High-throughput sample analysis is critical to the drug discovery process. Mass spectrometry (MS) based methods can achieve label-free, universal mass detection of a wide range of analytes with exceptional sensitivity, selectivity, and specificity. As a result, there is significant interest in improving the throughput of MS-based analysis for drug discovery. In particular, a number of sample introduction systems for MS-based analysis have been improved to provide higher throughput. Acoustic droplet ejection (ADE) has been combined with an open port interface (OPI) to provide a sample introduction system for high-throughput mass spectrometry. When an ADE device and OPI are coupled to a mass spectrometer, the system can be referred to as an acoustic ejection mass spectrometry (AEMS) system. The analytical performance (sensitivity, reproducibility, throughput, etc.) of an AEMS system depends on the performance of the ADE device and the OPI. The performance of the ADE device and the OPI depends on selecting the operational conditions or parameters for these devices.
AEMS technology brings fast, precisely controlled, low-volume sampling to the direct highflow liquid transferring to the ESI without carry-over, to achieve this high-throughput analytical platform with high reproducibility, and wide compound coverage. The analytical throughput of an AEMS system is determined by the delay time between ejections from different sampling events, with the consideration of being able to accurately identify and quantify the compounds from the potential interference of adjacent ejections.
In one aspect, the technology relates to a method of ejecting a plurality of samples from a well plate, the method including: receiving a first sample intensity prediction associated with a first sample in a first well of the well plate; receiving a second sample intensity prediction associated with a second sample in a second well of the well plate, wherein the second sample intensity prediction is less than the first sample intensity prediction; determining an ejection time delay value for a subsequent analysis of the first sample and the second sample, based at least in part on the second sample intensity prediction; acoustically ejecting the first sample from the first well; and after acoustically ejecting the first sample, acoustically ejecting the second sample from the second well. In an example, the method includes receiving the well plate in a mass spectrometry device. In another example, the method includes calculating an adjusted time period, wherein the adjusted time period is based at least in part on a reference time period and the ejection delay time value. In yet another example, acoustically ejecting the second sample is performed after the adjusted time period elapses. In still another example, the method includes receiving a third sample intensity prediction associated with a third sample in a third well of the well plate, wherein the third sample intensity prediction is at least one of greater than and equal to the second sample intensity prediction.
In another example of the above aspect, the method includes, after acoustically ejecting the second sample, acoustically ejecting the third sample from the third well, wherein ejecting the third sample is performed after the reference time period elapses. In an example, the method includes analyzing the first sample, the second sample, and the third sample with the mass spectrometry device to obtain, respectively, a first ion intensity signal, a second ion intensity signal, and a third ion intensity signal. In yet another example, the method includes storing, in a memory coupled to the mass spectrometry device, the first sample intensity prediction with the first ion intensity signal.
In another aspect, the technology relates to a method of ejecting a plurality of samples from a well plate, the method including: acoustically ejecting a first timing sample from a first well of a well plate; analyzing the first timing sample with a mass spectrometry device to determine a first ion intensity signal; subsequent to ejecting the first timing sample, acoustically ejecting a second timing sample from a second well of the well plate; analyzing the second timing sample with the mass spectrometry device to determine a second ion intensity signal, wherein the second ion intensity signal is less than the first ion intensity signal; and determining an ejection time delay value for performing a subsequent analysis of a first analysis sample from the first well and a second analysis sample from the second well based at least in part on the second ion intensity signal. In an example, the method includes calculating an adjusted time period, wherein the adjusted time period is based at least in part on a reference time period and the ejection delay time value. In another example, the method includes: acoustically ejecting the first analysis sample from the first well; and after acoustically ejecting the first analysis sample, acoustically ejecting the second analysis sample from the second well, wherein acoustically ejecting the second analysis sample is performed after the adjusted time period elapses. In yet another example, acoustically ejecting the second timing sample is performed after a timing time period elapses. In still another example, the timing time period is less than the reference time period.
In another example of the above aspect, the method includes: subsequent to ejecting the second timing sample, acoustically ejecting a third timing sample from a third well of the well plate; analyzing the third timing sample with the mass spectrometry device to determine a third ion intensity signal, wherein the third ion intensity signal is at least one of greater than and equal to the second ion intensity signal. In another example, the method includes, after acoustically ejecting the second analysis sample, acoustically ejecting the third analysis sample from the third well, wherein acoustically ejecting the third analysis sample is performed after the reference time period elapses.
In another aspect, the technology relates to a mass analysis instrument including: an open port interface (OPI); a fluid pump configured to pump a transport fluid into the OPI; an electrospray ionization (ESI) source, in fluid communication with the OPI; a detector configured to detect ions emitted from the ESI source; a movable stage for receiving a well plate, wherein the movable stage is configured to selectively align individual wells of the well plate with the OPI; a processor; and memory storing instructions that when executed by the processor cause the mass analysis instrument to perform a set of operations including: with the movable stage positioned at a first position relative to the OPI, acoustically ejecting a first sample from a first well of the well plate; moving the movable stage to a second position relative to the OPI; with the movable stage positioned at the second position relative to the OPI, acoustically ejecting a second sample from a second well of the well plate, wherein a time period between the acoustic ejection of the first sample and the acoustic ejection of the second sample is based at least in part on at least one of a predicted or an actual intensity of the first sample and at least one of a predicted or an actual intensity of the second sample. In an example, the set of operations further includes receiving the predicted intensity of the first sample and the predicted intensity of the second sample. In another example, the set of operations further includes detecting with the detector, in a timing operation preceding the acoustic ejections of the first sample and the second sample, the actual intensity of the first sample and the actual intensity of the second sample. In yet another example, moving the stage includes performing a moving operation and a waiting operation during the time period, wherein the moving operation is longer than the waiting operation. In still another example, moving the stage includes performing a moving operation and a waiting operation during the time period, wherein the moving operation is shorter than the waiting operation.
The system 100 includes an ADE 102 that is configured to generate acoustic energy that is applied to a liquid contained within a reservoir 110 that causes one or more droplets 108 to be ejected from the reservoir 110 into the open end of the sampling OPI 104. A controller 130 can be operatively coupled to the ADE 102 and can be configured to operate any aspect of the ADE 102 (e.g., focusing structures, acoustic ejector 106, automation elements 132 for moving a movable stage 134 so as to position a reservoir 110 into alignment with the acoustic ejector 106, etc.). This enables the ADE 106 to inject droplets 108 into the sampling OPI 104 as otherwise discussed herein substantially continuously or for selected portions of an experimental protocol by way of non-limiting example. Controller 130 can be, but is not limited to, a microcontroller, a computer, a microprocessor, or any device capable of sending and receiving control signals and data. Wired or wireless connections between the controller 130 and the remaining elements of the system 100 are not depicted but would be apparent to a person of skill in the art.
As shown in
It will be appreciated that the flow rate of the nebulizer gas can be adjusted (e.g., under the influence of controller 130) such that the flow rate of liquid within the sampling OPI 104 can be adjusted based, for example, on suction/aspiration force generated by the interaction of the nebulizer gas and the analyte-solvent dilution as it is being discharged from the electrospray electrode 116 (e.g., due to the Venturi effect). The ionization chamber 118 can be maintained at atmospheric pressure, though in some examples, the ionization chamber 118 can be evacuated to a pressure lower than atmospheric pressure.
It will also be appreciated by a person skilled in the art and in light of the teachings herein that the mass analyzer detector 120 can have a variety of configurations. Generally, the mass analyzer detector 120 is configured to process (e.g., filter, sort, dissociate, detect, etc.) sample ions generated by the ESI source 114. By way of non-limiting example, the mass analyzer detector 120 can be a triple quadrupole mass spectrometer, or any other mass analyzer known in the art and modified in accordance with the teachings herein. Other non-limiting, exemplary mass spectrometer systems that can be modified in accordance with various aspects of the systems, devices, and methods disclosed herein can be found, for example, in an article entitled “Product ion scanning using a Q-q-Q linear ion trap (Q TRAP) mass spectrometer,” authored by James W. Hager and J. C. Yves Le Blanc and published in Rapid Communications in Mass Spectrometry (2003; 17: 1056-1064); and U.S. Pat. No. 7,923,681, entitled “Collision Cell for Mass Spectrometer,” the disclosures of which are hereby incorporated by reference herein in their entireties.
Other configurations, including but not limited to those described herein and others known to those skilled in the art, can also be utilized in conjunction with the systems, devices, and methods disclosed herein. For instance, other suitable mass spectrometers include single quadrupole, triple quadrupole, ToF, trap, and hybrid analyzers. It will further be appreciated that any number of additional elements can be included in the system 100 including, for example, an ion mobility spectrometer (e.g., a differential mobility spectrometer) that is disposed between the ionization chamber 118 and the mass analyzer detector 120 and is configured to separate ions based on their mobility difference between in high-field and low-field). Additionally, it will be appreciated that the mass analyzer detector 120 can comprise a detector that can detect the ions that pass through the analyzer detector 120 and can, for example, supply a signal indicative of the number of ions per second that are detected.
During use of the mass spectrometry device of
Among other things, the present technology provides solutions to reduce the delay time between acoustic ejections while reducing the likelihood of interference between samples. To do so, the present technology utilizes predicted or actual intensity relationship between sample signals and adjusts the delay time between ejections accordingly. Predicted intensity signals may be based on one or more factors, such as measured concentrations, relative concentrations (e.g., high or low), sample ejection volume, or sample matrix. This information may be delivered to the mass spectrometry device, for example, by a technician who prepares a sample tray, prior to mass spectrometry analysis. Based on such information about the samples, the delay times between samples may be adjusted. For example, the delay time between ejecting a known high-concentration sample and a known low-concentration sample may be greater than the delay time between ejecting a low-concentration sample and a high-concentration sample. For samples where predicted characteristics are not available, actual intensity signals may be determined by quickly ejecting and analyzing samples to determine if additional delay time is required. Even though these quick ejections may require additional time, by adjusting the required time between ejections needed for a complete mass spectrometry analysis, overall processing time may be saved.
An example signal plot is depicted in
However, there are often conditions where the intensity signals of adjacent or consecutive ejections can deviate significantly. Such conditions can be particularly challenging when the signal dynamic range between adjacent ejections is 50, 100, 1000 times, or more. Such a condition is depicted in
In standard AEMS systems, the acoustic ejection method is the same for an entire run, including the acoustic calibration, carrier solvent flowrate, ejection volume, the ejection delay time, etc. In such a configuration, the delay time is set based on the worst case, as the required delay time for the widest expected signal dynamic range within the assay.
The technologies described herein contemplate methods for adjusting dynamically the delay time between samples, so as to reduce or eliminate unnecessary wait times. This can greatly improve the analytical throughput of a mass analysis system. The delay time may be dynamically adjusted according to predicted or predetermined signal information of adjacent ejections which relate to concentrations of the samples within each well. For example, a longer ejection delay time will be added between the sequence of high expected signal followed by the low expected signal. A shorter ejection delay time would be enough for the adjacent ejections with similar concentrations, and for a low expected signal followed by a higher expected signal. A longer ejection delay time may also added due to the presence of a tailing peak shape of a signal.
Several approaches to achieve the results are contemplated to generate the information about the predicted or actual signal relationship of adjacent ejections. For example, for some stability assays and dose-response activity screenings, the sample plate is arranged typically from low concentration to high concentration, or at least similar concentration within a given group.
A method 500 of controlling ejections from samples of a well plate is described in more detail in
The method 500 begins with receiving a first sample intensity prediction associated with a first sample in a first well of the well plate, operation 502. This sample intensity prediction may be input by a user of a mass spectrometry device that will be used to analyze the samples in the well plate. In operation 504, a second sample intensity prediction associated with a second sample in a second well of the well plate is received. Again, as noted above, for purposes of illustration, the second sample intensity prediction is less than the first sample intensity prediction. With this high first sample intensity prediction and a comparatively low second sample intensity prediction, an ejection time delay value for a subsequent analysis of the first sample and the second sample may be determined, operation 506. This determination is based at least in part on the first and second sample intensity predictions. Further optional operations may be performed, for example, if three, four, or more samples are utilized. For example, operation 508 includes receiving a third sample intensity prediction associated with a third sample in a third well of the well plate and, as noted above, the third sample intensity prediction is greater than or equal to the second sample intensity prediction. With these three sample intensity predictions known, subsequent analysis of the first, second, (and third or more samples, if present) may be performed.
That analysis begins with operation 510, where the well plate containing the first, second, and third samples (in first, second, and third wells, respectively) is received in a mass spectrometry device. In preparation for a processing of the well plate having the first, second, and third samples, operation 512 includes calculating an adjusted time period. The adjusted time period is based at least in part on a reference time period and the determined ejection delay time value from operation 506. The reference time period may be the shortest required delay time between sample ejections where additional delay time (the determined ejection delay time) is not required; that is, a sample of a known sample intensity followed by a sample of a sample intensity greater than or equal to the known sample intensity. The method 500 continues with operation 514, acoustically ejecting the first sample from the first well. As an increased delay time is required before ejecting the second sample, the method includes operation 516, waiting for the adjusted time period to elapse. Thereafter, operation 518, acoustically ejecting the second sample from the second well, is performed. In this example, since it is known that the third sample has a sample intensity greater than or equal to the second sample intensity, operation 520, waiting for the reference time period to elapse, is performed. Thereafter, the method 500 continues with operation 522, acoustically ejecting the third sample from the third well, is performed.
Further ejections may be repeated for additional samples from additional wells of the well tray. The delay time between each is dictated by factors that include the relative sample intensities between adjacent ejections, or the presence of a tailing peak shape. With regard to the latter, as an example, high concentration ejections followed by low concentrations require a delay characterized by the adjusted time period, while low concentration ejections followed by equal or greater concentration ejections require a delay only characterized by the reference time period. Continuing with method 500, the first, second, and third samples (as well as any additional samples) are then analyzed, operation 524. Respective ion intensity signals may be obtained from the analysis. Operation 526 may also be performed, which includes storing information obtained from the analysis. This information may include the sample intensity prediction(s), reference time period, ejection time delay, adjusted time period, resulting ion intensity signal(s), etc. All of this information may be saved and stored for future reference and quality control. For example, if the information indicates a discrepancy between the received information (e.g., between a low second sample intensity prediction and a high third sample intensity prediction) and the analyzed ion intensity signal (indicative of high second sample intensity prediction and a low third sample intensity prediction), the well plate may be re-analyzed.
While the method 500 of
Additional timing samples may be ejected and analyzed, though only acoustically ejecting a third timing sample from a third well of the well plate, operation 714, and analyzing the third timing sample with the mass spectrometry device to determine a third ion intensity signal, operation 716, are depicted. As with the method 500 of
The analysis aspect of the method 700 begins at operation 718, acoustically ejecting the first analysis sample from the first well. A subsequent ejection is delayed until the adjusted time period elapses, operation 720, after which the second analysis sample is acoustically ejected from the second well, operation 722. Thereafter, the method 700 waits for the reference time period to elapse, operation 724, before acoustically ejecting the third analysis sample from the third well. The various ion intensity signals for each analysis sample may then be generated, analyzed, evaluated, stored, etc.
The mass spectrometry device depicted in
In its most basic configuration, operating environment 900 typically includes at least one processing unit 902 and memory 904. Depending on the exact configuration and type of computing device, memory 904 (storing, among other things, instructions to control the eject the samples, move the stage, or perform other methods disclosed herein) can be volatile (such as RAM), non-volatile (such as ROM, flash memory, etc.), or some combination of the two. This most basic configuration is illustrated in
Operating environment 900 typically includes at least some form of computer readable media. Computer readable media can be any available media that can be accessed by processing unit 902 or other devices having the operating environment. By way of example, and not limitation, computer readable media can include computer storage media and communication media. Computer storage media includes volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data. Computer storage media includes, RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, solid state storage, or any other tangible medium which can be used to store the desired information. Communication media embodies computer readable instructions, data structures, program modules, or other data in a modulated data signal such as a carrier wave or other transport mechanism and includes any information delivery media. The term “modulated data signal” means a signal that has one or more of its characteristics set or changed in such a manner as to encode information in the signal. By way of example, and not limitation, communication media includes wired media such as a wired network or direct-wired connection, and wireless media such as acoustic, RF, infrared and other wireless media. Combinations of the any of the above should also be included within the scope of computer readable media. A computer-readable device is a hardware device incorporating computer storage media.
The operating environment 900 can be a single computer operating in a networked environment using logical connections to one or more remote computers. The remote computer can be a personal computer, a server, a router, a network PC, a peer device or other common network node, and typically includes many or all of the elements described above as well as others not so mentioned. The logical connections can include any method supported by available communications media. Such networking environments are commonplace in offices, enterprise-wide computer networks, intranets and the Internet.
In some examples, the components described herein include such modules or instructions executable by computer system 900 that can be stored on computer storage medium and other tangible mediums and transmitted in communication media. Computer storage media includes volatile and non-volatile, removable and non-removable media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules, or other data. Combinations of any of the above should also be included within the scope of readable media. In some examples, computer system 900 is part of a network that stores data in remote storage media for use by the computer system 900.
This disclosure described some examples of the present technology with reference to the accompanying drawings, in which only some of the possible examples were shown. Other aspects can, however, be embodied in many different forms and should not be construed as limited to the examples set forth herein. Rather, these examples were provided so that this disclosure was thorough and complete and fully conveyed the scope of the possible examples to those skilled in the art. In addition, some aspects of the present disclosure are described above with reference to block diagrams and/or operational illustrations of systems and methods according to aspects of this disclosure. The functions, operations, and/or acts noted in the blocks may occur out of the order that is shown in any respective flowchart. For example, two blocks shown in succession may in fact be executed or performed substantially concurrently or in reverse order, depending on the functionality and implementation involved.
Although specific examples were described herein, the scope of the technology is not limited to those specific examples. One skilled in the art will recognize other examples or improvements that are within the scope of the present technology. Therefore, the specific structure, acts, or media are disclosed only as illustrative examples. Examples according to the technology may also combine elements or components of those that are disclosed in general but not expressly exemplified in combination, unless otherwise stated herein. The scope of the technology is defined by the following claims and any equivalents therein.
This application is being filed on Dec. 22, 2021 as a PCT International Patent Application and claims the benefit of and priority to U.S. Provisional Application No. 63/128,940, filed on Dec. 22, 2020, which application is hereby incorporated herein by reference.
Filing Document | Filing Date | Country | Kind |
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PCT/IB2021/062200 | 12/22/2021 | WO |
Number | Date | Country | |
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63128940 | Dec 2020 | US |