The invention relates to a particle detection device, comprising a droplet flow generator arranged to generate a flow of separated and subsequent flying liquid droplets at a droplet dispensing frequency, wherein each droplet may either contain zero solid particles or one or more solid particles, and a mass spectrometer which is arranged for detecting particles of at least one specific predetermined species.

When analyzing a stream of free flying liquid droplets for determining the nature of solid particles inside said droplets, such as in a single-cell MALDI mass spectrometer, there may be a need for droplet selection. Such a stream of droplets may be formed from an existing aerosol or from a (sample) liquid which is separated into droplets.

In a single-cell MALDI mass spectrometer, such as described in WO 2010/021548, cells may be separated from each other by suspending them in a liquid containing all the additives required by the MALDI process and dispensing this liquid in the form of very’ small (30 pl to 100 pl) ‘free- flying’ droplets. Depending on the details in the dispensing technique, the initial velocity of these droplets is one to a few meters per second. To prepare the cells for the MALDI process these droplets are allowed to evaporate in such a way that proteins are extracted from the cell and are co- crystalized with the MALDI matrix. Once the volatile part of the droplets has evaporated, particles remain comprising of (remains of) the and MALDI-matrix cry stals. After transporting these particles into the mass-spectrometer, the particles are individually illuminated by means a pulsed UV laser to ablate and ionize (part of) the MALDI matrix material.

The analysis rate of single-cell MALDI mass spectrometer, such as the BiosparQ® Cirrus® D20, may be limited by several processes and/or components:

1) The droplet evaporation capacity’. To evaporate all adherent fluid from the droplet, the droplet stream needs to be surrounded by a sufficiently large volume flow of air. Since this air is used to transport the particles into the mass spectrometer itself, this volume is limited by the capacity’ of the pumps that maintain the mass spectrometer vacuum. Given this limited volume flow, the number of droplets that can evaporate per unit of time is limited as well.

2) Since every’ particle is illuminated individually by the ionization laser, the number of particles that can be analyzed is limited by the maximum firing frequency of the ionization laser.

3) In case delayed extraction is used to improve the mass focusing of the mass spectrometer, the (owing to the finite recovery’ time of the high-voltage switch assembly) finite switching capacity-’ of the high-voltage electronics limits the number of single -particle spectra that is produced per unit of time.

In some clinically important cases tire concentration of bacteria in certain clinical samples may be extremely low (e.g. 1 CFU/ml to 10 CFU/ml may be clinically relevant for whole blood samples). Clearly, when the cell-analysis rate of the single-cell MALDI mass spectrometer is not extremely high, the time required to process a sample volume sufficiently large obtain a statistically reliable result (e.g. > 10 ml for whole blood) may become impractical, since the number of droplets that need to be processed is extremely large (10 ^A 8 if no pre-concentration is applied). At the same time, the fraction of relevant particles (particles that contain (remains of) a cell) is extremely low. Hence, a large part of the capacity of the instrument is spent on irrelevant particles.

To that end the particle detection device may comprise a droplet separation device, comprising a droplet separator which is arranged to separate said flow of droplets into droplets considered to have zero solid particles and droplets considered to have at least one solid particle, wherein the droplet separator of the droplet flow separation device is arranged to direct the droplets considered to have at least one solid particle to an inlet of the mass spectrometer and to direct droplets considered to have zero particles away from said inlet. A mean frequency of droplets that are directed to the inlet of the mass spectrometer is, or results in, a variable droplet frequency dependent process parameter of said mass spectrometer.

The consequence of droplet selection is that the number of droplets that is selected for analysis varies with the concentration of cells in the suspension. In clinical samples this concentration may vary strongly, e.g. for urinary’ tract infections the concentration of viable cells may vary from 10 ^A 3 CFU/ml to more thanl0 ^A 5 CFU/ml.

Thus the number of droplets that are passed to the drying section of the inlet varies as well. This in turn results in a vary ing ‘solvent-vapor load’ for the dry ing section.

As described in WO 2018/211112 1 (MALDI Mass Spectrometry’ method), the process parameters, such as the temperature and the (partial) vapor pressure of the solvent components in the droplet surrounding gas, required to obtain properly crystalized MALDI matrices and sufficient extraction of proteins form cells are bound by certain limits. The consequence of large variations in the ‘solvent-vapor load’ is that the process parameters required for the proper operation of the drying section do not suffice anymore at certain cell concentrations.

The invention aims to address the above problem.

To that end, according to the invention the value of the droplet dispensing frequency of the droplet flow generator is changeable and the device further comprises a system control unit which is arranged to set the value of the droplet dispensing frequency in dependence of said droplet frequency dependent process parameter of said mass spectrometer. To accommodate for the consequences of the vary ing particle concentration of the sample, the dispensing frequency may thus be adjusted according to reciprocal value of the particle concentration.

Preferably the system control unit is arranged to determine the mean frequency of droplets which are directed to the inlet of the mass spectrometer and set the value of the droplet dispensing frequency such that said mean frequency of droplets which are directed to the inlet of the mass spectrometer is equal to a predetermined target frequency.

Preferably the system control unit is connected to the droplet separator and determines the mean frequency of droplets that are directed by said droplet separator to the inlet of the mass spectrometer from signals obtained form said droplet separator.

In another preferred embodiment the system control unit is connected to the mass spectrometer and is arranged to set the value of the droplet dispensing frequency in dependence of the droplet frequency dependent process parameter which is derived from signals obtained from said mass spectrometer.

Preferably said droplet separation device comprises a light source arranged to cast light on each droplet in said flow; an image capturing device arranged to capture a digital optical image of each droplet in said flow while said droplet is being lit by said light source; and an image analyzer which is coupled to said image capturing device and which is arranged to analyze each captured image based on pixel values of the captured digital optical image and to output a signal which represents whether a captured digital optical image is at least one of an image of a droplet considered to have zero solid particles or an image of a droplet considered to have at least one solid particle. Preferably, the light source is arranged to cast monochromatic light on a first side of each droplet in said flow, such that a light interference pattern is generated by diffraction of the monochromatic light passing through said droplet; wherein the image capturing device is arranged to capture a digital optical image of said interference pattern of said droplet at a second side of each droplet in said flow while said droplet is being lit by said light source, said second side being opposite to said first side; and wherein the image analyzer is arranged to analyze each captured image based on pixel values of the captured digital optical image of the interference pattern. Preferably the light source is a laser light source. Preferably , for each captured image, said image analyzer subtracts stored pixel values of either a stored reference image of a droplet having zero solid particles or of a preceding captured image of an interference pattern of a preceding droplet, from corresponding pixel values of each captured image before analyzing said each captured image based on the resulting pixel values. Preferably, for each captured image, said image analyzer aligns and/or resizes the captured image where necessary’ to match the stored image or the preceding captured image, preferably based on the interference patterns in said images, before subtracting said pixel values from the corresponding pixel values in the so aligned and/or resized image. Preferably, for each captured image, after said subtraction said image analyzer determines the sum of the resulting pixel values of all pixels in the captured image and compares said sum to a threshold value, and determines to output said signal based on said comparison.

Preferably said droplet flow generator comprises a droplet dispenser arranged to generate said flow of droplets from a liquid sample. Preferably said droplet dispenser comprises a drop-on-demand unit, such as a piezoelectric DOD or thermal DOD unit. Preferably said drop-on-demand unit is arranged to receive a replaceable drop-on-demand cartridge having a container for holding a liquid sample and a single nozzle for dispensing said droplets.

Preferably said droplet dispenser is connected to at least one of said light source and said image capturing device so as to timely trigger at least one of pulses of said light source or shutter openings of said image capturing device in order to capture the images of the droplets in said generated flow of droplets.

Preferably said image analyzer is further arranged to analyze said each captured image based on pixel values of the captured digital optical image of the interference pattern such that said output signal represents whether a captured digital optical image is at least one of an image of a droplet considered to have zero or more than one solid particles or an image of a droplet considered to have exactly one and not more than one particle, and said droplet separator is arranged to separate said flow of droplets based on said output signals into droplets considered to have zero or more than one solid particles and droplets considered to have exactly one and not more than one solid particle. Preferably said solid particles are biological particles, in particular single-cell particles such as bacteria or amoebae, or viruses. Preferably said liquid comprises a solvent such as water and/or ethanol and preferably said liquid comprises a MALDI matrix material.

The invention furthermore relates to a method for detecting particles of at least one specific predetermined species, said method comprising: generating a flow of separated and subsequent flying liquid droplets, wherein each droplet may either contain zero solid particles or one or more solid particles; wherein, in said flow of flying liquid droplets, liquid droplets comprising zero solid particles are separated from liquid droplets comprising at least one solid particle, and wherein the droplets considered to have at least one solid particle are directed to an inlet of a mass spectrometer, in particular a MALDI mass spectrometer, which is arranged for detecting particles of at least one specific predetermined species and wherein droplets considered to have zero particles are directed away from said inlet, wherein a mean frequency of droplets that are directed to the inlet of the mass spectrometer is, or results in, a variable droplet frequency dependent process parameter of said mass spectrometer; and wherein the value of the droplet dispensing frequency of the droplet flow generator is changeable and the value of the droplet dispensing frequency is set in dependence of said droplet frequency dependent process parameter of said mass spectrometer.

Preferably said method further comprises casting light on each droplet in said flow; capturing a digital optical image of said droplet; analyzing each captured image based on pixel values of the captured digital optical image and generating a signal which represents whether a captured digital optical image is at least one of an image of a droplet considered to have zero solid particles or an image of a droplet considered to have at least one solid particle; and separating said flow of droplets based on said output signals into droplets considered to have zero solid particles and droplets considered to have at least one solid particle.

Preferably casting the light on each droplet in said flow comprises casting monochromatic light on a first side of each droplet in said flow, such that a light interference pattern is generated by diffraction of the monochromatic light passing through said droplet; wherein capturing the digital optical image comprises capturing a digital optical image of said interference pattern of said droplet at a second side of each droplet in said flow while said droplet is being lit by said light source, said second side being opposite to said first side; and wherein analyzing each captured image based on pixel values of the captured digital optical image comprises analyzing each captured image based on pixel values of the captured digital optical image of said interference pattern.

Preferably analyzing each captured image based on pixel values of the captured digital optical image comprises subtracting stored pixel values of either a stored reference image of an interference pattern of a droplet having zero solid particles or of a preceding captured image of an interference pattern of a preceding droplet, from corresponding pixel values of each captured image before analyzing said each captured image based on the resulting pixel values.

These and other aspects of the invention will be further elucidated with reference to the figures, which are purely diagrammatical and not drawn to scale, wherein:

Fig. 1 shows a schematic representation of a droplet separation device;

Fig. 2a and 2b show captured images of droplets using a general light source;

Fig. 3 a and 3b show captured images of droplets using a laser light source and

Fig. 4 shows a schematic representation of an apparatus for MALDI mass spectrometry’ with the droplet separation device of Fig. 1.

According to Fig. 1 a droplet separation device comprises a droplet flow generator in the form of a piezoelectric or thermal drop-on-demand (DOD) cartridge 1. Preferably the cartridge 1 is a releasable/replaceable cartridge. Piezoelectric or thermal drop-on-demand (DOD) technology’ as such is well known, for instance in the form inkjet or bubble jet printing technology-’. The cartridge 1 is filled with a liquid sample, such as a sample prepared from a whole blood, sputum or saliva sample, which may contain solid particles, such as bacteria, to be determined. For applying MALDI MS the cells of the sample are dispersed in a clean liquid. The original fluid phase (including dissolved interferents such as salts) is removed from the whole blood, sputum or saliva sample and is replaced by a MALDI solution. When triggered by a system control unit the DOD cartridge 1 generates and exhausts a flow of droplets 10a, 10b from the liquid sample at a fixed frequency.

Downstream from the cartridge 1 the droplets 10a, 10b pass a droplet inspection stage comprising a laser source 2 at one side and an image capturing device 3 at the opposite side of the stream of droplets 10a, 10b, which may be CCD or CMOS camera chip. The laser source 2 is arranged to cast a beam of monochromatic light having a single wavelength on the droplets 10a, 10b, and the image capturing device 3, which may comprise optical lens(es) 31, is arranged to capture an images of the resulting interference pattern from the other side of the droplets 10a, 10b. The shutter of the image capturing device 3 is triggered by the system control unit at the same frequency as the droplet generation, with an appropriate delay caused by trigger signal delay unit 4a, or in the alternative the laser source is triggered by the system control unit to flash the light beam at the same frequency as the droplet generation, with an appropriate delay caused by trigger signal delay unit 4b.

When either the light source 2 or the image capturing device 3 is triggered with a suitable delay 4a, 4b, an image can be produced that characterizes the droplet, as shown in Figs. 3a, 3b. Since modem microfluidics are capable of dispensing highly identical droplets and since droplets of a sufficiently small size have a perfect spherical shape, the images created from identical droplet match to a high degree, provided that the timing of either the camera or the light source is sufficiently accurate. Thus, it appears that images produces from droplets that contain particles (such as cells), differ from those that do not. Analyzing this difference with a microprocessor based image processing unit 5, cell containing particles can be detected and a proper trigger signal can be send to a droplet manipulating device 6 that separates the stream of droplets into a stream of relevant droplets 10a and irrelevant droplets 10b.

An example of the resulting image of a droplet 10b without a solid particle is shown in Fig. 3a, and an example of the resulting image of a droplet 10a with solid particles is shown in Fig. 3b. For comparison, corresponding images of respective droplets 10b, 10a which are captured by the image capturing device 3 while using a white light source having a multitude of wavelengths, instead of the laser source 2, are shown in Fig. 2a and Fig. 2b.

With reference to Fig. 2b, which is an image of a droplet 10a with two solid particles as indicated by arrows, when using a white light source it is observed that whether or not the solid particles appear (clearly) in the image depends on the location of the particles within the droplet 10a. At some locations they may appear clearly and at other locations they may not appear clearly, or not at all. Thereby it may be difficult or impossible to determine the difference between the image of Fig. 2a and the image of Fig. 2b. On the contrary’, with reference to Fig. 3b, when a laser source 2 is used it is observed that the droplet 10a and the solid particles therein produce clearly recognizable interference patterns of the laser light in the image, regardless of the location of the solid particles within the droplet 10a, and which recognizably differ from the interference patter in an image of a droplet 10b which does not contain solid particles, as shown in Fig. 3a.

The droplet separation device comprises a droplet selector control unit 5. The unit 5 comprises memory means designated as reference image storage 52, in which an image of the interference pattern of a droplet without solid particles, as shown in Fig. 3a, is stored. For applications where the vast majority of droplets do not contain solid particles, also the (temporarily) stored image of any preceding droplet (for instance the immediately preceding captured image) may serve as reference image stored in reference image storage 5 , as this is very likely an image of a droplet without a solid particle. The unit 5 further comprises memory’ means designated as captured image storage 51, in which the subsequent images captured by the image capturing device are temporarily stored in order to be compared in real time, using image processing technology’, to the reference image stored in reference image storage 52 by a processor, which forms an image comparison and trigger signal generation unit 53.

An example of a simple and effective algorithm for generating a trigger signal by the unit 53 is: calculate reduced pixel values for every’ pixel in the stored captured image by subtracting from every’ pixel (brightness) value of the stored captured image the corresponding pixel (brightness) value of the stored reference image (as shown in Fig. 3a); calculate the sum of the calculated reduced pixel values of all the pixels of the image; produce a trigger signal in dependence on whether or not the calculated sum is higher than a predetermined threshold value.

If the calculated sum of the calculated reduced pixel values of all the pixels of the image is higher than said predetermined threshold value the droplet in the image is considered to contain one or more solid particles, if the calculated sum is equal to or lower than said predetermined threshold value the droplet in the image is considered not to contain a solid particle.

This algorithm may also be effective if the captured images of the droplets are not generated by using an interference pattern of monochromatic light.

If required, for instance depending on the purpose of the droplet separation, a more complex image processing algorithm may be used, for instance to determine the number of solid particles in the droplet. Alternative methods to detect particles in droplets are for instance evaluating the symmetiy of the image intensity with respect to the droplet center or more elaborate image processing techniques.

In order to separate the flow of droplets, the trigger signal produced by the unit 53 is input to a droplet manipulator 6, which is arranged to either change the flow path of droplets 10a which are considered to contain solid particles or (as shown in Fig. 1) to change the flow path of droplets 10b which are considered to not contain solid particles, or both. As shown in Fig. 4, the droplet manipulator 6 may change the path of the droplets 10b considered not to have solid particles, for instance by using a mechanical shutter, such that they are guided towards a waste liquid container 9.

Alternative means for deflecting droplets may include dielectrophoretic charge, magnetic force (for magnetic particles), acoustic excitation or pneumatic excitation. In order to separate the flow of droplets, as an alternative to changing the path of unwanted droplets to a waste location, also annihilation of the unwanted drople ts may be applied, for instance by using laser ablation.

Fig. 4 shows an apparatus for MALDI mass spectrometry with the droplet separation device 1, 2, 3, 4, 5, 6, wherein the droplets 10a which are considered to contain at least one (biological) solid particle, such as a bacterial particle, flow to the mass spectrometer inlet 81 of the mass spectrometer 8, and droplets 10b considered not to have solid particles are guided towards the waste liquid container 9. Thereby the ratio of relevant particles to irrelevant particles is increased. This increases the cell-analysis rate, and subsequently decreases the time to result for a complete microbial analysis.

For each droplet 10a, created by droplet dispenser 1, that is directed towards the mass spectrometer inlet 81 by the droplet manipulator 6, a pulse is sent to the system control unit 7. The system control unit 7 analyses the mean frequency of these pulses in analyzing unit 7a, compares the mean frequency with a predetermined target frequency 7c and adjusts the frequency of a pulse generator 7b that drives the droplet dispenser 1 such that said mean frequency of droplets 10a which are directed to the inlet of the mass spectrometer is equal to a predetermined target frequency 7c.

As described in WO 2018/211112 1 (MALDI Mass Spectrometry method), the process parameters, such as the temperature and the (partial) vapor pressure of the solvent components in the droplet surrounding gas, required to obtain properly crystalized MALDI matrices and sufficient extraction of proteins form cells are bound by certain limits. Therefore, alternatively or additionally the system control unit 7 may be connected to the mass spectrometer 8 and set the value of the droplet dispensing frequency of the pulse generator 7b in dependence of one or more process parameters obtained directly from said mass spectrometer 8.

The invention has thus been described by means of preferred embodiments. It is to be understood, however, that this disclosure is merely illustrative. Various details of the structure and function were presented, but changes made therein, to the full extent extended by the general meaning of the terms in which the appended claims are expressed, are understood to be within the principle of the present invention. The description and drawings shall be used to interpret the claims. The claims should not be interpreted as meaning that the extent of the protection sought is to be understood as that defined by the strict, literal meaning of the wording used in the claims, the description and drawings being employed only for the purpose of resolving an ambiguity’ found in the claims. For the purpose of determining the extent of protection sought by the claims, due account shall be taken of any element which is equivalent to an element specified therein. An element is to be considered equivalent to an element specified in the claims at least if said element performs substantially the same function in substantially the same way to yield substantially the same result as the element specified in the claims.

Index of reference numerals:

1. Droplet dispenser

2. Laser source

3. Image capturing device

31. Optical lens(es)

4. 4a, 4b Trigger signal delay unit

5. Droplet selector control unit

51. Captured image storage

52. Reference image storage

53. Image comparison and trigger signal generation

6. Droplet manipulator

7. System control unit

8. Mass spectrometer . Mass spectrometer inlet Waste liquid containera. Droplet with solid particleb. Droplet without solid particle