# US 11,307,128 B2 · System and method for the optical measurement of stability and aggregation of particles > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,307,128 B2 - Document ID: US11307128B2 - Status: Granted - Kind: B2 - Application number: 17/123,863 - Filed: 2020-12-16 - Published: 2021-04-08 - Granted: 2022-04-19 - Earliest claimed: 2015-10-01 - Assignees: Nanotemper Technologies GmbH - Applicants: NanoTemper Technologies GmbH - Inventors: Philipp Baaske; Stefan Duhr; Dennis Breitsprecher; Jonathan Derix - Web page: https://patentgrep.com/patent/US11307128B2 ## CPC - G01N 15/00: Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials - G01N 21/51: inside a container, e.g. in an ampoule (G01N21/53 takes precedence) - G01N 21/64: Fluorescence; Phosphorescence - G01N 21/645: {Specially adapted constructive features of fluorimeters} - G01N 2015/0092: {Monitoring flocculation or agglomeration} - G01N 2021/6482: {Sample cells, cuvettes} - G01N 2021/6491: {Measuring fluorescence and transmission; Correcting inner filter effect} - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract The present invention relates to a method for the optical measurement of at least the stability and the aggregation of particles in a liquid sample which is located in a sample container, wherein the method comprises the following steps: irradiating the sample with light of at least one first wavelength in order to fluorescently excite the particles, irradiating the sample with light of at least one second wavelength in order to examine the scattering of the particles, measuring the fluorescence light which is emitted by the sample; and measuring the extinction light at the second wavelength, wherein the irradiated light of the second wavelength runs through the sample container, is reflected back, runs again through the sample container in the opposite direction and exits as extinction light, wherein the stability is determined based on the measured fluorescence light and the aggregation is measured based on the measured extinction light. The invention further relates to a corresponding apparatus. ## Claims ### Claim 1 A method for optically measuring the thermal stability of viruses in a liquid sample, which is in a sample container, wherein the method comprises the following steps: irradiating the sample with light of at least a first wavelength, to fluorescently excite the viruses, irradiating the sample with light of at least a second wavelength to examine the scattering of the viruses, measuring the fluorescence light emitted by the sample; and measuring the extinction light at the second wavelength, wherein the irradiated light of the second wavelength runs through the sample container, is reflected back, runs again through the sample container in opposite direction and exits as extinction light, wherein the stability of the viruses is measured on the basis of the measured fluorescence light and the aggregation on the basis of the measured extinction light, wherein the sample container is shifted during a measuring period relatively to the irradiated light of the first and/or second wavelength and/or to the detector and is driven back and forth several times; and wherein a plurality of sample containers or a plurality of capillaries are scanned by said relative movement. ### Claim 2 (depends on 1) The method according to claim 1, wherein the sample container is tempered and the stability measurements are preferably performed at different temperatures. ### Claim 3 (depends on 2) The method according to claim 2, wherein the sample container rests on a tempering element and is tempered by a contact. ### Claim 4 (depends on 3) The method according to claim 3, wherein the tempering element further reflects back the irradiated light of the second wavelength, again runs through the sample container in opposite direction and exits as extinction light. ### Claim 5 (depends on 3) The method according to claim 3, wherein the tempering element is made of a material i) which has little autofluorescence <1%, and/or ii) which has a high reflectivity >30% in the wavelength range of the second wavelength and preferably comprises silicon or consists of pure silicon. ### Claim 6 (depends on 3) The method according to claim 3, wherein at the surface of the tempering element at least one groove is configured, the sample container is arranged above the groove and the irradiated light of the second wavelength is reflected back from the bottom of the groove. ### Claim 7 (depends on 6) The method according to claim 6, wherein the groove has a width between 1-10 mm and a depth of more than half of the coherence length of the light of the second wavelength. ### Claim 8 (depends on 2) The method according to claim 2, wherein during a measuring period the temperature of the samples is changed, preferably increased to determine the thermal stability. ### Claim 9 (depends on 2) The method according to claim 2, wherein the temperature is continuously increased by a rate of at least 0.5° C. per minute, preferably 1° C. per minute. ### Claim 10 (depends on 1) The method according to claim 1, wherein the thermal stability of the viruses is determined by identification of inflection points, preferably by analysis of the first or second derivation of the raw data or by other mathematical processes. ### Claim 11 (depends on 10) The method according to claim 10, wherein the inflection points are used to determine if the virus contain RNA or DNA. ### Claim 12 (depends on 1) The method according to claim 1, wherein the fluorescence light and the extinction light are measured with a common optical system. ### Claim 13 (depends on 1) The method according to claim 1, wherein the irradiation of the sample i) is not conducted simultaneously with the first and second wavelengths; or ii) the irradiation with the second wavelength is conducted continuously, whereas the irradiation with the first wavelength is conducted intermittently, preferably periodically. ### Claim 14 (depends on 1) The method according to claim 1, wherein the fluorescence light and the extinction light are measured simultaneously. ### Claim 15 (depends on 1) The method according to claim 1, wherein i) the extinction light and the fluorescence light are measured by a common detector; ii) the extinction light is measured by a first detector and/or a second detector and fluorescence light of a first fluorescence wavelength is measured by the first detector and fluorescence light of a second fluorescence wavelength is measured by the second detector; or iii) the extinction light is measured by a first detector, fluorescence light of a first fluorescence wavelength is measured by a second detector and fluorescence light of a second fluorescence wavelength is measured by a third detector. ### Claim 16 (depends on 1) The method according to claim 1, wherein the sample container is a capillary. ### Claim 17 (depends on 1) The method according to claim 1, wherein i) a fluorescence value is determined by integrating the intensity of the fluorescence light via the shifting and/or ii) an extinction value is determined by integrating the intensity of the extinction light via the shifting. ### Claim 18 (depends on 1) The method according to claim 1, wherein during a measuring period a plurality of sample containers and/or the optical system are continuously driven back and forth several times and the measurements of the fluorescence light and/or the extinction light are conducted during the movement. ### Claim 19 (depends on 1) The method according to claim 1, wherein the second wavelength is chosen such that less than 1%, 0.1%, 0.05% is absorbed by the sample or the particles in the sample. ### Claim 20 (depends on 1) The method according to claim 1, wherein the light of the first wavelength and the light of the second wavelength are united to a collinear ray which is irradiated into the sample container. ### Claim 21 (depends on 1) The method according to claim 1, wherein the extinction light of the second wavelength, which is reflected back and leaves the sample container in the opposite direction to the irradiation direction, deviates from the irradiation direction 5° at most, preferably less than 2°, further preferred less than 1°. ### Claim 22 (depends on 1) An apparatus for the optical measurement of the thermal stability of viruses in a liquid sample which is located in a sample container, in particular according to claim 1, wherein the apparatus comprises: a first light source for irradiating light of a first wavelength into the sample container to fluorescently excite the particles to be examined, a second light source for irradiating light of a second wavelength into the sample container to measure the scattering of the particles, a first detector for measuring the excited fluorescence light which is radiated from the sample, a second detector for measuring extinction light at the second wavelength wherein the irradiated light of the second wavelength runs through the sample container, is reflected back, runs again through the sample container in the opposite direction and exits as extinction light and an evaluation means which determines the bonding based on the measured fluorescence light and based on the measured extinction light. ### Claim 23 (depends on 22) The apparatus according to claim 22 comprising a tempering element with a reflecting surface at which the irradiated light of the second wavelength is reflected back, and wherein the apparatus is preferably configured to arrange at least one sample container on the surface for measurement purposes. ### Claim 24 (depends on 22) The apparatus according to claim 22, wherein the at least one sample container is a capillary. ### Claim 25 (depends on 22) The apparatus according to claim 22, wherein the reflective surface consists of silicon, preferably of crystalline silicon. ### Claim 26 (depends on 22) The apparatus according to claim 22, wherein at least one groove is configured at the surface of the tempering element, the sample container is arranged above the groove and the irradiated light of the second wavelength is reflected back from the bottom of the groove. ### Claim 27 (depends on 26) The apparatus according to claim 26, wherein the groove has a width between 1-10 mm and a depth of more than half of the coherence length of the light of the second wavelength.