Volume measurement of microdroplets

Abstract

Improvements to acoustically dispensed samples injected into an open port probe (OPP) are described. Apparatus and methods are described to calibrate volumetric dispense determination and mechanisms in an acoustic dispenser to produce accurate and precise volume delivery.

Description

Technical Field

[0001] The present application relates to the field of micro-droplets. In particular, the present application relates to a system and method for measuring the volume of dispensed micro-droplets using mass spectrometry. Background Art

[0002] Microfluidic dispensing involves the control and manipulation of fluids to extract small volumes of fluids from bulk fluid samples for examination. Microfluidic dispensing emerged in the early 1980s and has been used in a variety of fields such as, among others, inkjet printing, DNA microarrays, lab-on-a-chip technology, 3-D print heads, reformatting of drug libraries and microtiter plate replication, dispensing of individual cells and cell lysates.

[0003] Microfluid distribution continues to grow and develop, and is now able to distribute smaller and smaller volumes of fluids, usually via a method of delivering highly accurate volumes by non-contact methods. Microfluid distribution is particularly useful in areas where reagents are expensive or available in limited quantities, and in applications where high speed and high throughput are desired. For example, for these reasons, drug development and discovery including high throughput screening (HTS) and characterization of pharmacologically related dosing / distribution / metabolism / excretion (ADME) properties, as well as areas related to next generation gene sequencing, have included microfluid distribution. Recently, the inventors have combined microfluid distribution technology to introduce samples into analytical measurement tools such as mass spectrometers.

[0004] The basic operation of microfluidic distribution involves separating a small volume of sample material from a relatively large "bulk" sample. The sample material can be distributed in different forms, for example, as a single discrete droplet, a group of droplets, a mist, or other physical arrangement of the sample material. Depending on the specific mechanism used to separate the sample material, different distribution forms can be more or less reproducible at each distribution.

[0005] For example, dispensing by droplet has been used to dispense discrete droplets as small as the picoliter range. Some of the most common types of systems for delivering small volume droplets from a sample are broadly characterized as jet or dynamic devices, examples include, for example: acoustic technology; piezoelectric technology; pressure-driven technology; pneumatic pump / valve technology; electric field-driven technology, etc. These dispensing devices all transfer a measured amount of energy directed into the bulk sample in order to separate a desired sample volume in the form of one or more droplets from the bulk sample fluid.

[0006] The amount of energy required to dispense a droplet from a bulk sample fluid is related to the fluid properties of the bulk fluid, with viscosity and surface tension being primary considerations. The dispensing parameters that control the energy generated by the dispensing device and transferred to the bulk sample fluid need to be specifically tailored to the fluid properties of the bulk sample fluid in order to deliver a target droplet volume with sufficient energy to break away from the bulk sample fluid.

[0007] Due to the complexity of the problem, the selection of specific dispensing parameters corresponding to the desired droplet volume for a given bulk sample fluid is accomplished through an empirical adjustment process of one or more dispensing parameters, dispensing one or more droplets, measuring the dispensed volume of the one or more droplets, adjusting the dispensing parameters, and iteratively repeating the sequence until dispensing parameters that consistently deliver the desired droplet volume for the bulk sample fluid are identified. Regardless of the dispensing technique used, the dispensing parameters are often interdependent, making the adjustment process highly parameterized since adjustments to one dispensing parameter may affect adjustments to other dispensing parameters.

[0008] Acoustic droplet dispensing is used commercially to transfer liquid samples from one microtiter plate to another, which is called plate replication and reformatting. Dispensers have also been developed for transferring samples from test tubes of various configurations into microtiter plates. The inventors used acoustic droplet dispensing to direct controlled volumes of sample into capture probes for collection and transfer for mass analysis by a mass spectrometer.

[0009] As an example of liquid dispensing, acoustic droplet dispensing (ADD) is a technique for contactlessly, volumetrically, and precisely transferring droplets from a sample well in a microtiter plate to a corresponding sample well in a second microtiter plate. The use of energy in the form of acoustic waves allows the transfer of fluids in the form of discrete droplets to be contactless, volumetrically accurate, and precise when conditions are highly controlled. Typical well densities in microtiter plates are 96, 384, and 1536 wells per microtiter plate, and typical droplet volumes for dispensing are in the range of 1-20 nL.

[0010] Volumes greater than 20 nL can be dispensed, with the expectation that these droplets will fragment after desorption due to fluid instabilities. Multiple droplets can be dispensed sequentially into target wells to cumulatively reach the desired dispense volume. Pharmaceutical research and development organizations use this method extensively to dispense small volumes of compounds, typically dissolved in DMSO, from their large drug libraries for further testing in HTS assays screening for biological activity and ADME assays to determine drug properties.

[0011] For example, an acoustic dispenser generates sound waves through a piezoelectric vibrator powered by RF power, and is transferred to the bottom of a sample well through a metal lens via a coupling fluid. The metal lens is connected to the bottom of the sample well using a coupling fluid because air gaps must be avoided because sound waves are rapidly decelerated by gaseous media. The sound waves propagate through the bottom of the sample well, which can be made of a variety of compatible plastics or other materials and have various thicknesses and shapes, and then travel through the fluid of the sample to reach the curved liquid surface at the surface. At this point, pressure disturbances occur when the sound waves decelerate at the liquid-gas interface, which under appropriate conditions will accurately launch droplets of known volume to a few centimeters above the surface in a precise and reproducible manner.

[0012] The operation of other droplet dispensers, such as pneumatic or pressure based droplet dispensers, may similarly vary in droplet dispensing based on physical parameters of the liquid sample being dispensed.

[0013] The operation of the dispenser can be controlled by adjusting a number of physical parameters that are closely related to the type of dispenser. For example, an acoustic dispenser can vary frequency, power, direction, duration or burst rate, focus position, etc., in order to generate droplets from a liquid sample. Determining what values ​​to use for these physical parameters when setting the operating parameters of the dispensing device to repeatedly deliver the desired droplet volume from liquids with different fluid properties and depths is a process known as calibration. Variations in sample, plate, and environmental properties require adjustment of the physical parameters to compensate. Determining what values ​​to use for the physical parameters to deliver the desired droplet volume is a process known as "calibration".

[0014] For example, in the case of an acoustic dispenser, it is necessary to calibrate the acoustic parameters so that accurate and precise droplet volumes can be reproducibly dispensed. Droplet volume measurement is primary for the calibration process. Samples with different fluid properties require unique calibration files, and unique calibration files are required at different depths of the sample. However, the acoustic wave frequency, power, energy duration (repetition or burst rate), focus, and individual characteristics of the piezoelectric transducer and lens elements in the instrument all affect the volume of the dispensed droplets. The values ​​of these parameters for delivering a specified droplet volume strongly depend on the viscosity and surface tension properties of the overall solution. For this reason, calibration files for different liquids with different viscosities are required. Calibration files for specific fluids at different depths in the sample well may also be required, because when energy travels through the fluid to reach the surface where the energy is deposited to emit droplets, the energy dissipates. Due to the variations in the manufacture of piezoelectric transducers and associated lens assemblies, calibration files are specific to each individual instrument.

[0015] Calibration is a multi-step, iterative process that involves adjusting physical parameters (e.g., acoustic power, frequency, repetition or "burst" rate, and focusing the wave to a point close to the sample liquid surface with a lens). Its value is affected by the viscosity of the sample fluid, which changes the surface tension of the sample liquid and thus the power required to launch a droplet. Changes in sample, plate, and environmental properties require adjustment of physical parameters to compensate.

[0016] After each volume measurement, the physical parameters of acoustic power, frequency, and repetition or "burst" rate are iteratively adjusted and the volume measurement is repeated. The closer one gets to the correct volume, the fewer adjustments are needed. Eventually, only one parameter needs to be fine-tuned, while all other remaining parameters are held at fixed values, thereby reducing the parametric nature of the process. Some methods provide for holding frequency and power fixed and adjusting only burst rate and focus distance, but all of these parameters can be adjusted to achieve a target droplet volume.

[0017] Additionally, the distance the wave must travel to reach the surface will affect the location of the wave's focus, which is controlled by positioning the focusing lens. This distance varies with the volume of the sample in the well, which changes over time as the sample is dispensed, evaporated, or varies from sample to sample due to the nature of the analysis. This distance must be accurately determined for each well and adjusted by the lens position. This is done by measuring the time it takes for the sound wave to reflect back to the piezoelectric transmitter (which also serves as a detector). If the speed of sound in the sample fluid is known, the distance to the surface can be calculated. For many types of fluid mixtures, the speed of sound is unknown and must therefore be measured by the instrument. Determining the speed of sound in any sample fluid is the first step in the calibration process. Once the speed of sound is determined for a known overall sample composition, the data is stored and used to determine the fluid depth in all wells to be analyzed. Once the speed of sound in a particular fluid has been determined, it does not have to be repeated.

[0018] Typically, conventional calibration operations are performed in the factory or in the field by a service engineer using a series of iterative protocols, standardized reference solutions of UV absorbing compounds or luminescent fluorophores, and a spectrophotometer that measures the transmission, absorption, or emission of light to determine the concentration of the dispensed droplets, which can be converted into droplet volume. A predetermined number of droplets, typically 10-200 droplets, are emitted from a sample that includes a light emitting or absorbing reference solution, and the sample is then diluted to a known volume much larger than the total volume of the droplets, and the concentration is determined using a spectrophotometer. Calibration is not typically a problem when using acoustic dispensing for plate replication or reformatting applications because the sample fluid composition is well-defined and uniform, typically 100% DMSO and little else. The concentration of drug library compounds in this solvent is not high enough to substantially affect fluid viscosity.

[0019] In cases where the fluid composition is not clear and may vary between samples, it will be necessary to empirically determine the parameters for creating a new calibration file whenever a sample with sufficiently different fluid properties is encountered. Small changes may have large effects, for example, due to fluid properties introduced by biological solutes in aqueous solvents, plasma from different patients or samples from fermentation media taken at different times of incubation. Samples with different solvent ratios require different calibration files, for example, different combinations of alcohol to water. A change of a few percentage points may require a different calibration file. Commercially available acoustic distribution plate replicator instruments provide calibration files that have been created at the manufacturing site for a limited number of liquids.

[0020] New calibration files are also required when using different types of sample plates with different material compositions and dimensions, where the thickness and composition of the bottom of the hole through which the sound waves travel is particularly important. In addition, separate calibration files are required at different depths of the fluid in the hole. Typically, for some dispensers, 100 separate calibration files will be recorded at different depths of the fluid in the 384 plate wells.

[0021] The calibration files are stored on the instrument, reused and not necessarily replaced as long as the sample composition and plate type remain constant. Due to slight differences in the piezoelectric generator and the metal (usually aluminum) transmission / focusing lenses, each instrument will have unique settings.

[0022] In situations where sample composition can vary widely and unpredictably, calibration issues are a serious obstacle. Summary of the invention

[0023] Embodiments of the present application relate to the field of droplet generation and analysis, where a measured liquid sample can be dispensed from a bulk liquid in the form of small sample droplets, and the volume of each of the individual liquid sample droplets needs to be accurately determined. In some embodiments, the volume determination can be used to calibrate a droplet generator that dispenses sample droplets from a bulk fluid. In some embodiments, the droplet generator can be calibrated to provide consistent, repeatable droplet dispensing. Accurate and precise volume measurements are needed for droplets designated for additional sample manipulation, for deposition on surfaces, and for analytical measurements of the composition of the droplets.

[0024] In some embodiments, methods and apparatus are provided for calibrating an acoustic droplet dispenser for accurate and precise droplet volume delivery.

[0025] For this reason, a system that can accurately measure the volume of a droplet in a real-time manner for the purpose of rapidly calibrating deposition parameters is highly desirable. This is particularly important for biological samples where fluid properties can vary in unpredictable ways from sample to sample. For applications where the gravimetric mass of a solute is being measured in a droplet from a sample, a means is needed to simultaneously determine the volume of the droplet in order to know its concentration.

[0026] In some embodiments, a method for calibrating a droplet dispenser is provided, which dispenses sample droplets from a liquid sample, the method comprising: providing a liquid sample, the liquid sample comprising a calibrant with a known calibrant concentration; dispensing droplets of an assumed droplet size from the liquid sample; ionizing the droplets of the assumed droplet size; measuring the calibrant mass of the calibrant in the ionized droplets using a mass spectrometer; and determining the actual droplet size by comparing the expected calibrant mass based on the assumed droplet size with the measured calibrant mass.

[0027] In some aspects, the method may include: determining that the difference between the actual droplet size and the assumed droplet size is greater than a threshold size; and adjusting at least one droplet dispenser parameter of the droplet dispenser based on at least one of the magnitude and sign of the difference in droplet size. In some aspects, the method may include repeatedly dispensing subsequent droplets of a subsequent assumed droplet size from the liquid sample using the at least one previously adjusted droplet dispenser parameter, ionizing the subsequent droplets, measuring a subsequent calibrant mass of a calibrant in the ionized subsequent droplets, determining an actual subsequent droplet size by comparing an expected subsequent calibrant mass based on the assumed subsequent droplet size with the measured subsequent calibrant mass, and adjusting the at least one droplet dispenser parameter of the droplet dispenser until the difference between the actual subsequent droplet size and the assumed droplet size is less than the threshold size.

[0028] In some embodiments, a system for measuring the volume of fluid dispensed by a fluid dispenser is provided, the system comprising: a capture probe for receiving and capturing a dispensed volume of fluid from the fluid dispenser, the dispensed volume of fluid comprising a calibration standard; an ionization source for ionizing the captured volume of fluid; and a mass spectrometer for measuring the mass of the calibrant contained in the ionized volume of fluid.

[0029] In some aspects, the system further includes a reference compound of known concentration introduced into the transport fluid of the capture fluid.

[0030] In some aspects, the system further operates to determine the volume of the measured fluid based on the mass of the calibrant.

[0031] In some aspects, the system further operates to determine the measured volume of the fluid based on a ratio of the measured mass of the calibrant to the measured amount of the reference compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Those skilled in the art will understand that the drawings described below are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.

[0033] Figure 1A and Figure 1B is a schematic diagram of an embodiment of the present teachings.

[0034] Figure 2 An embodiment of a system of the present teachings is illustrated.

[0035] Figure 3 A. Figure 3 B and Figure 3 C illustrates various orientations and configurations of embodiments of the present teachings.

[0036] Figure 4 A. Figure 4 B. Figure 4 C and Figure 4 D illustrates various embodiments of capture probes of the present teachings.

[0037] Figure 5 and Figure 6 Alternative embodiments of the present teachings are illustrated.

[0038] Figure 7 Depicted is a cross-sectional view of an acoustic distributor coupled to a sample hole.

[0039] Figure 8 Another embodiment of a droplet dispenser for dispensing droplets into capture probes is illustrated.

[0040] Fig. 9 The measured signal of a sample including a calibrant is shown.

[0041] Fig.10 The calibration curve is shown graphically.

[0042] Fig.11 The signal of a reference compound in a mass spectrometer is shown.

[0043] Fig.12 Another calibration curve is shown.

[0044] Fig.13 Another calibration curve is shown.

[0045] Fig.14 An example of an in situ kinetic measurement is illustrated.

[0046] Fig.15Data from example in situ kinetic measurements are shown.

[0047] Fig.16 Illustrated are measurement data from an example of high frequency dispensing from a sample well for mass analysis.

[0048] Fig.17 Measurement data illustrating the absence of ion suppression for plasma injected into the capture probes is shown graphically.

[0049] Fig.18 Graphed are measurement data illustrating the absence of ion suppression for a surfactant (Tween 20) injected into the capture probe.

[0050] Fig.19 is a simplified schematic diagram illustrating droplets in a sample processing area being dispensed into capture probes.

[0051] Fig. 20 An example of a calibration curve is illustrated. DETAILED DESCRIPTION

[0052] In some embodiments, systems and methods for dispensing sample droplets from a bulk sample fluid and performing volume measurements on dispensed droplets are presented. In many fields, accurate and precise volume measurements are required for droplets designated for additional sample manipulation, for deposition on surfaces, and for analytical measurements on the composition of droplets.

[0053] For the purpose of illustrating the above problems, the present application describes in detail an implementation using an acoustic-based contactless dispenser. Although the present application and examples are primarily directed to an implementation using an acoustic microfluidic dispensing device, the present application contemplates a variety of dispensing technologies, including but not limited to acoustic technology; piezoelectric technology; pressure-driven technology; pneumatic pump / valve technology; and electric field-driven technology.

[0054] In some embodiments, systems and methods for accurately and precisely measuring the volume of individual droplets in the picoliter to microliter range are presented. In some embodiments, the systems and methods can include capturing micro-dispensed droplets in a flowing stream of liquid and delivering them to an atmospheric pressure ion source of a mass spectrometer for measurement. The volume of each droplet can be determined by determining the gravimetric mass of calibration and reference compounds in the droplet and the transport fluid. The measurement is fast enough to perform real-time volume measurements when the droplets are dispensed at rates up to 10 Hz. The systems and methods can operate independently of solvent and sample components and are applicable to biological fluids from a variety of sources.

[0055] Method invention described herein relates to using mass spectrometer to measure the specific gravity mass of reference standard added to sample with known concentration to determine and calibrate the volume of dispensed droplets.Whenever droplets are emitted, calibration parameters can be adjusted, and its volume is measured by mass spectrometer, and then parameter adjustment is performed to make the parameter reach the target value.Since mass spectrometer is also an analytical measurement tool, if calibration is required in the case where the sample viscosity is highly unpredictable as pointed out about fermentation example, each sample can be calibrated before analytical measurement, which is otherwise referred to as "calibration on the fly".If the calibration parameters only slightly deviate from, resulting in a small deviation from the target dispensed volume, an alternative method to recalibrate each sample is to measure the volume with a mass spectrometer during analytical measurement, record slight volume differences, and consider the volume differences in subsequent concentration calculations.

[0056] The embodiments described herein can be implemented using a capture probe as an interface with a mass spectrometer to capture the allocated droplets for volume measurement. Examples of suitable capture probes include "open port probes" (OPP), which are the subject of multiple Sciex licensed patents and have been successfully used to capture the allocated droplets for analytical measurements. See, for example, US 9,632,066BS-Open Port Sampling Interface, US 2016 / 0299041A1-Capture Probes, and US 9,153,425B2-Equipment for High Spatial Resolution Chemical Analysis of Samples and Methods for High Spatial Resolution Chemical Analysis. The present invention describes new uses of capture probes including OPP.

[0057] In some embodiments, the capture probe can be modified to introduce an internal standard into the transport fluid. The mass of the reference standard in each droplet can be calculated using the response ratio between the reference standard in the sample and the internal standard in the transport fluid, and then its volume. Since the mass spectrometer performance changes over time, the response ratio will remain constant without experiencing drift. This eliminates the need to construct a calibration curve of the reference standard concentration and MS response (peak area) for continuously calculating the droplet volume. This method of using an internal standard in the transport fluid also improves measurement accuracy.

[0058] The ability to quickly determine the volume of an individually dispensed droplet provides a means to accurately and precisely determine the concentration of solutes in a sample when their gravimetric weight is determined by a mass spectrometer. It also provides a means to calibrate the droplet dispenser, and the system is applicable to a variety of droplet generators. This is particularly important when the fluid properties of the sample to be dispensed are unpredictable and the parameters of the dispenser need to be recalibrated in real time. This is also important when measurements are to be made on components within the droplet and the concentrations of those components in the droplet need to be determined.

[0059] Unlike plate replication, acoustic dispensing as a means of introducing samples into a mass spectrometer for analytical measurements may encounter samples of varying and often unknown composition and, therefore, viscosity. Since mass spectrometry, in contrast to plate replication tools, is an analytical measurement tool for a variety of applications and sample types, it would be advantageous to have a means to quickly and automatically adjust physical parameters as needed on a sample-by-sample basis. This ability to calibrate on a sample-by-sample basis is referred to as "on-the-fly calibration."

[0060] In particular, the inventors have proposed the use of acoustic distribution for organisms and fermentation products that may vary widely in composition from sample to sample. Examples of situations where the sample composition will vary are common. The sample to be distributed can come from a preparation scheme involving liquid-liquid or solid phase extraction techniques, which involves extraction fluids with different concentrations of alcohol, acetonitrile, or solvents that are immiscible with water such as chloroform or hexane. Protein precipitation in plasma samples is common and involves adding large amounts of organic solvents to an aqueous medium to cause the protein to leave solution. In situations like these, some understanding of the resulting solvent composition is understood and predictable to a certain extent, but calibration processes for these various media must be available to the user.

[0061] It is common that the sample composition and thus the viscosity can be radically changed in an unpredictable manner. Monitoring the biochemical reaction products from mammals, yeast and bacterial cell cultures is an example. During the life cycle changes of the cells, the overall chemical composition of the culture medium has substantial changes. It is reported that the culture medium present at the beginning and end of the fermentation or incubation time can be dispensed acoustically, but the distribution fails during the intermediate time period. It seems that a large amount of complex carbohydrates are produced during these failure periods, resulting in sufficiently high viscosity changes to cause the original calibration parameters to be insufficient. The only way to handle this type of situation is "instant calibration".

[0062] Typically, a spectrophotometer is used to measure the transmission, absorption or emission of light from known concentrations of dispensed calibration compounds. It utilizes a standardized reference solution with known concentrations of UV absorbing or luminescent fluorophore calibration compounds dissolved in the sample solvent or matrix to be calibrated.

[0063] Since spectrophotometers are concentration-sensitive detectors, they use relationships such as those expressed by Beer Lambert's Law to measure the concentration of the sample dispensed. The process involves dispensing a predetermined number (usually 100-200) of droplets in a well, followed by diluting the total collection of droplets in the well with a known volume of diluent. The diluent far exceeds the accumulated droplets to make it possible to measure with a spectrophotometer-based plate reader. In some cases, pipetting errors may be introduced into the measurement at this point.

[0064] It will be clear from this description that conventional spectrophotometric calibration schemes measure the average volume from a collection of droplets, and therefore do not measure individual droplet volumes.

[0065] In order to be able to calculate the volume of the accumulated droplets, the total volume of the droplets plus the diluent needs to be accurately measured. Using this method introduces additional errors because it assumes that the total volume is equal to the volume of the added diluent. This is because the volume contributed by the droplets is unknown because the system has not been calibrated. If the volume of the diluent is much larger than the volume of the dispensed droplets, the error is minimized, but there is still an error. If the volume of the overall solution (i.e., the total volume of the dispensed droplets plus the diluent) is brought to a known volume rather than by adding a known volume of diluent, this source of error can be theoretically eliminated, but this brings trouble, requiring the droplets to be captured in a bottle tube with an accurate volume measurement mark, and changes in the position of the mark and the aiming of the liquid meniscus are large sources of error. As the calibration proceeds, each step is repeated, and the volume of the target droplet is gradually converged, thereby reducing this source of error because the total volume can be more accurately determined as the volume of the diluent plus the volume of the dispensed droplets.

[0066] A means for avoiding these sources of error and the time taken to correct for them by performing iterative recalibration is to directly measure the mass of the calibration compound in each droplet to calculate the droplet volume from the known calibrant concentration. With spectrophotometric methods, this is not possible. The proposed mass spectrometry-based calibration system directly measures the mass of the calibration compound in each droplet.

[0067] Spectrophotometric determination of the concentration of accumulated droplets diluted to a larger volume requires the construction of a standard curve prepared from dyes of known concentrations. Although the dispensed samples are sufficiently diluted, best practice is to prepare different calibrant concentrations in the same solvent as the unknown being measured. This is because attenuation or amplification of the emission or absorption of light by components in the sample can occur. Quenching phenomena will attenuate the signal, and luminescence phenomena will amplify the signal, both of which will change the linear equation of the standard curve.

[0068] For this reason, a new standard curve is required for each new liquid type and sample matrix to be calibrated. This increases the time and labor involved and prevents the automation of rapid calibration on a sample-by-sample basis. The proposed mass spectrometry-based calibration system introduces the concept of a master standard curve, which conveniently provides a single standard curve that can be used to calculate droplet volume regardless of the sample type or volume to be dispensed.

[0069] Spectrophotometric determination of the concentration of the dye in the total diluted sample allows calculation of the mass of calibrant dispensed to achieve that concentration value. Knowing the original concentration of the calibrant in the dispensed droplets, the cumulative volume of the dispensed droplets can be calculated. The calculated cumulative volume divided by the number of dispensed droplets provides the average droplet volume. This value does not provide an indication of volume variation within that population of dispensed droplets. If repeated measurements provide the same average, there is still no indication of intra-batch error. Droplets can be either large or small around the mean.

[0070] The present mass spectrometry-based calibration system measures the volume of each individual droplet immediately after dispensing, providing an accurate assessment of the variability of each droplet and a means to correct for it.

[0071] As described below, the depth of a fluid in a hole can be quickly determined. The method is based on the principle of sonar.

[0072] When it is understood that different calibration files are required for different liquid levels in the hole for a single sample type, the time and labor required for the calibration task increase exponentially. The energy of the sound wave dissipates when it travels through the fluid, so as the depth increases, it is necessary to increase the power and / or duration of the sound wave. The power is adjusted by changing the voltage applied to the transducer or alternatively changing the length of time (sometimes referred to as burst rate) for energy deposition on the surface. Since the surface immediately stores the deposited energy, more energy will be deposited over a longer period of time. In practice, both voltage and burst rate are finely adjusted to provide the power for achieving the target volume.

[0073] Typically, for as many depth positions as in a 384-well plate, up to 100 separate calibration files are required. Since the calibration process outlined above is caused at each liquid level, that is, between each iteration of the acoustic parameters, 100-200 droplets are allocated for collection, dilution, and their concentration is measured by fluorescence, there is an inherent quantum mechanics problem, that is, the measurement distorts what is being measured. Attempts are being made to establish calibrations at different depths, while the depth is changing to change the calibration parameters. Droplet volume measurement reduces the liquid level by a sufficient amount of more than 100-200 allocated droplets, so that different power settings are required at the beginning of the distribution series and at the end. In order to correct this, the calibration process at each liquid level is repeated several times to average out errors.

[0074] The mass spectrometry-based calibration system avoids quantum mechanical issues because volume measurements and parameter adjustments are performed on individual nanoliter-sized droplets, since the level changes caused by a single droplet are negligible.

[0075] Acoustic droplet dispensing requires that the depth of the liquid to be dispensed can be measured quickly and dynamically both for calibration purposes and for routine dispensing of samples. Measuring depth does not require dispensing of droplets and therefore does not involve measuring the volume of the emitted droplets. Therefore, regardless of the method used to measure and calibrate the droplet volume, the process for measuring depth is the same. This aspect of calibration or routine dispensing is the same as the spectrophotometry or mass spectrometry described for droplet volume measurement. However, mass spectrometry has an advantage when considering the relationship between liquid depth and liquid volume in the sample well, so a detailed description of depth measurement is included below.

[0076] Due to the loss of distribution, evaporation or sampling method, the liquid level in each sample well can vary, so the volume of the sample in each well can vary significantly in normal laboratory operation. It is necessary to know the distance of the meniscus to set the focus of the sound wave to within a few hundred microns at or near the surface to concentrate the energy at the emission point of the droplet for each sample. The acoustic power requirement also increases with the increase of depth. Therefore, it is necessary to measure the depth of each sample well before the distribution event and for the correct acoustic calibration file to be called to allow the lens on the acoustic transducer assembly to be mechanically positioned to the appropriate focal length. The method requires determining or knowing the speed of sound through the sample liquid and the bottom of the plate containing the liquid. The speed of sound can be directly measured by calculating the time it takes for the sound wave to be reflected from the surface back to the piezoelectric transmitter that also serves as an acoustic wave detector.

[0077] The depth of the fluid in the sample well must be determined for two reasons.

[0078] First, according to typical depth variation, for different liquid depths, different calibration files are usually required.For example, usually the calibration files after the fluid of 200nL of 384-hole density plates is completed.This will be equivalent to about 100-200 calibration files in each hole, to cover the full range of possible fluid depth in the sample well.Because the power of sound waves gradually dissipates when they pass through fluid, the surface at different fluid depths needs higher energy for greater depth by the same power transfer.The voltage applied to the transducer by variation or the length (sometimes referred to as burst rate) of the time that energy is deposited on the surface is regulated by variation.Because the surface stores the deposited energy immediately, more energy will be deposited in a longer time.In practice, voltage and burst rate are both finely regulated, to provide the power for realizing target volume, but when the correct value converges, a parameter can remain fixed, and a remaining parameter is used for fine adjustment setting.

[0079] Second, for conventional sampling, lens focusing is required for different liquid depths. In order to effectively eject sample droplets, it is necessary to focus the calibrated sound waves with a lens to a point at or near the surface of the sample liquid, usually within a few hundred microns of the surface. A common practice is to focus the sound waves to a point 200 μm above the surface. The focus is controlled by the physical position of the focusing lens. The distance is a function of the volume of the sample in the hole and the hole geometry. When the sample is distributed, evaporated or changed sample by sample due to the nature of laboratory operations, the fluid level of the sample can change over time. The distance must be accurately determined for each hole and adjusted for the lens position. This can be determined immediately before dispensing each sample, or it can be performed as a batch mode by sequentially processing all sample wells on the plate before dispensing from the selected one or more holes.

[0080] The depth measurement is performed by measuring the time it takes for a sound wave transmitted upward through the bottom of the sample well to reflect back to the piezoelectric transducer that serves as both the transmitter and detector of the sound wave. Reflection of part of the emitted sound wave occurs at each point where the transmission medium changes state (from solid to liquid to gas). Reflections occur from the bottom of the orifice plate, from the transition from the solid orifice plate bottom to the sample liquid, and from the meniscus at the surface of the liquid sample where the strongest reflection is observed. This can determine the speed of sound through both the plate bottom and the liquid sample when testing a known plate bottom thickness and a known liquid depth.

[0081] Once the speed of sound is determined through a plate of a certain material and thickness, those values ​​remain constant for all plates of this type and the information is stored. Once the speed of sound through a liquid medium is determined, it remains constant for all samples of that bulk composition and the information is stored. The entire process, including the mechanical refocusing of the lens, takes on the order of tens of milliseconds to allow this measurement to be made for each sample well prior to dispensing.

[0082] Plate replicator applications typically encounter a limited number of different solvent types, 100% dimethyl sulfoxide being the most common solvent for storing drug libraries, and therefore the speed of sound through the sample is generally consistent. Plates with well features are also standard. Therefore, the requirement to create a large number of calibration files and measure the speed of sound in many different systems is not as strong in this application area.

[0083] The proposed application is quite different and involves using a mass spectrometer to analyze biological fluids and measure the amount of compounds dissolved therein and identify the compounds based on their molecular weight and chemical bond fragmentation pattern. An almost infinite number of samples with different fluid properties are encountered in biological and industrial processes, all of which require unique calibration files.

[0084] For example, the inventors have performed ADD-OPP on fermentation products collected over time during the fermentation process and observed that the measurements were inconsistent because the fluid properties changed sufficiently during the fermentation to deviate from the assumed fluid properties of the calibration. Similar problems arise when measuring biological products such as serum, where acoustic droplet ejection may be useful for some samples, while exhibiting enough deviation to produce erroneous measurements for other samples.

[0085] For completeness, below is an example of a protocol for determining the speed of sound in a system and the depth of a fluid in a hole. To perform this protocol, no droplets are dispensed, only the time for the sound wave reflection from the phase change is measured.

[0086] (a) The reflection time is measured for a known sample volume (known distance or path length), sample composition where the speed of sound is known, and a plate for which this measurement has been established. This establishes and checks the baseline parameters.

[0087] (b) Measure the reflection time for a known volume (e.g., a known depth and known path length for a given sample well geometry), a known sample composition, and an unknown plate. By comparing the results from (a) and (b), this establishes the speed of sound and the time of flight of the sound wave through the bottom of the plate. This will remain constant for all remaining plates of this type (i.e., same manufacturer, same style (plate thickness), same material (plastic type)). The plate is now a known plate that is permanently recorded in the database.

[0088] (c) Measure the reflection time for a known volume (e.g., known depth and known path length for a given sample well geometry), unknown sample composition, and known plate. By comparing the results from (b) with (c), this provides the time of flight of the sound wave through that depth of the new fluid, and thus the speed of sound in that medium. From this point on, the overall fluid composition will remain constant. Now, this depth measurement can be made for each well, followed by loading the correct calibration file, adjusting the lens position appropriately as needed, and desorbing the droplet within a time frame of tens of milliseconds per well.

[0089] (d) Establishing the fluid volume and fluid depth relationship of the sample reservoir. Different models and styles of orifice plates have different sample reservoir geometries, which translate into different volumes of liquid, which occupy different depth levels of the liquid. This relationship between volume and depth is particularly important for knowing when a sample well is approaching an empty state. Knowing the remaining volume from the depth measurement enables one to know how many droplets can be redistributed before the well is so close to empty that further distribution is considered unreliable. In some embodiments, the proposed system can be operated to maintain an estimate of the sample volume in each well, and provide an indication of whether the sample volume / fluid height estimated for a particular well is too small (i.e., below a threshold for the sample well geometry) to allow for an expected droplet ejection from the well.

[0090] After calibration parameters have been established to deliver reliable droplet volumes, tables of this relationship can be prepared for different well plate designs. A defined number of drops of known volume are dispensed and the depth is remeasured. This process is continued until the sample reaches the minimum feasible volume for dispensing. Typically, approximately 100 drops are dispensed per depth measurement, totaling between 100–500 nl per level.

[0091] Since the present mass spectrometry-based calibration system accurately measures the volume of each droplet, establishing the fluid volume to fluid depth relationship does not require the system to accurately dispense a known volume to each droplet. Instead, the present mass spectrometry-based calibration system only needs to count the number of dispensed droplets captured by the capture probe and measure / confirm the volume of each droplet. Therefore, the present mass spectrometry-based calibration system does not require a fully calibrated dispensing system to be in place in order to perform fluid depth measurements.

[0092] The goal of acoustically dispensing the sample into a mass spectrometer system will be to achieve the following performance metrics.

[0093] 1. High speed. Achieved a sample introduction rate that is 10-100 times faster than the current HPLC method. Requires a speed of about hundreds of milliseconds per sample.

[0094] 2. Avoid errors due to cross contamination of samples. The sample does not directly contact the surface. "Non-contact", for example, by avoiding additional pipetting.

[0095] 3. Consume small amounts of sample (preferably low nanoliter volumes) for a single analytical measurement.

[0096] 4. Flexible control of the sample introduction rate. Depending on the needs of mass spectrometry, the data acquisition time for each sample can be extended from hundreds of milliseconds to tens of seconds.

[0097] 5. Robust and reliable. Will be able to maintain high sample introduction rates for long periods of time without failure. Capable of continuous operation of at least 100,000 samples per day.

[0098] 6. No sample pre-purification is required. Blood, plasma, fermentation medium, etc. can be analyzed directly, although this will introduce new sensitivity limitations.

[0099] 7. Quantification is precise and accurate. The coefficient of variation of the dispensed volume and the variability of the mass spectrometry signal totaled <10%.

[0100] 8. Lower limit of detection and quantification. Low nM. Low parts per billion.

[0101] 9. Applicable to a wide range of chemical species. Molecular weight ranges from <50 amu to molecules exceeding 100,000 amu.

[0102] 10. Accept a wide variety of sample fluids whose properties are unpredictable and not necessarily known before the time of analysis. Aqueous and non-aqueous containing high concentrations of solutes that can alter the properties of the fluid (such as proteins, polysaccharides, alcohols, etc.). For this reason, the acoustic dispenser needs to be adjusted or calibrated on a sample-by-sample basis. To do so, a means of measuring the sample volume of each droplet is required during each dispense or at least within the sampling period.

[0103] Performance criteria 1-3. Acoustically driven plate replicator instruments have demonstrated the ability to dispense low nanoliter droplet volumes at a few Hz without any direct physical contact with the sample. To achieve the remaining six properties, methods for coupling the acoustically generated droplets to the mass spectrometer ion source will come into play.

[0104] Performance Indicators 4-9. A method has been developed to transfer acoustically generated droplets to an electrospray mass spectrometer ion source that delivers droplets through a liquid stream, known as an open port probe (OPP). The interface is described in more detail in WO 2019 / 104235 (PCT / US2018 / 062337), which is incorporated herein by reference. As described in more detail below, the interface with some additional modifications described below achieves the above performance indicators 4-9.

[0105] Performance Indicator 4. OPP can transform discrete digital signals from an acoustic dispenser into a continuous ion beam by capturing and merging individual dispensed droplets in a flowing solvent stream. This occurs when droplets are dispensed into the OPP at a rate of 10 Hz or more for typical capture probe solvent flow rates / volumes. In doing so, the sample introduction time can be adjusted to the mass spectrometer acquisition method. For example, signal averaging over a longer period of time can be used as a means to reduce detection limits. With dynamic control over the sample introduction time, ion mobility separation coupled to MS / MS of all components in a complex biological sample using scanning modes such as SWATH becomes feasible. Small amounts of sample are consumed even over relatively long periods of time using acoustic droplet dispensing. In addition, a range of pore sizes can be accommodated, with larger pore volumes supporting extended periods of sampling from the pores.

[0106] Performance Index 5. OPP delivery fluid is delivered using a gas pressure drop pump, which is highly reliable because it does not have any mechanical moving parts. In addition, the orifice of the fluid delivery pipe is large and inherently resistant to clogging.

[0107] Performance Indicator 6. OPP dilutes acoustically dispensed droplets by >100 times (or more), which allows sample purification steps to be avoided. When the sample is diluted to this extent and the dilution is instantaneous after sampling and before ionization, no inhibition of the electrospray ionization process occurs. Blood samples can be injected directly, and biochemical reactions can be monitored while the process is ongoing without disturbing the components of the reaction mixture.

[0108] Performance Specification 7. The OPP passes the sample to a standard electrospray ion source which has historically been shown to have high precision and accuracy, providing CVs of less than 10%. The accuracy and precision of the acoustic partitioning also plays a role here.

[0109] Performance Indicator 8. The OPP delivers the sample into a standard electrospray ion source that has historically demonstrated high sensitivity, efficient conversion of molecules to ions and delivery of the ions into the vacuum system. The flow rate supports both high sensitivity and robust operation and is in the microflow range, tens to low hundreds of microliters per minute.

[0110] Performance Specification 9. The OPP delivers the sample to a standard electrospray ion source which has historically been shown to have the highest compound coverage of any ion source used for mass spectrometry.

[0111] However, current methods for calibrating acoustic dispensers cannot cope with performance indicators 10. Biological systems being measured by mass spectrometers are usually analyzed, in which fluids have a wide range of fluid properties and viscosities and can change over time in unpredictable ways during reactions or incubations. In contrast to plate replication tools, mass spectrometry is an analytical measurement tool for a variety of applications and sample types, and therefore will require available means for quickly and automatically adjusting physical parameters as needed sample by sample. In order to make ADD-MS a viable technology, when encountering samples of different viscosities, a means for calibrating acoustic parameters to automatically deliver accurate and precise volumes is used. This ability to calibrate sample by sample is called "instant calibration."

[0112] Examples of situations where sample composition varies generally require immediate calibration. Blood samples can have widely varying viscosities depending on the hematocrit. Where preservatives have been added to blood or plasma or a degree of purification has been performed to preserve the sample, additives such as alcohols, acids, salts or other diluents that change viscosity will affect their fluid properties. The viscosity of urine samples is widely affected by the health status of the individual. The differences in properties of biological fluids from different species are large.

[0113] It is common that the sample composition and thus the viscosity can be completely changed over time in an unpredictable manner. Monitoring fermentation and cell culture media in the incubation time period is a good example. During the change of the life cycle of the cell, the overall chemical composition of the culture medium has substantial changes. It is reported that the culture medium present at the beginning and end of the fermentation or incubation time can be dispensed acoustically, but the distribution fails during the intermediate time period. It seems that a large amount of complex carbohydrates are produced during these failure periods, thereby causing sufficiently high viscosity changes to cause the original calibration parameters to be insufficient. The only way to handle this type of situation is instant calibration.

[0114] The present invention proposes a method and apparatus for measuring the volume of individual droplets dispensed using microdispensing techniques. It enables on-the-fly calibration, which directly measures the mass of calibrants in individual droplets using a mass spectrometer, and utilizes a modified OPP to capture droplets, transport them into an atmospheric pressure ionization mass spectrometer, and deliver reference compounds to improve the accuracy and precision of the resulting droplet volume measurements.

[0115] Mass-Sensitive vs. Concentration-Sensitive Methods. A mass spectrometer is a mass-sensitive detector and a device that measures the molecular weight of a molecule based on its mass-to-charge ratio. Determination of the amount or mass of a compound in a sample by a mass spectrometer is performed by counting the number of molecules after a net charge has been imparted to the molecules of the compound in the sample by inducing a deficiency or excess of electrons, a process known as ionization. Similarly, radioisotope detectors directly measure the mass of a compound in a sample by counting the number of nuclear decay particles per unit time. In most cases, spectroscopy determines the concentration of a molecule in a solution directly from the absorption, transmission, or emission of light, using, for example, the relationship expressed in Beer-Lambert's law.

[0116] Direct measurement of the mass of a calibration compound of known concentration in a droplet directly provides the volume of the droplet. Measuring the concentration of a calibration compound of known concentration in a droplet does not directly provide information about its volume. Its volume can be estimated indirectly by diluting the droplet to a much larger known volume to which the volume contribution of the droplet is negligible, and reading the concentration of the diluted sample from a calibration curve of concentration versus spectrophotometric signal. This will provide the mass of the calibration compound in the original droplet. This method is indirect and requires additional droplet manipulation and is therefore not amenable to real-time volume measurement. In practice, several droplets are counted and collected, and a calculation of the overall mass is performed, thereby providing an average droplet volume, rather than the exact volume of each droplet.

[0117] The calibrant in each droplet is captured, transported and ionized. The present system and method provide for directly determining the volume of individual droplets using atmospheric pressure ionization and mass spectrometry to count the ions generated from the calibration. This is done by measuring the mass (amount) of the calibrant compound of known concentration in a droplet of unknown volume, thereby directly determining the volume of the droplet. The droplets are introduced into an atmospheric pressure ion source in the liquid phase for ionizing the calibrant molecules and then counting them with a mass spectrometer. Preferred methods of ionization are electrospray and atmospheric pressure chemical ionization, but are not limited thereto. Figure 1A An exemplary mass analysis instrument 100 according to various embodiments of the present teachings is presented. The mass analysis instrument 100 is an electromechanical instrument for separating and detecting ions of interest from a given sample. The mass analysis instrument 100 includes a computing resource 130 to both perform control of system components and receive and manage data generated by the mass analysis instrument 100. Figure 1AIn the embodiment of the present invention, the computing resource 130 is illustrated as having separate modules: a controller 135 for directing and controlling the system components and a data handler 140 for receiving and compiling data reports of detected ions of interest. As desired, the computing resource 130 may include more or fewer modules than those depicted, may be centralized, or may be distributed across system components as desired. Typically, based on control information and other processing information from various system components, the detected ion signals generated by the ion detector 125 are formatted in the form of one or more mass spectra. Subsequently, data reports (e.g., mass spectra) may be processed using a data analyzer (in Figure 1A Subsequent data analysis performed (not shown) in order to interpret the results of the mass analysis performed by the mass analysis instrument 100.

[0118] In some embodiments, the mass analysis instrument 100 may include Figure 1A For the purposes of this application, although the computing resource 130 may not directly control the sample separation / delivery component 105 or provide data processing to the sample separation / delivery component 105, the mass analysis instrument 100 may be considered to include all of the illustrated components.

[0119] In the context of the present application, the separation / delivery system 105 includes a delivery system capable of delivering a measurable amount of a sample (typically a combination of an analyte and an accompanying solvent sampling fluid) to an ion source 115 disposed downstream of the separation system 105 to ionize the delivered sample. A mass analyzer 120 receives the generated ions from the ion source 115 for mass analysis. The mass analyzer 120 operates to selectively separate ions of interest from the generated ions received from the ion source 115 and deliver the ions of interest to an ion detector 125, which generates a mass spectrometer signal indicative of the detected ions to a data handler 140.

[0120] It will also be appreciated that the ion source 115 may have a variety of configurations known in the art.The present application is primarily directed to ionization sources that operate by ionizing a sample in droplet form, such as an electrospray process.

[0121] For the purposes of this application, the components of the mass analysis instrument 100 may be considered to operate as a single system. Conventionally, the combination of the mass analyzer 120 and the ion detector 125 together with the associated components of the controller 135 and the data handler 140 is often referred to as a mass spectrometer, and the sample separation / delivery device may be considered to be a separate component. However, it will be understood that although some of the components (such as the separation system 105) may be considered to be "separate", all of the components of the mass analysis instrument 100 operate in conjunction in order to analyze a given sample.

[0122] Figure 1B 1 is a block diagram illustrating an exemplary computing resource 130 on which embodiments of the present teachings may be implemented. The computing resource 130 may include a single computing device, or may include multiple distributed computing devices that are in operative communication with components of the mass analysis instrument 100. In this example, the computing resource 130 includes a bus 152 or other communication mechanism for transmitting information and at least one processing element 150 coupled to the bus 152 for processing information. As will be understood, the at least one processing element 150 may include multiple processing elements or cores, which may be packaged as a single processor or in a distributed arrangement. In addition, in some embodiments, multiple virtual processing elements 150 may be provided to provide control or management operations for the mass analysis instrument 100.

[0123] Computing resource 130 also includes volatile memory 150 coupled to bus 152 for use by at least one processing element 150, which may be a random access memory (RAM) or other dynamic memory component as illustrated. Computing resource 130 may also include static non-volatile memory 160, such as a read-only memory (ROM) or other static memory component as illustrated, coupled to bus 152 for storing information and instructions for use by at least one processing element 150. Storage component 165, such as a storage disk or storage memory, is provided and illustrated as coupled to bus 152 for storing information and instructions for use by at least one processing element 150. As will be appreciated, in some embodiments, storage component 165 may include a distributed storage component such as a networked disk or other storage resource available to computing resource 130.

[0124] Optionally, the computing resource 130 may be coupled via the bus 152 to a display 170 for displaying information to a computer user. An optional user input device 175, such as a keyboard, may be coupled to the bus 152 for communicating information and command selections to the at least one processing element 150. An optional graphical input device 180, such as a mouse, trackball, or cursor direction keys, is used to communicate graphical user interface information and command selections to the at least one processing element 150. As illustrated, the computing resource 130 may also include input / output (I / O) components 185, such as serial connections, digital connections, network connections, or other input / output components that enable intercommunication with other computing components and various components of the mass analysis instrument 100.

[0125] In various embodiments, the computing resource 130 can be connected to one or more other computer systems across a network to form a networked system. The network may include a private network or a public network such as the Internet. In a networked system, one or more computer systems can store data and supply data to other computer systems. In a cloud computing scenario, one or more computer systems that store and supply data can be referred to as servers or clouds. For example, one or more computer systems may include one or more network servers. For example, other computer systems that send data to a server or cloud and receive data from a server or cloud can be referred to as clients or cloud devices. Various operations of the mass analysis instrument 100 can be supported by the operation of a distributed computing system.

[0126] The computing resource 130 can be operated to control the operation of the components of the mass analyzer instrument 100 through the controller 135, and to handle the data generated by the components of the mass analyzer instrument 100 through the data handler 140. In some embodiments, the computing resource 130 is responsive to at least one processing element 150 executing the instructions contained in the memory 160 or 165 and performing operations on the data received from the mass analyzer instrument 100 to provide the analysis results. The at least one processing element 150 executes the instructions contained in the memory 155, 160, 165 so that the mass analyzer instrument 100 operates to perform the methods described herein. Alternatively, the present teachings can be implemented using hard-wired circuitry instead of software instructions or in combination with software instructions. Therefore, the implementation of the present teachings is not limited to any specific combination of hardware circuitry and software.

[0127] According to various embodiments, instructions configured to be executed by the processing element 150 to perform a method or to cause the mass analysis instrument 100 to operate to perform a method are stored on a non-transitory computer-readable medium accessible to the processing element 150 .

[0128] Based on different principles, devices and methods for acoustically distributed droplets interfaced to mass spectrometers for analytical measurement purposes have been developed. For example, US 9,664,647 B2 delivers acoustically generated droplets to the inlet of a mass spectrometer by atmospheric pressure gas, and evaporates them during transportation. U.S. patent application US 2017 / 0243729 A1 is another variation on this theme that applies voltage to the entire liquid sample. None of these methods have been proven to have the precision or accuracy to measure the volume of the distributed droplets. None of these disclosures provide all the performance indicators described in the previous section that are expected from a high-speed, non-contact method for transferring a sample to a mass spectrometer for analytical measurement purposes.

[0129] A preferred method of delivering droplets into a mass spectrometer ion source is by capturing each droplet in a flow stream of a delivery fluid passing through a conduit or tube. Due to the laminar flow characteristics of the capture and delivery device, which minimizes the diffusion and merging of droplets, each droplet remains separated from the rest.

[0130] Figure 2 An embodiment of the present invention is depicted that illustrates five major components, each of which has a different function. The first component is a sample dispensing device whereby the amount of sample delivered can be controlled. In this embodiment, sample delivery is achieved by imparting energy to the surface of a fluid sample through a burst of sound waves, thereby ejecting droplets of known and controllable volume. The amount of sample entering the processing area can be varied by varying the power, frequency, or duration of the sound wave pulses. The dilution of the sample in the processing area is varied by varying the injection volume. As described above, other sample delivery devices are contemplated.

[0131] The second component is a sample processing area where the sample is received and the concentration of the sample is adjusted to the capture probe that is optimal for electrospray ionization. In this embodiment, the sample processing component includes an OPP, which includes a fluid transfer pump for providing fluid to carry out sample processing and transportation. The flow of the conveying fluid in this area can be changed by pump change, thereby changing the dilution degree of the sample and the rate of transportation. The volume of the sample processing area can also be changed by changing its geometry, which will affect the amount of dilution that the sample will suffer. This is an effective way to increase or decrease the dilution ratio, but it will require replacement of physical parts or additional mechanical connections, which may not be easily applicable to quickly modify the dilution ratio online in real time.

[0132] The third component includes an ionization component that provides a facility for generating charged droplets from the processed sample, including a gas expansion region to form a pressure drop to attract the sample from the processing region to a charged droplet generation region where a high electric field is applied. Applying a high electric field to the charged droplets converts the discharged sample droplets into sample ions. Controlling this gas flow will enable the flow of liquid leaving the processing region to be varied, thereby providing an additional means for changing the degree of dilution in the processing region.

[0133] The fourth component is an atmospheric pressure ionization mass spectrometer, which is used to receive sample ions, filter the sample ions by m / z, and measure the amount of ions produced.

[0134] The system is supported by a computing element equipped with data and algorithms for interpreting the generated signals and control communication links with the sample dispensing device, the fluid delivery pump, and the pressurized gas source. After measuring the signals and determining the volume of the dispensed droplet based on the generated analysis results, the system can be operated to adjust one or more parameters of the sample dispensing device to dispense subsequent droplets of known volume for analysis by the fourth component, the mass spectrometer.

[0135] In some aspects, embodiments may further include a moving part for moving a sample well plate including a plurality of sample wells to position the expected sample wells to align with the sample processing area. In some embodiments, the time required for positioning the sample wells and acoustically emitting them into the processing chamber is about tens of milliseconds per sample. Individual samples may be stacked in a transfer line between the processing chamber and the ion generation point, where their temporal intervals are limited only by the diffusion of molecules in the solution in the conduit, typically about hundreds of milliseconds in the prototype of the present invention. This enables the sample to be emitted, its signal detected, compared with a reference to estimate suppression, and a certain amount of re-emission to provide appropriate dilution in the processing chamber to provide the correct conditions in the ion emission droplet for a linear analyte response and avoid suppression effects.

[0136] Figure 3 A. Figure 3 B and Figure 3 C depicts different geometric relationships between components. Figure 3 A shows a sample processing component oriented vertically upward and a charged droplet generation component oriented vertically downward. This allows the sample to be deposited in the processing component under the influence of gravity or other forces. Figure 3 B shows the same two compartments oriented in opposite vertical directions. Figure 3 C shows two compartments in a horizontal configuration. Any angle between vertical and horizontal can be used if the sample can be introduced into the processing region and the charged droplet generation components are oriented so that the ions can reach the entrance orifice of the mass spectrometer by some means.

[0137] If the microdispensing technology naturally operates by firing droplets in an upward direction against gravity, the sample handling area can be positioned with the fluid downward. The preferred mode of operation with this type of dispenser is to have the flow balanced, i.e., the inlet flow to the capture reservoir is equal to the outlet flow, so it does not overflow from the reservoir onto the sample. It can also operate with the inlet flow slightly less than the outlet flow.

[0138] For a micro-droplet dispenser that dispenses droplets downwardly in the general direction of gravity or at a certain angle, the sample processing area can face upward. In this case, the operating mode can be flow balanced, that is, the inflow is less than the outflow, or the inflow is greater than the outflow, so that the overflow is captured in a moat or drain.

[0139] The operating mode can be manually controlled by visually setting the flow rate while observing the condition of the reservoir. The mode can also be automatically controlled by monitoring the state of the reservoir with any of a variety of methods such as machine vision, light deflection, conductivity, etc. and feeding the information back to the transport fluid delivery pump.

[0140] Figure 4 A. Figure 4 B. Figure 4 C and Figure 4 D shows an additional embodiment of a capture probe presenting a similar sample processing region, with fluid inlet and outlet tubes that are not coaxially arranged. Figure 4 A and Figure 4 B shows a linear arrangement or a curved single tube or 2 tubes butted together with openings to allow sample entry into the processing region. In some aspects, a processing chamber can include a single tube with an orifice that exposes a processing fluid flowing through the tube. Figure 4 C shows the inlet and outlet pipes arranged parallel to each other. Figure 4 D shows two tubes arranged in a co-linear manner with a gap therebetween to define and thereby produce a sample processing area. In some aspects, a planar grooved surface can be provided to confine the fluid by coating the surface with a hydrophobic material. In some aspects, the processing area may not be defined by a wall and is limited only by the surface tension of the liquid deposited on the surface when it is transported between the two tubes. Other embodiments of the capture probe presenting the processing area are also contemplated, such as the use of two tubes that are close or far from each other and are enclosed in a processing chamber. The processing chamber in the form of an open groove has a supply pipe for supplying a processing fluid and a discharge pipe for discharging the processing fluid from the groove.

[0141] A preferred embodiment of a droplet capture / transport device is referred to herein as an open port probe (OPP), which involves an arrangement of two concentric coaxial tubes, the inner tube being a conduit for transporting droplets to the ionization region, the outer tube guiding and limiting the transport fluid (usually a solvent) delivered from the pump to the inlet of the inner conduit tube at the sample processing region in order to capture the sample, and transporting the captured sample to the ion source through the inner conduit tube. The sample processing region at the inlet of the inner conduit is filled with a fluid for capturing droplets deposited in the region. The diameter of the droplet capture reservoir is preferably in the range of 0.5 to 5 mm, but may be larger or smaller. The volume of the reservoir is preferably in the range of 0.2 to 2.0 uL, but may be larger or smaller. The transport fluid is supplied to the sample processing region by a pump through an external supply conduit, and the sample processing region is emptied through the internal transport conduit at approximately the same rate. This steady-state condition results in a permanent vortex located in the droplet capture reservoir, which rapidly mixes and sweeps away the incoming droplets. For the entire volume of the capture region to be purified, the flushing rate of the droplet capture reservoir in the sample processing region is preferably <1 second. Depending on the volumetric flow rate of the delivery fluid through the sample processing region, droplets dispensed into the reservoir in the low nanoliter range will be instantaneously diluted by a factor of 100-1000. This large dilution and efficient rapid mixing are important factors for normalizing the mass spectrometer signal for a variety of sample solvents and matrices, as well as for normalizing the mass spectrometer signal for a range of droplet volumes, both of which are important requirements to enable the generation of the standard curves described below.

[0142] At the distal end of the internal transport conduit is the ionization region inside the ion source. The atomizer tube surrounds the droplet transfer conduit in this region, and high-pressure gas (atomizing gas) is delivered to the nozzle orifice restriction around the outlet of the transport conduit through the atomizer tube. When the high-pressure gas (usually air or nitrogen) leaves the nozzle, it accelerates to a speed close to MACH 1. The shear force of the gas breaks the liquid into a plume of droplets. The expanded gas at the outlet also produces a local pressure drop at the end of the transfer conduit, thereby pulling the fluid from the sample processing area through the transport conduit. The speed of the fluid can be between 1-1000uL / minute, but preferably between 50-500uL / minute. The flow rate is preferably set to a constant rate determined by the pressure of the gas, the inner diameter of the transfer conduit, and the viscosity of the fluid. In order to balance the inlet and outlet fluid flows from the sample processing area, the pump that delivers the transport fluid is adjusted to match the flow rate generated by the suction of the fluid at the outlet at the distal end of the transport conduit.

[0143] Figure 5 and Figure 6 An alternative embodiment of delivering sample droplets to a mass spectrometer for analysis is illustrated. Figure 5A droplet dispenser is shown dispensing droplets onto a conveyor that delivers the dispensed droplets to an ionization source for ionization. Figure 6 Illustrated is a linear capture probe having a pinhole orifice sample processing region ("linear OPP") where dispensed droplets are received for capture and transport to an ionization source.

[0144] Figure 7 is a simplified cross-sectional view of an acoustic dispenser coupled to a sample well dispensing droplets into the sample processing region of a coaxial capture probe (OPP). Figure 8 is an alternative embodiment of a droplet dispenser that uses measured force to force a liquid sample through a small pinhole orifice.

[0145] Volume measurements are made based on mass using the calibration compound signal. With a reliable means of capturing, transporting, and ionizing solutes in droplets, the droplet volume can be determined based on the mass (amount) of the calibrant measured in each droplet by the mass spectrometer. This is done by preparing a calibration curve that relates the mass (amount) of the calibrant in the droplet to the signal from the mass spectrometer. Fig. 9 The mass signal (intensity) measured for multiple captured and analyzed samples including calibrants is shown. The peak area of ​​each measurement varies relative to the amount of calibrant contained in each sample. For example, by plotting the signal peak area against the known calibrant concentration of the sample, the peak area from Fig. 9 The mass measurement data are converted into a calibration curve ( Fig.10 ).

[0146] The volume of the droplets used to generate the standard curve must be known and within the approximate range of the size of the droplets to be measured. For example, if the droplet volumes to be measured are in the range of 1-100 nL, a calibration curve droplet volume of 5 nL is sufficient. The mass spectrometer measures the mass of the calibrant in the droplet, regardless of its volume and overall solvent composition. This measurement is greatly aided by the large dilution factor in the OPP capture reservoir, ensuring that slight variations in the signal due to droplet volume and composition are normalized. In the OPP droplet capture reservoir, phenomena such as suppression or enhancement of ionization efficiency due to overall sample composition are eliminated.

[0147] The volume of the droplet for constructing the calibration curve can be transmitted and determined in a variety of ways. For example, the droplet of acoustic distribution in the low nL volume range (1-10ug) can be weighed on an analytical balance to determine its volume. This can also be done using conventional spectroscopy. Other types of pre-calibrated micro-dispensers can also be used for this purpose, such as piezoelectric or pressure-driven devices. Once a single calibration curve is constructed, the single curve can be used to calibrate the acoustic distribution parameters for various sample compositions with different viscosities and for a series of different droplet distribution volumes. The droplet volume of each sample assigned for analytical measurement can also be checked, as long as the sample contains calibration compounds.

[0148] The calibration curve will be specific to a particular mass spectrometer because a mass spectrometer can have different signal levels for a single concentration of a calibration compound. The signal detected in the mass spectrometer can also vary over time as the ion optics accumulate contamination from the sample. Depending on the sample load, drift in the signal can occur over the course of a day. This will not affect the ability of the calibrated acoustic parameters to deliver accurate and precise volumes using parameters developed for a specific sample composition, but will have an impact on the real-time measurement of the volume of each sample dispensed because the calibration curve will change. The purpose of real-time volume measurement is that if the sample viscosity changes in an unpredictable way from sample to sample, the dispensed volume will vary under a single set of acoustic dispensing parameters. In the worst case, some samples will not even dispense under those acoustic parameters. This will be detected and noted because of the no-signal condition, but nothing can be done to correct it at this time.

[0149] Volume measurement is performed based on mass using a master standard curve, which is a ratio of calibration compounds to reference compounds. The construction of a master standard curve is a preferred embodiment because it is a correction for the signal drift of the mass spectrometer for the use of different mass spectrometers, and enables real-time volume measurement (hereinafter referred to as instant volume measurement). Real-time volume measurement not only corrects for slight errors when dispensing droplets each time, but also enables instant calibration. If a sample cannot be dispensed because its fluid properties are beyond the calibration parameter range, recalibration can be performed quickly to establish the correct conditions to emit droplets with the correct volume.

[0150] The standard curve prepared using the ratio of calibration compound to reference compound is called the master standard curve. It is prepared in a similar manner to the curve using calibration compound of known concentration in the sample to be dispensed. As described above, the exact volume of calibration compound must be dispensed roughly within the large range of the final target volume that the dispensing parameters will be adjusted to achieve. The difference is that the reference compound of known concentration is added to the transport fluid of the capture probe. The reference compound can be added to the transport fluid in the reservoir supplying the capture probe, or injected as a supplement to the supply of transport fluid supplied to the sample processing area. The relationship to be established is the ratio of the calibration compound signal divided by the reference compound signal [C / R] and the mass of the calibration compound dispensed (see Fig.11 ). The ideal situation is that the calibration compound and the reference compound are stable isotope variants of each other. This corrects for any variability that may be introduced due to differences in their chemical properties (see Fig.12 ).

[0151] The master standard curve is so represented because it only needs to be prepared once. When constructed with stable isotope variants of calibration compounds and reference compounds, it is applicable to mass spectrometers at different stages of performance degradation during the entire operating life of the mass spectrometer and to all mass spectrometers of different types. This single master standard curve is applicable to all solvent combinations and different sample compositions and the change of droplet volume when performing analytical measurements. One reason for doing so is, for an embodiment employing a capture probe, the normalization effect of the ionization process caused by the large dilution volume of the capture probe (e.g., OPP) mentioned above. Another reason is that the ratio of the signal from the calibrant and the reference is used to measure volume, isotope variant or other.

[0152] Since the master standard curve does not have to be reconstructed sample by sample, it is suitable for instant volume measurement that allows deviations in sample distribution volume to be considered when calculating sample concentration. The ratio of the calibration compound to the reference compound can be used to determine the volume very quickly (usually tens of milliseconds) from the master standard curve. It also enables instant calibration. When encountering a sample of unknown composition, the volume distributed using an existing, suboptimal calibration file can be quickly measured without resorting to the generation of a new calibration file. If the distributed volume is outside the target range of the acoustic parameter, the power and duration are usually adjusted and the volume is remeasured. Repeated volume measurements and parameter adjustments are made to individual droplets until the target volume is achieved. If no droplets are distributed at all because the acoustic parameters are incorrect for the sample, a lack of signal will be detected and it is possible to recalibrate quickly at this time. The process can be very fast, with multiple iterations of measurement and adjustment per second and is automatic. The time it takes will depend on how many parameter adjustment iterations are needed to achieve the target volume. In most cases, this will take less than 1 second. Very few samples are required. It is easy to achieve calibration files associated with very specific depths at very tight intervals.

[0153] When it is necessary to analyze and measure samples, it is common that the fluid properties of the samples can be changed by situation. For example, blood from patients experiencing different physiological states will have different hematocrits and widely different sample viscosities. Human urine from different disease states shows similar variability, and urine from different species will represent extreme values ​​on the viscosity scale. Fermentation media and bioreactors for the production of valuable pharmaceuticals and industrial chemicals will have different concentrations of solutes (such as polysaccharides) that have a profound effect on fluid properties at different time periods in the culture medium life cycle.

[0154] For the reasons already described, a master standard curve can be prepared from a single solvent. Ideal microdispensing solvents can be used as standards to increase the practicality and practicability of this method. An ideal solvent is dimethyl sulfoxide (DMSO), which has sufficiently high viscosity and surface tension to produce highly stable droplets in the picoliter to nanoliter range. It has a high vapor pressure, thereby minimizing errors due to evaporation. Stable and reproducible test solutions and protocols are essential for verification and validation of instruments and methods.

[0155] The reason why this single master standard curve is unknown for sample type, sample distribution volume, mass spectrometer and mass spectrometer situation is twofold.The first reason is because the change of ionization efficiency and mass spectrometer response caused by the change under these situations is considered by using the response ratio of calibration compound and reference compound.When situation changes, these two are equally affected.When calibration compound and reference compound are stable isotope variants of each other, all chemical effects are eliminated because they are only different in their nuclear properties, and are not different in electronic / chemical properties.In practice, because consumption is large, the most abundant isotope will be used as the reference compound in the transport fluid.The least abundant isotope will be the calibrant in the sample, because a very small amount of this more expensive reagent will be used.

[0156] The second reason is because of the nature of the capture probe and mass spectrometer interface. For example, acoustically partitioned samples are in the low nanoliter range. Capture probes such as OPP instantaneously mix 100 to 500 times the transport fluid as they capture it. For all practical purposes, it is the transport fluid that is being delivered to the electrospray ionization region, regardless of the composition of the sample or the droplet volume in the typical range.

[0157] Fig.13 An alternative standard curve example derived by introducing samples with the same concentration (eg, 5 uM) and varying the total dispense volume (1-100 nL) is illustrated. Higher dispense volumes can be achieved, for example, by high frequency multiple dispenses as described above.

[0158] The embodiments of the present application can be usefully used in novel applications of ADE-OPP such as fermentation analysis and kinetic analysis. Below, the description of kinetic analysis is described.

[0159] The device and method inventions described herein are directed to systems comprising several components designed to perform in situ kinetic measurements, including measurements taken when a sample solution reaches static equilibrium and no longer changes with time.

[0160] Understanding the chemical properties of solutions and how they change over time is a fundamental object in the fields of chemistry, biochemistry, and clinical chemistry. Methods and devices have been developed for monitoring the chemical composition of solutions as it changes over time in a controlled manner and as it remains static and in terminal equilibrium. Reference is made to the method enabled by the present invention, "In Situ Kinetics".

[0161] In one embodiment, a chemical or biochemical reaction can be monitored over time intervals by, for example, dispensing discrete nanoliter volume droplets from at least one reaction well at regular sampling intervals. In this embodiment, a reaction occurs within a reaction well, and as the reaction continues, the product within the well is sampled. This is in contrast to the conventional method of preparing a number of reaction wells, each representing a different measurement time point, and analyzing the series of wells after quenching the reaction in each well at an appropriate time. This approach multiplies the number of sample wells to be prepared, thereby increasing the cost of expensive reagents and consumables, limiting the frequency and duration of time point measurements, and increasing the human element of the experiment due to the quenching process. Fig.14 This difference is illustrated in the figure. Fig.15 Data from in situ kinetic studies are shown in Fig.15 Kinetic data from human liver microsomal analysis are shown, illustrating the disappearance of two drugs due to metabolic consumption. Reactions were performed in the presence and absence of NADPH, an essential cofactor for cytochrome P-450 enzymes.

[0162] In order to fully realize this new in situ kinetic method, several conditions must be met. The apparatus and method we have developed meet all of these criteria. It involves using a mass spectrometer as a detector, which has a sample inlet interface for the mass spectrometer called an open port probe (OPP) described previously in the calibration section of this document. The sample is dispensed into the open port probe using acoustic sound waves also described previously in the calibration section of this document. The open port probe captures droplets dispensed from a well or test tube, and internal standards can be added to the transport fluid of the OPP to normalize the signal, which is also described in the calibration section of this document.

[0163] The conditions that must be met and how they are achieved are as follows.

[0164] 1. The dispensed volume must be small relative to the overall reaction to avoid depleting the reaction or altering the kinetics due to volume / diffusion rate change phenomena. With this approach, the dispensed volume is in the low nL range, typically 1-20 nL, while the reaction volume is in the μL range, typically tens to hundreds of μL.

[0165] As described in the calibration section of this document, physical parameters are established to distribute the sound waves within this volume.

[0166] 2. For each consecutive time measurement, the dispensing should be contactless in order not to be affected by the previous measurement due to cross contamination of the sample. The device and the method it enables perform dispensing in a contactless manner, resulting in a zero sample carryover situation.

[0167] Launching droplets with acoustic energy avoids the use of physical surfaces such as needles or pipettes to retrieve the sample. Surfaces are always a problematic source of carryover. Droplets are caught in a fluid vortex at the entrance of an open-port probe and are immediately swept away. Figure 5 The flushing vortex is shown in . The fluid selected is one in which the sample is highly miscible and will not precipitate out of solution. The sample never contacts a solid surface.

[0168] 3. Sample dispensing must be fast and can be dispensed at any time close to the start of the reaction or at any time interval thereafter. The device and the method it enables can dispense discrete samples at a rate greater than 1 Hz, typically in the range of 0.1-10 Hz, and a wide range of 0.01 to 100 Hz can be used. Fig.16 is data from a sample running at about 0.6 Hz.

[0169] Acoustic sound waves can dispense droplets at frequencies up to 1kHz, but transferring these droplets as discrete entities to a mass spectrometer is problematic. When they are fired into an open-port probe, they are stacked in a transfer line that delivers them in a transport fluid to the mass spectrometer. Diffusion phenomena limit how tightly these droplets can be stacked in space and time. The flow is limited to roughly 3 per second before they begin to merge.

[0170] 4. Continuous sampling where the measurement is uninterrupted for the duration of the reaction is a desired option. A continuous uninterrupted flow of sample must be possible even when sampling at high frequencies where information may be lost. The present invention will dispense a continuous flow of sample for a short period of time or as long as there is sample left in the well.

[0171] To achieve an uninterrupted, continuous signal, droplets are fired into the open port probe at a rate at which they merge in the vortex. This occurs at a frequency of approximately 5 Hz or higher. The steady-state signal is the result of a period of time as short or long as is required to continuously monitor the reaction kinetics. See Fig.16 , Fig.16 The continuous mass spectrometer signal is shown, which is caused by the droplet being dispensed at 30Hz, resulting in the droplet merging in the transport conduit of the capture probe. This technique is also useful for many other purposes such as the adjustment of mass spectrometer lenses, mass analyzers, or using data acquisition methods that benefit from having a longer time period. Such examples are signal averaging to increase the signal-to-noise ratio and accurate mass measurements that benefit from better ion statistics achieved with longer integration times.

[0172] When the time of sample introduction can be increased to any desired value, data with differential ion mobility compensation voltage values ​​for each compound in the sample will be obtained. Data acquisition methods such as SWATH obtain product ion spectra of each precursor ion present in the sample. Through the present invention, a huge amount of information about the precise chemical composition of the sample is obtained, and the sample introduction time can be adjusted to be long enough to achieve a good signal-to-noise ratio for all components of the sample. Combining differential ion mobility scanning with SWATH will provide deep chemical coverage of the sample by combining orthogonal separation technology. However, these powerful technologies can only be realized when the sampling time can be automatically adjusted and controlled. With this acoustic injection method, the sampling time can be automatically adjusted for each individual sample by measuring S / N in real time when the signal averaging gradually improves S / N, and stopping the acoustic distribution when the target value is achieved.

[0173] 5. The reaction should be dispensed in its original form, without the measurement tool requiring purification or modification of components in any way. For example, direct analysis of biological fluids, enzymes, cofactors, buffers, and surfactants without pre-purification is necessary to preserve the integrity of the original reaction in the medium. Likewise, the requirement to modify physiological buffer conditions to make them compatible with the measurement tool is to be avoided, as it is likely that the results will no longer represent a living system.

[0174] Due to ionization suppression, there is currently no way to perform direct analysis of complex biological samples without some degree of sample purification before the sample is introduced into the mass spectrometer. Ionization suppression is the result of the physical-chemical processes involved in the ionization event. When there are very high concentrations of endogenous materials, ionization by electrospray is suppressed because the surface of the charged droplets containing the analyte is shielded by an impermeable layer, thereby preventing field emission of the analyte from the droplets. Similarly, when there are very high concentrations of endogenous materials, a phenomenon known as reagent ion depletion suppresses ionization by atmospheric pressure chemical ionization. Endogenous materials with high gas phase acidity and basicity will consume all the charge present in the reagent ion population, leaving no charge for the analyte that is present in trace amounts compared to the bulk solution components.

[0175] Fig.17 The data in are examples that demonstrate that no signal suppression occurs when plasma is injected directly into the capture probe. As shown, the analyte signal is similar despite the ion suppression and subsequent signal loss that would normally occur with the plasma matrix. Fig.18The data in is an example that demonstrates that no signal suppression occurs when a high throughput screening assay containing Tween 20 surfactant is injected directly into a mass spectrometer. As shown, for each group, the left column without detergent has similar peak areas as the right column with detergent (Tween 20).

[0176] The device and the methods it enables eliminate the need to purify the sample prior to injection into a mass spectrometer by the following means. Fig.19 A 2.5 nL droplet dispensed into the vortex inlet of an open port probe is shown. The vortex volume is approximately 1 uL, which results in an instantaneous 400-fold dilution, which is then immediately transferred to the ionization region. The dilution of endogenous materials counteracts their ionization suppressing effect.

[0177] 6. Adding compounds that are not relevant to the requirements of the reaction for the purpose of enabling or improving the measurement must be avoided so as not to affect the results. An example is the addition of an internal reference standard to the reaction to improve measurement accuracy and precision. This runs the risk of interfering with the activity of the enzyme or catalyst of interest.

[0178] A second example is the use of light absorbing or emitting labels associated with reaction components to provide a secondary indirect means of monitoring the progress of a reaction. These also run the risk of confounding the results because they are indirect measurements and the labels are often found to alter the activity of key components of the biological system such as enzymes, transporters or receptors.

[0179] The present invention directly measures the amount of a component of interest by mass spectrometry without the use of labels. Fig. 20 The data in and the corresponding calibration curves demonstrate the inherently quantitative nature of this approach. Different amounts of sample injected into the capture probe sample processing region result in proportionally different signals from which a calibration curve can be constructed.

[0180] This option can be used to normalize the signal from the sample using an internal standard without adding an internal standard to the sample (where the internal standard may interfere with the result). The internal standard is added to the transport fluid of the OPP interface. Initially, the response ratio between the internal standard and the target analyte is measured and recorded. This relationship is then used to determine the amount of the analyte in the sample where the concentration of the analyte is unknown. By pre-establishing a calibration curve of mass spectrometry signal and analyte concentration and then reading the concentration of the unknown sample from the graph, the amount of the analyte can be determined without an internal standard in the transport fluid.

[0181] The device and the methods it enables enable enzymatic or catalytic reactions commonly encountered in biology or chemistry to be measured without modification. For example, high throughput screening assays for known biological targets, enzymes, receptors, based on living cells or otherwise are examples of direct analysis of plasma or other biological fluids. Measurements do not require the addition of internal standards to the reaction, and measurements are made directly on reactants and / or products without reliance on secondary indirect detection such as encountered when using, for example, fluorophores.

[0182] 7. Reaction conditions must be kept consistent, which means maintaining control over the critical parameter of temperature. Temperature control is often used, especially when only careful kinetic measurements are being made. Each sample well must be maintained at exactly the same temperature as the next sample well to generate meaningful data.

[0183] Methods to transfer heat uniformly to all wells (e.g., microtiter plates) have been used that heat the entire bottom or top of the plate to transfer heat uniformly. In the case of acoustic distribution of wells to OPPs, both the top and bottom areas of the plate are occupied by the acoustic transducer at the bottom and the OPP at the top. This makes the classical approach to solving this problem inappropriate.

[0184] In order to effectively couple the sound waves from the piezoelectric transducer to the bottom of the orifice plate, a liquid medium such as water is used to avoid air gaps. Current embodiments of acoustic distributors all flood the area between the lens of the transducer and the bottom of each individual hole with a small amount of coupling liquid. The elements of this device used to solve this problem are to flood the entire bottom of the orifice plate with the coupling fluid and control the temperature of the fluid. In this way, uniform temperature control, both heating and cooling, can be maintained evenly and consistently. Fig.11 This element of the invention is shown in FIG.

[0185] The ability to accurately and precisely control sample temperature with acoustic distribution has the benefit of transcending dynamic measurement. As described in the calibration section of this provisional patent application, the acoustic distribution parameters are specific to solutions with specific viscosity values. When viscosity changes, the distribution parameters also change. Keeping the temperature constant stabilizes the viscosity, and therefore helps to prevent the distribution parameters from drifting beyond the calibration.

[0186] Additionally, heating samples causes their viscosity to decrease. The maximum viscosity solution that can be acoustically dispensed is approximately 100 cp. The ability to effectively increase the temperature will increase the range of solution types that can be dispensed by reducing the viscosity of solutions whose viscosity is greatest when heated.

[0187] 8. The type of measurement performed on each well must remain flexible, unconstrained, and able to switch quickly from sample to sample. This is because in situ kinetics enables the analysis of a large number of different reactions while different reactants, products, and cofactors need to be monitored. The ability to analyze a variety of different compounds during the allocation of each well must be available. For example, the compound monitored for each well of a 96, 384, 1536, or 3456-well microtiter plate is changed because different analyses are performed in adjacent wells.

[0188] This approach meets this requirement by synchronizing the mass being monitored by the mass spectrometer with the well position. If the sample wells are judiciously ordered to monitor different mass channels in adjacent wells, this capability also increases the speed of analysis by allowing some adjacent peak overlap between sample wells. Fig.12 is an example of peak overlap occurring but there is no signal interference from adjacent samples because different masses are being monitored. The idea is similar to the "planned MRM" technique designed for chromatographic separations. In the present invention, the MS data acquisition method is not synchronized with the chromatographic retention times, but with the vial position. Using this approach, data from multiple samples, typically all samples in a microtiter plate, can be acquired in a single data file. In addition, faster switching speed methods (sub-second resolution) are needed.

Claims

1. A method for calibrating a droplet dispenser, the droplet dispenser dispensing sample droplets from a liquid sample, the method comprising: providing a liquid sample comprising a calibrant having a known calibrant concentration; dispensing a droplet of assumed droplet volume from a liquid sample; capturing the dispensed droplets using a transported liquid from a capture probe; Ionize a droplet of the assumed droplet volume; measuring the calibrant mass of the calibrant in the ionized droplets using a mass spectrometer; determining an actual droplet volume by comparing an expected calibrant mass determined based on an assumed droplet volume and a known calibrant concentration to the measured calibrant mass; determining that a difference between an actual droplet volume and an assumed droplet volume is greater than a threshold volume; as well as At least one drop dispenser parameter of the drop dispenser is adjusted based on at least one of a magnitude and a sign of the difference between the actual drop volume and the assumed drop volume.

2. The method according to claim 1, further comprising: Repeatedly dispensing a subsequent droplet of an assumed subsequent droplet volume from the liquid sample using the at least one droplet dispenser parameter previously adjusted, ionizing the subsequent droplet, measuring a subsequent calibrant mass of a calibrant in the ionized subsequent droplet, determining an actual subsequent droplet volume by comparing an expected subsequent calibrant mass determined based on the assumed subsequent droplet volume and a known calibrant concentration with the measured subsequent calibrant mass, and adjusting the at least one droplet dispenser parameter of the droplet dispenser until the actual subsequent droplet volume differs from the assumed subsequent droplet volume by less than a threshold volume.

3. The method according to any one of claims 1 to 2, further comprising: The dispensed droplets are transported together with a transport liquid to an ionization chamber of a mass spectrometer.

4. The method according to any one of claims 1 to 2, wherein: The droplet dispenser includes an acoustic droplet dispenser.

5. The method according to any one of claims 1 to 2, wherein: The capture probe includes a coaxial open-port probe OPP, which supplies a transport liquid to a sample processing region through an external conduit for capturing a sample, and receives and transports the captured sample and the transport liquid to an electrospray ionization source.

6. The method according to any one of claims 1 to 2, further comprising introducing a reference compound into the transport liquid.

7. A system for measuring the volume of a fluid dispensed by a fluid dispenser, the system comprising: a capture probe for receiving and capturing a dispensed volume of fluid from a fluid dispenser into a transport liquid, the dispensed volume of fluid comprising a calibrant, wherein a reference compound of known concentration is also introduced into the transport liquid of the capture probe; an ionization source for ionizing the captured volume of fluid; and A mass spectrometer is used to measure the mass of a calibrant contained in an ionized volume of fluid.

8. The system of claim 7, further operative to determine the volume of the measured fluid based on the mass of the calibrant.

9. A system according to claim 7 or claim 8, the system further operative to determine the volume of the measured fluid based on a ratio of the measured mass of the calibrant to the measured mass of the reference compound.

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