Characterization of particles in solution

Abstract

The present invention relates to a method for measuring characteristics of particles in solution and to a device for performing the same, wherein said method comprises the steps of providing a vessel comprising a sample of said particles in solution, wherein the sample has preferably a volume between 0.1 μL and 15 μL, providing a monochromatic light source and a light detector, transmitting light from the monochromatic light source to the vessel comprising the sample, detecting light emitted from the vessel with the light detector, and determining characteristics of said particles in solution comprised in the sample based on a dynamic light scattering (DLS) measurement.

1 . Method to measure characteristics of particles in solution, said method comprising the steps of:
 providing a vessel comprising a sample of said particles in solution, wherein the sample has preferably a volume between 0.1 μL and 15 μL;   providing at least a monochromatic light source and at least a light detector;   transmitting light from the monochromatic light source to the vessel comprising the sample;   detecting light emitted from the vessel with the light detector;   measuring fluorescence, preferably of said particles in solution comprised in the sample and/or of the material of the vessel, wherein said fluorescence is preferably an autofluorescence of said particles and/or of the material of the vessel; and   determining characteristics of said particles in solution comprised in the sample based on a dynamic light scattering (DLS) measurement.   
     
     
         2 . Method of  claim 1 , the method further comprising the step of measuring back-reflection of the vessel comprising the sample. 
     
     
         3 . Method of  claim 1 , the method comprising the steps of
 determining the position of the vessel based on the measured fluorescence and/or based on the measured back-reflection, and   optionally positioning the vessel based on the measured fluorescence and/or back-reflection and the determined vessel position.   
     
     
         4 . Method of  claim 1 , the method further comprising the step(s) of
 performing a nano differential scanning fluorimetry (nano-DSF) measurement; and/or   measuring back-reflection of the vessel comprising the sample.   
     
     
         5 . Method of  claim 1 , wherein
 a fluorescence measurement for each vessel is followed by a DLS measurement for each vessel; or   a DLS measurement for each vessel is followed by a fluorescence measurement for each vessel; or   a fluorescence measurement and a DLS measurement is performed for one vessel of the plurality of vessels followed by a fluorescence measurement and a DLS measurement for another vessel of the plurality of vessels.   
     
     
         6 . Method of  claim 1 , wherein the light from the monochromatic light source is coherent and has preferably a wavelength between 350 nm and 500 nm, preferably of 405 nm, 445 nm, or 488 nm. 
     
     
         7 . Method of  claim 1 , wherein the monochromatic light source is a laser, preferably a diode laser,
 preferably a diode laser selected from the group consisting of frequency stabilized diode laser, DPSS laser, PPLN doubled diode laser, frequency multiplied DPSS laser, diode pumped fiber laser, multiplied diode pumped fiber laser, and diode pumped upconversion fiber laser.   
     
     
         8 . Method of  claim 7 , wherein the laser has a coherence length of at least 0.1 mm. 
     
     
         9 . Method of  claim 7 , wherein the laser has a power between 1 mW and 200 mW, preferably between 10 mW and 180 mW, more preferably between 50 mW and 150 mW, even more preferably between 75 mW and 120 mW, for example at 100 mW. 
     
     
         10 . Method of  claim 7 , wherein the monochromatic light is delivered from the monochromatic light source via a at laser wavelength single mode fiber, preferably a single mode fiber which is not maintaining the polarization. 
     
     
         11 . Method of any  claim 1 , wherein light from the monochromatic light source is transmitted to the vessel with an angle φL to a longitudinal axis of the vessel, wherein φL is between 0 degrees and 45 degrees. 
     
     
         12 . Method of  claim 11 , wherein light detected with the light detector is emitted from the vessel with an angle φD to a longitudinal axis of the vessel, wherein φD is between 0 degrees and 45 degrees, wherein the value of φL is preferably identical to the value of φD. 
     
     
         13 . Method of  claim 12 , wherein an angle φS between the light that is transmitted from the monochromatic light source to the vessel and the light emitted from the vessel that is detected with the light detector is between 0 degrees and 150 degrees, preferably between 10 degrees and 150 degrees, more preferably between 10 degrees and 60 degrees. 
     
     
         14 . Method of  claim 1 , wherein the transmitted monochromatic light is focused in the vessel comprising the sample using an objective lens, wherein the light emitted from the vessel is preferably also focused by said objective lens, preferably wherein the objective lens has a focal length between 10 mm and 200 mm, and/or wherein the transmitted monochromatic light is focused in the vessel with a focal spot having a full width at half maximum (FWHM) between 3 μm and 30 μm, preferably resulting in a measurement volume between 0.01 nl and 0.1 nl, preferably between 0.01 nl and 0.02 nl, more preferably about 0.016 nl. 
     
     
         15 . Method of  claim 1 , wherein the light detector is a photomultiplier tube (PMT), a silicon photomultiplier (SiPM), or an Avalanche photodiode (APD) photon counting detector, preferably a PMT or a SiPM. 
     
     
         16 . Method of  claim 1 , wherein the DLS measurement is obtained in less than 5 sec, preferably in less than 1 sec. 
     
     
         17 . Method of  claim 1 , wherein the DLS measurement is performed only once per sample. 
     
     
         18 . Method of  claim 1 , wherein the DLS measurement comprises the step of performing at least one correlation operation, preferably at least one autocorrelation operation. 
     
     
         19 . Method of  claim 1 , wherein the DLS measurement comprises the steps of
 obtaining an analog output signal obtained from the light detector; and   processing the obtained analog output signal, wherein the step of processing the obtained analog output signal comprises the step of digitalizing the obtained analog output signal into a digitalized output signal, wherein the digitalized output signal is further processed with the step(s) of   i) processing the digitalized output signal as a digitalized single photon pulse signal, preferably in case the intensity of the detected light emitted from the vessel is below 2 million detected photons per second;   and/or   ii) processing the digitalized output signal as discrete values of an analog signal, preferably in case the intensity of the detected light is above 2 million detected photons per second.   
     
     
         20 . Method of  claim 19 , wherein the step of processing the obtained analog output signal comprises either step i) or step ii), and wherein the time to decide whether to process the digitalized output signal as a digitalized signal according to step i) or ii) is less than 1 sec, preferably maximal 0.05 sec, preferably by using an FPGA, or
 the photon counting and analog output signal can be processed simultaneously such that the decision whether to process according to step i) or step ii) can be made after the measurement.   
     
     
         21 . Method of  claim 19 , wherein the step of processing the obtained analog output signal further comprises the step(s) of
 storing the processed digitalized output signal obtained from step i) or step ii); or   storing the processed digitalized output signals obtained from step i) and step ii); and   further processing one of the stored output signals.   
     
     
         22 . Method of  claim 1 , the method further comprising the step of
 tempering the vessel over time at least with a first temperature at a first time point and a second temperature at a second time point.   
     
     
         23 . Method of  claim 22 , wherein the step of tempering the vessel over time at least with a first temperature at a first time point and a second temperature at a second time point comprises tempering the vessel with a tempering rate between 0.01° C. per minute and 30° C. per minute, preferably between 0.1° C. per minute and 10° C. per minute, and/or wherein the first temperature and the second temperature are between −20° C. and 160° C. 
     
     
         24 . Method of  claim 1 , further comprising the steps of
 providing a further light detector, and   measuring static scattering light of the vessel comprising the sample using the further light detector, preferably with an angle φ to a longitudinal axis of the vessel, wherein y is preferably between 10 degrees and 150 degrees, more preferably between 10 degrees and 60 degrees.   
     
     
         25 . Method of  claim 1 , wherein the vessel is a capillary and/or multi-well plate, preferably a glass capillary with a round cross-section and an inner diameter between 0.1 mm and 1 mm, preferably between 0.15 mm and 0.5 mm, and preferably an outer diameter between 0.2 mm and 1.2 mm, preferably between 0.65 mm and 1 mm and a length between 5 mm and 70 mm, preferably between 32 mm and 50 mm, more preferably of about 50 mm. 
     
     
         26 . Method of  claim 1 , wherein a plurality of vessels is provided, wherein each vessel comprises a sample of particles in solution, and wherein characteristics of particles in solution are measured for each vessel. 
     
     
         27 . Method of  claim 1 , wherein the characteristics are selected from the group consisting of particle size distribution, aggregation temperature, melting temperature, transition temperature, unfolding temperature onset, temperature of liquid-liquid phase separation (T LLPS ) free folding energy, second virial coefficient (B 22 /A 2 ), self-interactions of particles, colloidal stability, hydrodynamic radius, repulsive or attractive interaction between particles (k D ), solubility, long-term protein stability and critical denaturant concentrations. 
     
     
         28 . A device for detecting characteristics of particles in solution, preferably in accordance with  claim 1 , wherein said device comprises:
 means for accommodating at least one vessel comprising a sample of said particles in solution, preferably for accommodating between 0.1 to 15 μL of said particles;   a monochromatic light source and a light detector;   means for performing a DSL measurement;
 means for measuring fluorescence, preferably of said particles in solution comprised in the sample and/or of the material of the vessel; and 
   control means adapted for   controlling the means for accommodating at least one vessel;   controlling the monochromatic light source for transmitting light from the monochromatic light source to the at least one vessel;   controlling the light detector for detecting signals from the at least one vessel;   controlling said means for measuring the fluorescence;   controlling said means for performing a DSL measurement.   
     
     
         29 . The device of  claim 28 , wherein said device further comprises:
 means for performing a correlation operation, preferably an autocorrelation operation, wherein said autocorrelation operation is preferably an autocorrelation logic embodied in hardware and/or software; and   
     
     
         30 . The device of  claim 28  or  29 , wherein said device further comprises:
 means for digitalizing signals obtained from the light detector, wherein said means preferably comprise a field programmable gate array (FPGA), wherein the control means are further adapted for controlling said means for digitalizing signals obtained from the light detector. 
 
     
     
         31 . The device of  claim 28 , wherein said device further comprises:
 positioning means for positioning the means for accommodating the sample of said particles in solution, wherein the control means are further adapted for controlling the positioning means for accommodating the sample.   
     
     
         32 . The device of  claim 28 , wherein said device further comprises:
 a temperature control system for tempering the vessel over time at least with a first temperature at a first time point and a second temperature at a second time point, wherein the control means are further adapted for controlling said temperature control system for tempering the vessel over time at least with a first temperature at a first time point and a second temperature at a second time point.   
     
     
         33 . The device of  claim 28 , wherein said device further comprises:
 means for performing a nano-DSF measurement and/or means for measuring back-reflection, wherein the control means are further adapted for controlling said means for performing a nano-DSF measurement and/or said means for measuring back-reflection.   
     
     
         34 . The device of  claim 28 , wherein said device further comprises:
 a further light detector; and   means for performing a static scattering light measurement, wherein the control means are further adapted for controlling said means for performing a static scattering light measurement.   
     
     
         35 . The device of  claim 28 , wherein said device further comprises:
 a single mode fiber; and   means for delivering monochromatic light from the monochromatic light source via said single mode fiber, wherein the control means are further adapted for controlling said means for delivering monochromatic light from the monochromatic light source via said single mode fiber.   
     
     
         36 . The device of  claim 28 , wherein said device comprises
 the at least one monochromatic light source for the DLS measurement; preferably   an LED light source for the fluorescence measurement; and preferably   a further LED for the back-reflection measurement.   
     
     
         37 . The device of  claim 28 , wherein said device comprises
 at the least one light detector for the dynamic light scattering (DLS) measurement; preferably   at least one additional, preferably two additional light detectors for the fluorescence measurement, and preferably   one additional light detector for the back-reflection measurement.