  402  of designated trigger ions  204 ,  404  and ion fragments  206 ,  406  are removed from the duty cycle listing  400 , as well the corresponding confirmatory couplet(s)  302  of confirmatory ion(s)  304 ,  404  and ion fragment(s)  306 ,  406  are removed from the duty cycle listing  400 . The process cycles through the various steps  514 - 526  until the analysis period is complete and ion emission is terminated. 
         [0056]    Thus, for example, by referring to both  FIGS. 4A and 4B , it is possible to compare the duty cycle listing  400  at or near the commencement of the analysis period to the duty cycle listing  400 ′ as it may appear at approximately 7 seconds into the analysis period for the exemplary data. As can be seen, since the scanning window  408  for the ion/fragment couplet pointed to by  402 ′″ has passed, this couplet  402 ′″ has been removed from the duty cycle listing  400 ′. Similarly, as the analysis time has moved into the range of trigger or scanning windows  208 , for ion/fragment couplets  202 ″ in the trigger data set  200 , such corresponding couplets  402 ″ have been added to the duty cycle listing  400 ′. As will be understood, the updating step of Block  526  will be unnecessary for applications which do not involve trigger or scanning windows. 
         [0057]    As will be understood, the controller  12  may generate a report identifying the quantities of the various designated ion/fragment couplets and hence the presence or absence of the corresponding analytes of interest (Block  528 ). Quantities of confirmatory couplets  302  should approximate the quantities of the corresponding trigger couplets  202 , confirming both the quantity and presence of the corresponding analytes of interest, as will be understood. 
         [0058]    Thus, while what is shown and described herein constitute preferred embodiments of the subject invention, it should be understood that various changes can be made without departing from the subject invention, the scope of which is defined in the appended claims.