Characterization of particles in solution
A method for measuring characteristics of particles in solution and to a device for performing the same, wherein the method includes the steps of providing a vessel containing a sample of the 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 containing the sample, detecting light emitted from the vessel with the light detector, and determining characteristics of the particles in solution 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;
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,
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, in case an intensity of the light detected by the light detector is below a predetermined number of detected photons per second;
and/or
ii) processing the digitalized output signal as discrete values of an analog signal, in case the intensity of the light detected by the light detector is above said predetermined number of detected photons per second.
2 . Method of claim 1 , wherein the vessel is a capillary and/or multi-well plate.
3 . Method of claim 1 , the method further comprising the step of
measuring fluorescence, and/or
measuring back-reflection of the vessel comprising the sample.
4 . Method of claim 3 , the method further comprising the steps of
determining a 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.
5 . Method of claim 1 , wherein the light from the monochromatic light source is coherent.
6 . Method of claim 1 , wherein the monochromatic light source is a laser.
7 . Method of claim 6 , wherein the laser has a coherence length of at least 0.1 mm.
8 . Method of claim 1 , wherein the DLS measurement is obtained in less than 5 sec.
9 . Method of claim 1 , wherein the vessel has a volume between 0.1 μL and 15 μL.
10 . Method of 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.
11 . Method of claim 10 , 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 degree.
12 . Method of claim 11 , 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.
13 . Method of claim 1 , wherein the transmitted monochromatic light is focused in the vessel comprising the sample using an objective lens.
14 . 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.
15 . Method of claim 1 , wherein the DLS measurement is performed only once per sample.
16 . Method of claim 1 , wherein the DLS measurement comprises the step of performing at least one correlation operation.
17 . Method of claim 1 , 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 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 in case the intensity of the detected light is above 2 million detected photons per second.
18 . Method of claim 17 , wherein the step of processing the digitalized 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 output signal according to step i) or ii) is less than 1 sec or
a 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 met after the DLS measurement.
19 . Method of claim 17 , 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.
20 . 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.
21 . Method of claim 20 , 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.
22 . 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.
23 . 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.
24 . 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.
25 . Method of claim 24 , 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.
26 . 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 ), 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.
27 . A device for detecting characteristics of particles in solution according to the method recited in claim 1 , wherein said device comprises:
means for accommodating at least one vessel comprising a sample of said particles in solution;
a monochromatic light source and a light detector;
means for performing a DSL measurement; 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; and
controlling said means for performing a DSL measurement.
28 . The device of claim 27 , wherein said device further comprises:
means for performing a correlation operation.
29 . The device of claim 27 , wherein said device further comprises:
means for digitalizing signals obtained from the light detector wherein the control means are further adapted for controlling said means for digitalizing signals obtained from the light detector.
30 . The device of claim 27 , wherein said device further comprises:
means for measuring the fluorescence of said particles in solution comprised in the sample, wherein the control means are further adapted for controlling said means for measuring the fluorescence of said particles in solution comprised in the sample.
31 . The device of claim 27 , wherein said device further comprises:
positioning means for positioning the means for accommodating at least one vessel comprising the sample of said particles in solution, wherein the control means are further adapted for controlling the positioning means.
32 . The device of claim 27 , wherein said device further comprises:
a temperature control system for tempering the at least one 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.
33 . The device of claim 27 , 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 27 , 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 27 , 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.