Methods and devices for analyzing therapeutic payloads carried by nanoparticle delivery carriers

Abstract

There is described a method of determining at least one characteristic of a therapeutic payload and / or a nanoparticle delivery vehicle, the therapeutic payload and the nanoparticle delivery vehicle forming a diffuse scattering liquid suspension sample, the method comprising: positioning the sample within an integration chamber; the probe light is guided to a first port of the integral cavity; receiving a portion of the probe light from the second port of the integration cavity after scattering from the sample; measuring one or more spectral characteristics of the probe light received at the second port; and determining at least one quantitative characteristic of the therapeutic payload and / or the nanoparticle delivery vehicle in the liquid suspension based on the measured one or more spectral characteristics of the probe light received at the second port. An apparatus is also described.

Description

Technical Field

[0001] Disclosures relate to methods and apparatus for determining the properties of therapeutic payloads (e.g., loading) in nanoparticle delivery carriers. Examples of such nanoparticle delivery carriers include, but are not limited to, lipid-based delivery carriers. Specifically, the apparatus and methods relate to the analysis of diffusely scattered suspensions containing nanoparticle delivery carriers. In addition to analyzing therapeutic payloads, the nanoparticle delivery carriers can also be analyzed. Background Technology

[0002] Lipid nanoparticle (LNP) drug products comprise drug payloads carried by nanoparticle delivery carriers composed of lipids. The drug is a type of drug that can be delivered into the body via LNPs. Recent advances in this and similar technologies mean that vaccines, such as mRNA COVID-19 vaccines, can also be delivered into the body in this manner. During research and development and manufacturing, it is necessary to quantify the amount of payload present in the LNPs.

[0003] Quantification of RNA, DNA, proteins, etc., has traditionally been performed using ultraviolet-visible (UV-Visible) spectroscopy, in which RNA, DNA, proteins, etc., are held in a buffer solution. RNA typically absorbs light at ultraviolet wavelengths. This type of UV-Vis spectroscopy is common, but often requires careful dilution to ensure the sample is within the instrument's dynamic range. Thermo Fisher Scientific's NanoDrop micro-spectrophotometer and C-Tech's SoloVPE CTech™ SoloVPE® system represent advancements in this field, adapting standard UV-Vis methods to avoid the need for sample dilution to the instrument's dynamic range.

[0004] Traditional UV-Vis spectroscopy and these adapted instruments cannot be used to measure RNA, DNA, and proteins carried or encapsulated by LNPs because lipid nanoparticles scatter strongly at UV wavelengths. This is at least partly due to their size, which is likely around 100 nm. Classical scattering varies with wavelength λ. -4 It is directly proportional. Therefore, for other nanoparticles, scattering may also dominate absorption at other wavelengths. Figure 1 This is a schematic diagram and schematic graph of traditional ultraviolet-visible spectroscopy, where ultraviolet or visible light is directed to the sample (e.g., a sample in a cuvette). The light transmitted through the sample is analyzed, the extinction spectrum is calculated, and absorption bands are identified. The wavelength and intensity or amplitude of the absorption bands allow for the determination of the type and quantity of substances such as RNA, DNA, and proteins. However, as... Figure 2 As shown, for LNP particles, the ultraviolet-visible spectral light is strongly scattered, and therefore the spectrum is dominated by scattering. This makes it impossible to determine the absorbance value of the LNP payload.

[0005] To be more detailed, Figure 3 The image shows the spectra obtained by applying conventional UV-Vis spectroscopy to LNP mixed with two different concentrations of RNA. Traditionally, RNA is quantified based on absorbance at 260 nm (known as A260). The Beer-Lambert law states that absorbance is proportional to concentration. Figure 3 In the diagram, the top curve represents a sample with twice the RNA concentration of the bottom curve, but the absorbance difference is not twofold because the extinction spectrum is dominated by LNP scattering. Therefore, UV-Vis spectroscopy cannot be used for reliable analysis of RNA, DNA, and protein payloads carried by LNP nanoparticles.

[0006] Such encapsulated RNA has traditionally been quantified using fluorescence assays (typically RiboGreen), a method that is time-consuming and prone to systematic and operator errors.

[0007] Therefore, an improved technique is needed to measure the RNA and other payloads carried by LNPs. Summary of the Invention

[0008] This invention provides a method for determining at least one property of a therapeutic payload and / or nanoparticle delivery carrier, the therapeutic payload and nanoparticle delivery carrier forming a diffusely scattering liquid suspension sample. The method includes: positioning the sample within an integrating cavity; directing probe light to a first port of the integrating cavity; receiving a portion of the probe light from a second port of the integrating cavity after scattering from the sample; measuring one or more spectral characteristics of the probe light received at the second port; and determining at least one property of the therapeutic payload and / or nanoparticle delivery carrier in the liquid suspension based on the measured one or more spectral characteristics of the probe light received at the second port. The inventors have recognized that the method described herein can be used to measure complex drug or vaccine delivery carriers when the delivery carrier is highly scattering. For example, as mentioned above, standard UV-Vis spectroscopy is typically used to analyze and quantify RNA. However, as mentioned above, nanoparticle delivery carriers can be highly scattering, thus hindering accurate measurement of RNA or other payloads. The method described herein corrects for scattering by using an integrating cavity to allow scattering effects to be significantly eliminated. Therefore, this technique may be referred to as scatter-corrected absorption (SCA). A unique aspect of this technique is that material highly scattering at UV-Vis wavelengths may surround or encapsulate the payload to be measured. Although in other cases, the payload may only be partially surrounded by the nanoparticle delivery carrier or be suspended outside the carrier. During the development phase, the payload and delivery carrier may be held together in suspension, but the payload is not yet loaded into the carrier. Therefore, due to the high scattering of the delivery carrier and its potential larger volume in the suspension than the payload, UV-Vis spectroscopy measurements of the payload are often difficult. The use of an integrating sphere and measurement technique in this invention overcomes the scattering difficulties, enabling accurate measurement of the payload. This technique also allows for the measurement of absorption from the highly scattering delivery carrier. Therefore, this technique can be used to measure the payload and / or delivery carrier, regardless of whether the payload is loaded into or onto the delivery carrier, in situations where the presence of the delivery carrier and its scattering renders conventional UV-Vis spectroscopy inaccurate. The scattering-corrected absorption technique is independent of particle size or distribution and can be used to quantify the payload. This method is particularly suitable for particles in the size range of 10–150 nm, especially 60–150 nm. For example, this method can be applied to LNPs, which are typically in the 60-80 nm range; adeno-associated viruses (AAVs), which may be in the 20-25 nm range or larger; lentiviruses or exosomes, which may be up to 150 nm; and antibody-drug conjugates (ADCs), which have particularly large size variations. This method is suitable for diffuse or turbid samples and does not require the use of fluorescent dyes.

[0009] A nanoparticle delivery carrier refers to a nanoscale transport unit that can carry a therapeutic agent forming a payload on or within the delivery carrier, although this measurement technique also measures the payload when it is suspended with the delivery carrier but not loaded onto it. Typically, the payload is highly efficient at encapsulating or loading the NPV (nanoparticle carrier), or may contain a buffer of unloaded payload that is replaced after manufacturing. Therefore, the encapsulation or loading efficiency is usually known, and the payload within the NPV can be easily quantified using measurements according to the invention. The delivery carrier can be lipid-based, such as lipid nanoparticles, and can carry drugs or vaccines. As we have described herein, other types of nanoparticle delivery carriers and payloads are possible.

[0010] The method of the present invention does not rely on knowledge of particle size, particle size distribution (Pdi), or the refractive index of the material to estimate scattering and / or scattering-corrected absorption. Furthermore, as described above, the method allows for characterization of the NPV itself, for example, by composition or concentration.

[0011] This method allows for the determination of the characteristics of the payload and / or NPV. The characteristics can be quantitative, such as the amount or concentration of the payload and / or NPV in the sample, or qualitative, such as the type of payload (e.g., the type of RNA carried) or the type of NPV.

[0012] The step of determining at least one characteristic of a therapeutic payload in a liquid suspension can be performed at a device containing a light source, or at a different location (e.g., remotely).

[0013] The step of determining at least one characteristic of a therapeutic payload and / or NPV in a liquid suspension may include determining one or more absorption spectral features of the therapeutic payload and / or NPV based on one or more spectral features of the measured probe light. The one or more absorption spectral features may be peaks or troughs detected in the received probe light and indicate absorption at a specific wavelength or wavelength range. The wavelength of the peaks or troughs may be characteristic of the therapeutic payload and / or NPV, thus indicating the presence of the payload and / or NPV.

[0014] The steps of determining one or more absorption spectral characteristics of the therapeutic payload and / or NPV may include applying a calibration transformation to one or more measured spectral characteristics of the probe light to determine the absorption spectral characteristics of the therapeutic payload and / or NPV. The calibration transformation can convert the original measured light or spectrum, which includes, for example, instrument characteristics, into a spectrum or value indicating the presence or absence of the therapeutic payload.

[0015] Methods may include performing linear decomposition to determine the uptake or quantity of LNPs.

[0016] The calibration transformation can be based on the calibration of the integrating sphere and / or the absorption coefficient of the therapeutic payload at the probe wavelength. This application refers to the absorption coefficient, but since this application focuses only on absorption, the term is used interchangeably with the extinction coefficient.

[0017] At least one characteristic of therapeutic payload and / or NPV can be a measure of the amount or concentration of payload and / or NPV in the sample, or related thereto.

[0018] At least one characteristic of the therapeutic payload and / or NPV can be the absorption spectrum of the payload and / or NPV.

[0019] The method may also include determining one or more features associated with the payload and / or NPV based on the absorption spectrum of the payload and / or NPV, and identifying the payload and / or NPV based on the features.

[0020] The probe light can enter the integrating cavity through the first port along a first axial direction and can be received at the second port along a second axial direction. The second axial direction can deviate from the first axial direction, such that the probe light received at the second port is scattered from the integrating cavity and / or the sample. This deviation can mean that the probe light cannot be directly transmitted from the first port to the second port (e.g., without scattering).

[0021] The method may also include guiding the probe light between a third port and a fourth port of the integrating cavity. The fourth port may be arranged opposite the third port such that the fourth port receives the probe light transmitted from the third port (e.g., transmitted directly without scattering), measures the transmitted light, and determines the extinction value of the probe light received at the fourth port. The extinction value may be a measure of the remaining light reaching the fourth port (after any scattering and absorption).

[0022] The method does not require moving the sample between measuring absorption and measuring extinction.

[0023] The method may also include quantifying the amount of scattering from the suspended sample based on extinction values ​​and determined absorption spectral characteristics. Scattering measurements can be used to determine the concentration or amount of the scattering nanoparticle delivery carrier.

[0024] The third port can be the same as the first port, so that the fourth port receives probe light transmitted directly from the first port (e.g., unscattered).

[0025] The fourth port can be the same as the second port, such that the second port receives probe light transmitted directly from the third port (e.g., unscattered).

[0026] The probe light can include one or more wavelengths in the ultraviolet-visible spectrum and can extend to the near-infrared (NIR), for example, including light covering the ranges of 240 nm to 750 nm, 240 nm to 650 nm, at approximately 260 nm, at one or more discrete wavelengths or spectral lines between 240 nm and 320 nm. Alternatively, the probe light can scan wavelength ranges of 240 nm to 750 nm, 240 nm to 650 nm, or between 240 nm and 320 nm, for example, by tuning the output wavelength of the probe light source. For example, RNA tends to absorb around 260 nm, but some payloads absorb at different wavelengths. For example, commercial liposomal cancer therapies encapsulating doxorubicin (actually nanocrystals) absorb in the red region, for example, up to approximately 600 nm. Polymer nanoparticles absorb at longer wavelengths, while metallic nanoparticles scatter strongly and absorb in the visible region. The absorption band produced by gold nanoparticles (NPs) with drug binding on the outside would otherwise be masked by scattering in the visible region. Silver nanoparticles absorb up to 750 nm. However, most xNA molecules absorb in the 240 to 320 nm range.

[0027] Nanoparticle delivery carriers carrying therapeutic payloads may include one or more of the following: lipid-based nanoparticles carrying RNA, antibody-drug conjugates, viral vectors (e.g., lentiviruses and AAVs), liposomes, exosomes, polymer nanoparticles, liposome-based systems (e.g., lipid complexes), metal nanoparticles, SPIONs, polymer micelles, dendritic polymers, nanoemulsions, silica particles, and nanogels.

[0028] Nanoparticle delivery carriers may include lipid-based nanoparticles, and the payload may include RNA, such as mRNA, siRNA, or ssRNA.

[0029] The payload may include one or more of the following: oligonucleotides, DNA, drugs, vaccines, cancer therapies, and gene-editing therapies. The payload may additionally or alternatively include small molecule drugs. Small molecule drugs are typically standard pharmaceutical products manufactured using chemical methods. Encapsulating small molecule drugs has many potential benefits, especially when combined with the other payloads described above. For example, they can prolong release profiles or minimize collateral damage (e.g., with highly toxic cancer drugs). An exemplary small molecule drug is doxorubicin.

[0030] Methods may include analyzing therapeutic payload and NPV to determine the ratio of NPV to payload.

[0031] This invention provides a method for analyzing or measuring / characterizing nanoparticle carriers, comprising: acquiring / receiving at least one or more scatter-corrected absorption measurements / data or scatter-corrected absorption spectroscopy measurements / data for a liquid sample or solution containing multiple nanoparticle carriers, the method comprising the method of any of the preceding claims, wherein the liquid sample or solution comprises a diffusely scattering liquid suspension sample, and wherein the step of acquiring / receiving at least one or more scatter-corrected absorption measurements / data or scatter-corrected absorption spectroscopy measurements / data includes measuring one or more spectral characteristics of a probe light. The characteristics of the therapeutic payload can be quantitative characteristics such as absorption spectra.

[0032] The step of determining at least one characteristic of a therapeutic payload in a liquid suspension may include using at least one scatter-corrected absorption measurement / data or scatter-corrected absorption spectroscopy measurement / data, and utilizing the extinction coefficient of the nanoparticle carrier's payload at the target absorption wavelength to determine the payload capacity of the nanoparticle carrier in the liquid sample or solution.

[0033] The step of determining at least one characteristic of a therapeutic payload in a liquid suspension may include using at least one scatter-corrected absorption measurement / data or scatter-corrected absorption spectroscopy measurement / data, and using the extinction coefficient of the payload of the nanoparticle carrier and the extinction coefficient of the carrier component of the nanoparticle carrier at the target absorption wavelength to determine the ratio of the payload of the nanoparticle carrier to the carrier component / part of the liquid sample or solution.

[0034] The step of determining at least one characteristic of a therapeutic payload in a liquid suspension may include using at least one scatter-corrected absorption measurement / data or scatter-corrected absorption spectroscopy measurement / data, and using the extinction coefficients of the constituent carrier elements of the carrier component / part of the nanoparticle carrier to determine the molar ratio of the constituent carrier elements of the carrier component / part of the nanoparticle carrier.

[0035] The step of determining at least one characteristic of a therapeutic payload in a liquid suspension may include determining the nanoparticle carrier concentration in a liquid sample or solution using at least one scatter-corrected absorption measurement / data or scatter-corrected absorption spectroscopy measurement / data and the measured or determined nanoparticle carrier size.

[0036] The step of determining at least one characteristic of a therapeutic payload in a liquid suspension may include determining the nanoparticle carrier concentration in a liquid sample or solution using at least one scattering spectrum or scattering spectrum data and the measured or determined nanoparticle carrier size.

[0037] This invention provides an apparatus for determining at least one characteristic of a therapeutic payload and / or nanoparticle delivery carrier forming a diffuse-scattering liquid suspension. The apparatus includes: an integrating cavity configured to receive a sample of the liquid suspension into a sample space; a light source arranged to guide probe light through a first port into the integrating cavity; and a detector for measuring the probe light received at a second port of the integrating cavity, the second port being arranged such that the light is received after scattering from the sample in the sample space. The apparatus may optionally include an analyzer configured to determine at least one characteristic of the therapeutic payload and / or nanoparticle delivery carrier in the liquid suspension based on one or more spectral characteristics of the probe light received at the second port. The therapeutic payload may be carried or encapsulated by an NPV in the liquid suspension.

[0038] The characteristics determined by the analyzer can be quantitative, such as the amount or concentration of the payload and / or nanoparticle delivery carrier in the sample, or they can be qualitative, such as the type of payload, the type of RNA carried, and / or the type of nanoparticle delivery carrier.

[0039] The analyzer may be further configured to determine one or more absorption spectral features of the therapeutic payload and / or nanoparticle delivery carrier based on one or more spectral features of the measured probe light, and at least one characteristic of the therapeutic payload and / or nanoparticle delivery carrier is determined based on one or more absorption spectral features of the therapeutic payload and / or nanoparticle delivery carrier. One or more absorption spectral features may be peaks or valleys detected in the received probe light and indicate absorption within a specific wavelength or wavelength range. The wavelength of the peaks or valleys may be characteristic of the therapeutic payload and / or nanoparticle delivery carrier, thus indicating the presence of the payload and / or nanoparticle delivery carrier.

[0040] The analyzer can be configured to determine the absorption spectral characteristics of a therapeutic payload and / or nanoparticle delivery carrier by applying a calibration transformation to one or more measured spectral characteristics of the probe light.

[0041] The calibration transformation can be based on the calibration of the integrating sphere and / or the absorption coefficient of the therapeutic payload and / or nanoparticle delivery carrier at the probe wavelength.

[0042] The light source can be configured to guide the probe light along a first axial direction through a first port into the integrating cavity, and a second port is arranged to receive the probe light scattered from the integrating sphere and / or the sample along a second axial direction, wherein the second axial direction deviates from the first axial direction.

[0043] The integrating sphere may also include a third port and a fourth port. The fourth port may be arranged opposite the third port to receive probe light transmitted directly from the third port (e.g., unscattered). The analyzer may be further configured to determine the extinction value of the probe light received at the fourth port.

[0044] The analyzer can be further configured to quantify the scattering of suspended matter samples based on extinction values ​​and determined absorption spectral characteristics.

[0045] The third port can be the same as the first port, such that the fourth port is configured to receive probe light transmitted directly from the first port (e.g., at least part of the light is unscattered).

[0046] The fourth port can be the same as the second port, such that the second port is configured to receive probe light transmitted directly from the third port (e.g., at least part of the light is unscattered).

[0047] The probe light may include one or more wavelengths in the ultraviolet-visible spectrum and may extend to the near-infrared (NIR) spectrum, for example including light covering the range of 240 nm to 750 nm, 240 nm to 650 nm, at about 260 nm, at one or more discrete wavelengths or spectral lines between 240 nm and 320 nm, or scan or tune the wavelength range of 240 nm to 750 nm, 240 nm to 650 nm, or between 240 nm and 320 nm.

[0048] The analyzer can be configured to determine at least one quantitative characteristic of a nanoparticle delivery carrier carrying a therapeutic payload, wherein the nanoparticle delivery carrier carrying the therapeutic payload may include one or more of the following: lipid-based nanoparticles carrying RNA, antibody-drug conjugates, viral vectors (e.g., lentiviruses and AAVs), liposomes, exosomes, polymer nanoparticles, liposome-based systems (e.g., lipid complexes), metal nanoparticles, SPIONs, polymer micelles, dendritic polymers, nanoemulsions, silica particles, and nanogels.

[0049] The analyzer can be configured to determine at least one quantitative characteristic of a nanoparticle delivery carrier carrying a therapeutic payload, wherein the nanoparticle delivery carrier may be a lipid-based nanoparticle and the payload includes RNA, such as mRNA, siRNA, or ssRNA.

[0050] The analyzer can be configured to determine at least one quantitative characteristic of a nanoparticle delivery carrier carrying a therapeutic payload, wherein the payload may include one or more of the following: oligonucleotides, DNA, drugs, vaccines, tumor therapies, and gene editing therapies.

[0051] The present invention further provides a nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device, such as the apparatus described herein, wherein the integrating cavity may include one or more reflective inner walls, the integrating cavity may be configured to receive a cuvette, the cuvette being configured to contain a liquid sample or solution comprising at least one or more nanoparticle carriers, the liquid sample or solution comprising a diffusely scattering liquid sample; wherein the integrating cavity may include at least one optical inlet port and at least one optical outlet port, the one or more optical inlet ports being configured to receive light from a light source, and the one or more optical outlet ports being configured to transmit light to a spectrometer, wherein the spectrometer may include a detector; wherein the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device may optionally be a lipid-based drug delivery carrier analyzer or a lipid-based drug delivery carrier measurement / characterization device, and is configured to operate in a diffuse reflection mode, in which light from the light source follows an optical path from one or more inlet ports into the integrating cavity, is incident on the reflective inner walls of the integrating cavity, and is diffusely reflected within the integrating cavity, such that the light from the light source illuminates the liquid sample before being transmitted through one or more optical outlet ports and received by the spectrometer for wavelength analysis to provide the absorption spectrum of at least one or more nanoparticle carriers contained in the liquid sample or solution.

[0052] Nanoparticle carrier analyzers or nanoparticle carrier measurement / characterization devices can be, or can be configured to, determine the effective payload or effective payload concentration of nanoparticle carriers, and / or determine the ratio of effective payload to carrier content / concentration of nanoparticle carriers, and / or determine the ratio or fractional content of the constituent carrier components of nanoparticle carriers, and / or determine the concentration of nanoparticle carriers in solutions or formulations. Nanoparticle carriers can refer to nanoparticle delivery carriers.

[0053] The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device can be a lipid-based nanoparticle analyzer or lipid-based nanoparticle measurement / characterization device, used or configured to determine the concentration of lipid-based nanoparticles in a solution or formulation, or used or configured to determine the concentration of lipid-based drug delivery carriers in a solution or formulation.

[0054] Lipid-based nanoparticle carriers may include or consist of (solid) lipid nanoparticles and / or liposomes.

[0055] The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device may also include an optical path modulator configured to selectively adjust the optical path through the integrating cavity, thereby providing at least two distinct optical paths. The optical path modulator may be configured such that, when in a first configuration, the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device is in transmission mode, in which light from the light source follows a first optical path from one or more optical inlet ports to the liquid sample, such that the light from the light source directly illuminates the liquid sample before the light transmitted by the sample is transmitted through one or more optical outlet ports and received by a spectrometer for wavelength analysis to provide the extinction spectrum of the liquid sample. The optical path modulator can be further configured such that, when the optical path modulator is in the second configuration, the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device is in diffuse reflection mode. In this mode, light from the light source follows a second optical path into the integrating cavity from one or more inlet ports, is incident on the reflective inner wall of the integrating cavity, and is diffusely reflected within the integrating cavity. This allows the light from the light source to irradiate the liquid sample before being transmitted through one or more optical outlet ports and received by the spectrometer for optical wavelength analysis, thereby providing the absorption spectrum of the liquid or liquid matrix contained in the liquid sample.

[0056] The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device may also include a controller, or a controller configured to control an optical path modulator to selectively adjust the optical path through the device.

[0057] The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device can be a (solid) lipid nanoparticle drug delivery carrier drug loading analyzer or (solid) lipid nanoparticle drug delivery carrier drug loading measurement / characterization device; or a (solid) lipid nanoparticle drug delivery carrier drug loading to lipid content / concentration ratio analyzer or (solid) lipid nanoparticle drug delivery carrier drug loading to lipid content / concentration ratio measurement / characterization device; or a (solid) lipid nanoparticle drug delivery carrier lipid component ratio analyzer or (solid) lipid nanoparticle drug delivery carrier lipid component ratio measurement / characterization device; or a liposome drug delivery carrier analyzer or measurement / characterization device; or a liposome nanoparticle drug delivery carrier drug loading analyzer or liposome nanoparticle drug delivery carrier drug loading to lipid content / concentration ratio analyzer or liposome nanoparticle drug delivery carrier drug loading to lipid content / concentration ratio measurement / characterization device; or a liposome nanoparticle drug delivery carrier lipid component ratio analyzer or liposome nanoparticle drug delivery carrier lipid component ratio measurement / characterization device.

[0058] The present invention further provides a method for determining at least one characteristic of a nanoparticle delivery carrier or a therapeutic payload carried by a nanoparticle delivery carrier, the nanoparticle delivery carrier forming a diffusely scattering liquid suspension sample, the method comprising: positioning the sample within an integrating cavity; directing probe light to a first port of the integrating cavity; receiving a portion of the probe light from a second port of the integrating cavity after scattering from the sample; measuring one or more spectral characteristics of the probe light received at the second port; and determining at least one characteristic of the nanoparticle delivery carrier or therapeutic payload in the liquid suspension based on the measured one or more spectral characteristics of the probe light received at the second port.

[0059] The invention may also include a computer program configured to perform the analysis methods described herein and executed by an analyzer or controller. The invention further provides a computer-readable medium having a computer program stored thereon, i.e., instructions that, when operated by a computer, cause the computer to perform the analyzer steps set forth herein. Therefore, the analyzer described herein may include a computer having a processor, memory, and a user interface.

[0060] This disclosure relates to nanoparticle carriers or nanoparticle delivery carriers configured to transport or deliver payloads or cargo.

[0061] Such payload delivery and transport can occur in media such as human or animal bodies.

[0062] Nanoparticle carriers or nanoparticle delivery carriers comprise a carrier component and a payload component. The payload is held or carried by the nanoparticle carrier or nanoparticle delivery carrier and can eventually be released from the nanoparticle carrier or nanoparticle delivery carrier, for example, when certain release conditions are met.

[0063] This disclosure relates to the characterization of nanoparticle carriers or nanoparticle delivery carriers.

[0064] This disclosure relates, for example, to a nanoparticle carrier analyzer or a nanoparticle carrier measurement / characterization device.

[0065] This disclosure relates, for example, to a nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device, for example, for: - Determine the effective loading amount or effective loading concentration of the nanoparticle carrier, and / or - Determine the effective load of the nanoparticle carrier as a ratio of carrier content / concentration, and / or - Determine the composition of the nanoparticle carrier, including the ratio or fractional content of the carrier components, and / or - Determine the concentration of nanoparticle carriers in the solution or formulation.

[0066] This disclosure also relates to, for example, an analytical method or measurement / characterization method for nanoparticle carriers.

[0067] This disclosure relates to an analytical or measurement / characterization method for nanoparticle carriers, for example, for: - Determine the effective loading amount or effective loading concentration of the nanoparticle carrier, and / or - Determine the effective load of the nanoparticle carrier as a ratio of carrier content / concentration, and / or - Determine the composition of the nanoparticle carrier, including the ratio or fractional content of the carrier components, and / or - Determine the concentration of nanoparticle carriers in the solution or formulation. Attached Figure Description

[0068] Embodiments of the present invention will be described by way of example only and with reference to the accompanying drawings, in which: Figure 1 This is a schematic diagram and a schematic curve of traditional ultraviolet-visible spectroscopy; Figure 2 This is a schematic diagram and schematic curve of traditional ultraviolet-visible spectroscopy applied to highly scattering LNP; Figure 3 The spectrum was obtained by applying traditional ultraviolet-visible spectroscopy to LNPs carrying two different concentrations of RNA. Figure 4 This is a schematic diagram of an apparatus according to an embodiment of the present invention; Figure 5 This is a schematic diagram of an apparatus according to another embodiment of the present invention; Figure 6 a and Figure 6 b is a graph of RNA and lipid nanoparticle liquid suspensions measured using conventional UV-Vis spectroscopy and the technique of this invention, respectively. Figure 7 a and Figure 7 b is a graph comparing the extinction and absorbance measurements of different amounts of LNP and RNA; Figure 8 It is a graph showing the linear relationship between RNA concentration and absorption at 260 nm; Figure 9a and Figure 9b It is a graph showing a further comparison between conventional ultraviolet-visible spectroscopy and the technology of the present invention, including a comparison showing the degree to which scattering dominates in conventional spectroscopy; Figure 10 It is a graph comparing the original measurement results, the calibrated measurement results, and the reference values; Figure 11 This is a table comparing the results obtained according to the present invention with the results of known measurement techniques; Figure 12 a and Figure 12 b are graphs comparing the extinction measurement results and the absorption measurement results of the three samples. Detailed Implementation

[0069] Figure 4 This is a schematic diagram of a device 100 according to the invention. The device includes an integrating cavity 110, the walls of which are coated with a diffuse reflective material. The integrating cavity can be spherical, but many other shapes are also possible. The integrating cavity has a first port 130 and a second port 140. A light source 150 is provided, which emits in a desired ultraviolet-visible wavelength band. As we will discuss below, this may be a limited wavelength range, such as only at 260 nm, a wider wavelength range, or may include a scanning / tuned wavelength light source. The light source 150 is configured to guide probe light to the first port 130 of the integrating cavity 110. The probe light is preferably guided to diffusely scatter on the surface of the integrating cavity. The device also includes a sample space 120, which may be a cuvette. The sample space is configured to hold a liquid suspension sample in the integrating cavity. In this invention, the sample is diffusely scattered, such that the probe light illuminates the sample. If the wavelength matches the absorption band, the sample will absorb some of the probe light. Most of the probe light will also be scattered by the sample, but the integrating cavity will return this sample-scattered light to the sample, allowing for more transmission and absorption by the sample. The second port is offset from the first port, so that light cannot propagate along a straight path from the first port to the second port, but must instead be scattered by the integrating cavity and / or the sample. Probe light arriving at the first port 130 can propagate in a straight line, such that it propagates along a first axial direction. Probe light received at the second port 140 can be received along a second axial direction, and the light is transmitted to the detector along that direction. The first and second axial directions are offset from each other. For example, they are not located directly on opposite sides of the integrating cavity. The integrating cavity may have a centerline, and at least one of the first and second ports is offset from the centerline. The sample space or support may be on the centerline.

[0070] Light exits the integrating sphere at the second port 140 and is directed to detector 160. The detector, which may be part of a spectrometer, may be configured with filters to scan the detection wavelength band. The apparatus also includes an analyzer 170, which receives the signal from the detector and determines quantitative or qualitative properties of the sample. The signal from the detector may represent one or more spectral features of the probe light received at the second port. For example, if a single wavelength (e.g., 260 nm) is used, the signal may be a representation of the amount of light received. Alternatively, the signal may form a spectrum of the amount of light received over a wavelength range. Based on the received signal, the analyzer determines the properties of the sample. These properties may be quantitative properties of the sample, such as the amount of RNA in the sample. Alternatively, the output may be the absorption spectrum of the sample in wavelength ranges such as 240 nm and 320 nm. Other wavelength ranges may also be used.

[0071] The analyzer can determine one or more spectral characteristics of a sample based on one or more spectral features of the probe light received by the detector at the second port. For example, the analyzer can store calibration data or calibration transformations. Calibration data may include calibration of the integrating sphere and / or calibration related to the relative absorbance of the sample substance at 260 nm. For example, a standard absorbance coefficient for RNA, such as 0.025 (μg / ml), is typically used. -1 × cm -1 However, accuracy can be improved by determining the absorption coefficient of a specific RNA preparation. Other calibration information may also be included. There are various possibilities for the size of the sample holder or cuvette; for example, a 10mm cuvette containing 1ml of sample or a 1mm cuvette containing 100μl of sample can be used.

[0072] Figure 4 The setup is designed to measure the absorption of diffusely scattered samples, which cannot be measured by traditional UV-Vis spectroscopy.

[0073] Figure 5An alternative embodiment is shown, which includes additional ports, namely a third port 130' and a fourth port 140'. The third port 130' can be configured to receive probe light from a light source 150 or a secondary light source, but is preferably substantially the same as light source 150. The fourth port 140' can be configured to receive probe light from an integrating sphere that has passed directly through the sample from the third port 130'. The probe light from the fourth port can be collected by detector 160 or a secondary detector substantially the same as detector 160. The arrangement of the third port 130' and the fourth port 140' allows for the measurement of the extinction value of light passing directly through the sample. This measurement is substantially the same as extinction measurements performed by conventional UV-Vis spectrometers. In this example, for diffusely scattering samples such as LNPs containing RNA, most of the probe light will be scattered, and the probe light absorbed by the RNA will dominate. Nevertheless, as we will describe below, including these additional ports allows for scattering measurements to confirm the device's functionality for diffusely scattering samples.

[0074] although Figure 5 The embodiment includes four ports, but other embodiments may include only three ports. For example, the second port 140 may be omitted, such as... Figure 5 The through-pass extinction measurement can be performed by guiding the probe light from the third port 130' to the fourth port 140'. Absorption measurement can be performed by guiding the probe light to the first port 130 and collecting the light at the fourth port 140'. In this way, the two ports used for absorption measurement are still offset, thus collecting light scattered around the integrating sphere. In another example, the first port 130 can be omitted. Similarly, the through-pass extinction measurement can be performed by guiding the probe light from the third port 130' to the fourth port 140'. Absorption measurement can be performed by guiding the probe light to the third port 130' and collecting the light at the second port 140. Again, the two ports used for absorption measurement are still offset, thus collecting light scattered around the integrating sphere. Figure 5 In the arrangement and related setup, the sample does not move between extinction measurement and absorption measurement.

[0075] For traditional UV-Vis spectrometers, according to Beer-Lambert's law, the absorbance A of the sample is given by the following formula: Log(I0 / I) = A = εlc Where I0 is the input light intensity of the sample, I is the light intensity after passing through the sample, ε is the molar attenuation coefficient or absorptivity of the substance, l is the optical path length through the sample, and c is the concentration of the absorbing substance. Therefore, as long as the attenuation coefficient and path length of a specific substance are known, the concentration of that substance in the sample can be determined.

[0076] For the integrating cavity method of this invention, the Beer-Lambert law can still be considered applicable. However, the effective path length used in the above equation needs to be determined through a calibration procedure. This procedure requires measuring a substance of known concentration at the target wavelength and applying curve fitting to obtain the effective path length of the device as a function of wavelength. The wavelength variation of the effective path length takes into account the possible variation of the reflectivity of the integrating cavity walls with wavelength. Once the effective path length is determined through calibration, the device can be used to measure diffusely scattered samples.

[0077] Figure 6 a and 6b show two graphs of RNA and lipid nanoparticle liquid suspensions. Figure 6 In Figure a, the measurement was an extinction measurement performed using conventional UV-Vis spectroscopy. The vertical axis shows the optical density (i.e., a measure of light loss as it passes through the sample), and the horizontal axis shows how this varies with wavelength between 240 nm and 320 nm. Figure 6 The graph for 'a' has two curves. The top curve represents empty LNPs (containing no RNA) in a buffer solution containing 20 μg / ml RNA, and the bottom curve represents empty LNPs (containing no RNA) in a buffer solution containing 10 μg / ml RNA. In other words, the sample with the top curve has twice the amount of RNA as the sample with the bottom curve. The conventional wavelength for measuring RNA concentration is 260 nm. At this wavelength, we expect to see that the absorbance of the sample with the top curve is twice that of the sample with the bottom curve due to the difference in concentration. However, as... Figure 6 As shown in figure a, the OD values ​​are 0.52 and 0.77, respectively. This is the result of strong scattering caused by LNP.

[0078] Figure 6 b. The apparatus and method of the present invention were used. The compositions of two identical samples were examined. At a wavelength of 260 nm, the respective OD values ​​were 0.27 and 0.54, consistent with doubling of the OD value upon doubling of the concentration. Figure 6 In diagram b, the dashed line represents other samples without LNPs, indicating that all RNA in the mixed sample has been correctly measured. Using the method of this invention, the effects of scattering have been eliminated. Therefore, Figure 6 These measurements in b can be considered to be related to scattering-corrected absorption (SCA).

[0079] In traditional techniques, extinction measures the sum of absorption and scattering, which are inseparable. Therefore, for conventional UV-Vis spectroscopy, extinction can be considered to be given by the following equation: Extinction = Absorption + Scattering Figure 7 a and Figure 7b shows a further comparison between conventional UV-Vis spectroscopy and the apparatus and method of this invention. As previously mentioned, most lipids do not absorb at 260 nm, which is a typical RNA absorption peak; therefore, if LNP scattering can be eliminated, we can directly measure the amount or concentration of RNA from the absorption peak. For Figure 7 a and 7b show the data for the following samples: A. 10 μg / ml RNA concentration and empty (unloaded) LNP (100%); B. 10 μg / ml RNA concentration compared to empty (unloaded) LNPs (50% mass reduction compared to A); C. 10 μg / ml RNA concentration and empty (unloaded) LNP (0%); D. 5 μg / ml RNA concentration compared to empty (unloaded) LNPs (same mass, 100% as A and B); and E. 5 μg / ml RNA concentration compared to empty (unloaded) LNP (200% increase in mass compared to A and B).

[0080] Therefore, the samples contained two different amounts of RNA. Sample AC had the most RNA, at a concentration of 10 μg / ml. Samples D and E contained 50% of the RNA of sample AC, at a concentration of 5 μg / ml.

[0081] Figure 7 a is with Figure 6 A similar curve, but Figure 7 a shows the extinction values ​​measured using conventional UV-Vis spectroscopy. Samples A, B, and C are shown in... Figure 7 The values ​​marked with an "x" are on the label. These samples have the same amount of RNA but different amounts of empty LNPs. The conventionally measured OD values ​​are different. This is a result of the different amounts of scattering caused by the different amounts of LNPs. Figure 7 In sample b, with OD values ​​measured according to the present invention, these three samples exhibit substantially the same absorbance at 260 nm, approximately 0.27 to 0.28. The same pattern can be observed for samples D and E, which are represented by triangles in the graph. Comparing ABC with DE, it can be seen that the sample with twice the RNA content has twice the absorbance. Therefore, the absorbance shows a linear relationship with the RNA content. In summary, based on these samples, it has been found that samples with the same RNA content, as measured according to the present invention, have OD measurements that differ by less than 3%.

[0082] Figure 8The nominal RNA concentration and absorbance at 260 nm were compared among nine samples with different amounts of RNA and LNP. The fit was highly linear, indicating that the absorbance can be considered proportional to the amount of RNA, but independent of the presence of LNP and the resulting scattering.

[0083] Figure 9a This is a graph showing a further comparison between conventional UV-Vis spectroscopy and the technique of this invention. The graph illustrates the degree to which scattering dominates in conventional spectroscopy. In conventional spectroscopy, extinction is measured, i.e., the top curve (B) in the graph. As previously mentioned, light scattering is known to be proportional to the sixth power of the particle diameter and the negative fourth power of the wavelength. The amount of scattering can be inferred from the fitting of the extinction measurement. The fitting can be performed using an equation of the following form: S(λ) = aλ -4 + b Furthermore, assuming no scattering occurs between 340 nm and 370 nm, scattering is deduced, and extinction E, absorption A, and scattering S are related by the following equation: E(λ) = A(λ) + S(λ) Using these equations, scattering is estimated and shown as the top dashed line C in the figure, and absorption can also be estimated and shown as the bottom dashed line E. Absorption is measured using this invention (line A, labeled SCA spectrum). As can be seen from the lower lines, the absorption estimated from conventional UV-Vis spectroscopy does not match the absorption measured by this technique, and there is a significant error, e.g., >25%.

[0084] Figure 9b Showing with Figure 9a Similar information, except the graph also shows a dashed line, which is a reference measurement for RNA lacking LNPs using conventional absorption / extinction methods. From Figure 9b It can be seen that the absorption calculated using scattering difference (III) underestimates the actual absorption of RNA (IV), while in contrast... Figure 9a It can be seen that SCA measurement provides a good estimate of RNA. However, the extrapolation of scattering using the conventional method described above does not account for the impact of absorption on scattering. Therefore, the conventional method contains inherent inaccuracies, while the SCA method of this invention provides a more accurate measurement.

[0085] Figure 10 This is a graph illustrating the calibration according to the method of the present invention. The curve in the graph is for RNA at 20 μg / ml in buffer. The dashed line at the top of the graph represents the measured (raw) data, which, after applying the calibration transformation discussed herein, resulted in the solid curve in the graph. The graph also shows measurements of the same sample measured using the conventional UV-Vis extinction method. Figure 10The measurements do not contain LNPs or other scattering substances. It can be seen that the calibration measurements according to the present invention match the reference measurements very well.

[0086] For some LNPs, lipids may exhibit absorption at a measurement wavelength of 260 nm. The absorption of empty LNPs and RNA-loaded LNPs can be measured using the method of this invention. Since absorption is additive, the absorbance of RNA can be found by subtracting the absorbance of the empty LNP (taking into account any concentration differences, etc.) from the loaded measurement. Therefore, the quantification of LNPs and RNA, as well as the LNP loading ratio, can be determined.

[0087] The technology of this invention has also been compared with other conventional technologies (such as RiboGreen fluorescence measurement technology). Figure 11 The table shows a comparison of the results. Figure 11 The results were obtained for two different LNP formulations (SM102-LNP and a proprietary absorbent LNP) and two different RNAs (RNA1 and RNA2). Results for the 260 nm absorbent LNP were determined by linear degradation as described above. All results were for a nominal concentration of 400 μg / ml RNA. The results of this invention are comparable to those of assay techniques. The results of this technique use a universally accepted standard extinction coefficient of 0.025 (μg / ml). -1 × cm -1 The results are obtained, but accuracy can be improved by using or determining the extinction coefficient of the RNA of actual interest.

[0088] The scattering-corrected absorption method of the present invention, applied to nanoparticle delivery carriers carrying payloads, differs from the analysis of other turbid or diffusely scattering samples. In many scattering samples, the scattering sample absorbs light in the liquid phase, while solids or semi-solids suspended in the liquid (e.g., suspensions or colloids) produce scattering. However, in the application of nanoparticle delivery carriers, the delivery carrier itself typically absorbs very little in the liquid. RNA absorbs both in the liquid and in the delivery carrier. Typically, formulations tend to achieve 100% encapsulation efficiency, therefore the amount of RNA measured by SCA is a good indicator of the actual RNA or other payload content.

[0089] We have already described this technique, which we refer to above as scatter-corrected absorption (SCA), with reference to lipid nanoparticles (LNPs) carrying RNA. This technique is also applicable to many other nanomedicine examples. For instance, it can be used to measure other nanoparticle delivery carriers carrying other therapeutic payloads, such as the following delivery carriers: Antibody-drug conjugates, • Viral vectors (such as lentiviruses and AAVs). Liposomes ·Exosomes, • Polymer nanoparticles • Liposome-based systems (e.g., lipid complexes) Metal nanoparticles, SPION Polymer micelles • Dendritic polymers, Nanoemulsions, and • Nanogel.

[0090] As we have discussed, the payload can include RNA. For example, this could be mRNA, siRNA, or ssRNA. The payload can also be one or more of the following: Oligonucleotides DNA ·drug, ·vaccine, Cancer treatment, and Gene editing therapy.

[0091] As supplementary information, we now provide further information regarding the background and relevance to this invention.

[0092] Nanoparticle carriers or nanoparticle delivery carriers can carry or transport payloads of various compositions, such as drugs, biologics, or bioactive compounds. The payload may, for example, include therapeutic agents, such as oligonucleotides like mRNA, siRNA, or DNA, or may include active agricultural agents or ingredients, or active pharmaceutical agents or ingredients.

[0093] Nanoparticle delivery carriers or systems are engineered technologies that use nanoparticles for targeted delivery and / or controlled release of payloads.

[0094] For example, nanoparticle drug delivery carriers or systems are widely used for targeted delivery and / or controlled release of therapeutic agents. One example of such nanoparticle delivery carriers or systems is a lipid-based delivery carrier. Lipid-based delivery carriers include lipid nanoparticles, such as (solid) lipid nanoparticles and liposomes.

[0095] Lipid nanoparticles are nanoparticles composed of lipids and have been developed as carriers for small molecule delivery. First approved in 2018 as a drug delivery carrier for the siRNA drug Patisiran (trade name Onpattro), lipid nanoparticles are now a key component of COVID-19 mRNA vaccines.

[0096] Lipid nanoparticles can be, for example, typically substantially spherical, with an average diameter between 10 and 1000 nanometers, typically in the range of 60 nm to 150 nm or 80 nm to 150 nm.

[0097] Solid lipid nanoparticles may have, for example, a solid lipid core matrix capable of dissolving lipophilic molecules. The lipid core may be stabilized by surfactants, such as emulsifiers. The lipid components may include one or more of the following, such as triglycerides, diglycerides, monoglycerides, fatty acids, steroids, or waxes.

[0098] Biomembrane lipids such as phospholipids, sphingomyelins, bile salts (sodium taurocholate), and sterols (cholesterol) can be used as stabilizers.

[0099] Drug payloads can, for example, be embedded inside solid lipid nanoparticles.

[0100] The lipid nanoparticles used in COVID-19 mRNA vaccines are typically composed of four types of lipids: ionizable cationic lipids (which bind to negatively charged mRNA with their positive charge), PEGylated lipids (for stability), phospholipids (for structure), and cholesterol (for structure).

[0101] The lipid portion and composition of lipid nanoparticles vary depending on the formulation, and typically include 3 to 4 components, such as cholesterol, a stable phospholipid, a PEGylated lipid, etc.

[0102] Liposomes can be viewed as “hollow” lipid nanoparticles, which can, for example, have a phospholipid bilayer and an interior composed of an aqueous substance. Drug payloads can, for example, be embedded within the interior of this lipid nanoparticle.

[0103] Liposomes can, for example, comprise basic spherical vesicles formed by the self-assembly of synthetic or natural phospholipids. These vesicles can have an aqueous core (surrounded by a hydrophobic membrane) and can load a variety of hydrophobic or hydrophilic molecules, for example, for therapeutic purposes.

[0104] Particle size, particle concentration, payload concentration, and payload-to-particle load are key parameters that need to be measured in the development and production of nanoparticle carriers (NPVs) or nanoparticle carrier systems, applicable to therapeutic, agricultural, and skincare fields. Nanoparticle carriers (nanoparticle delivery carriers) or NPV systems comprise (i) a particle or carrier component and (ii) a payload component.

[0105] The particulate or carrier component may be made of or contain various materials, including but not limited to: • Lipid preparations or lipid compositions, • Polymer formulations or polymer compositions, • Liposome formulations or liposome compositions, Other nanoparticle formulations or nanoparticle compositions; The payload portion or component may be made from or contain the following: mRNA, ·siRNA, DNA Other therapeutic payloads, • Active agricultural ingredients • Active pharmaceutical ingredients.

[0106] Practical, accurate, and cost-effective analytical techniques are needed to measure these characteristics. Optical methods, including ultraviolet-visible (UV-Vis) spectroscopy, are used to measure these parameters.

[0107] In standard UV-Vis instruments, when a sample scatters light, the intensity of the transmitted light, as a function of wavelength, is affected by the scattering and absorption of the sample. This produces a wavelength-dependent "extinction" spectrum, where extinction = scattering + absorption. Typically, absorption spectra are the primary focus when using UV-Vis spectroscopy because they can be used to quantify analyte concentrations via Beer-Lambert's law. When the sample scatters light, the scattering interferes with obtaining the sample's true absorption spectrum, rendering Beer-Lambert's law invalid. Consequently, concentration estimates will be inaccurate.

[0108] Nanoparticle carriers (NPVs) typically range in size from 60 nm to 80 nm to 150 nm in diameter. Due to their size and refractive index, NPVs significantly scatter light in the ultraviolet region (the area where the payload and NPV particles partially absorb light). Therefore, attempting to optically measure the components of an NPV system (e.g., NPV concentration, payload / NPV concentration, payload load) using conventional UV-Vis spectroscopy techniques is challenging because light scattering interferes with obtaining useful absorption spectra.

[0109] A method is needed to subtract / remove scattering contributions from extinction measurements to obtain accurate results. Such methods include: A. Background subtraction: The scattering of the sample is approximated by measuring the UV-Vis spectrum of the sample in a region where it does not absorb light, and the measured value is removed from the measured extinction spectrum as a “flat background”.

[0110] B. Approximate Model Subtraction: The wavelength-dependent light scattering spectrum of the sample can be approximated by a mathematical model (e.g., Rayleigh scattering) to produce a theoretical scattering spectrum, which can then be removed as background from the measured extinction spectrum.

[0111] C. Measuring sample scattering: The scattering spectrum of the sample can be measured orthogonally or separately, and then subtracted from the measured extinction spectrum.

[0112] However, these methods have significant drawbacks in certain situations, especially when: 1. The NPV scattering spectrum is significantly stronger than the absorption spectrum of the component. In this case, the absorption spectrum is almost indistinguishable against a large scattering background, and the background subtraction method will be insufficient to separate an accurate absorption estimate (similar to fluorescence interference in Raman spectroscopy).

[0113] 2. The NPV component and the payload / payload component have overlapping absorption spectra. In this case, background subtraction will be insufficient to deconvolve the relative contribution of each NPV component to the absorption.

[0114] Other techniques used to characterize NPV components include: • Fluorescence assay (Ribogreen assay): Requires multiple sample preparation steps, and each sample measurement takes at least 10 minutes.

[0115] • Field-current separation-multi-angle light scattering (FFF-MALS): Only measures particle size, not concentration, and is very complex.

[0116] • Dynamic light scattering (DLS): Measures only particle size, not concentration. It can only measure particles exceeding a certain size threshold and has a strong bias towards large particles in the sample.

[0117] • Particle tracking analysis (PTA): This method can measure particle size and concentration, but can only measure particles exceeding a certain size threshold. Samples need to be significantly diluted before measurement.

[0118] One object of the present invention and disclosure is to provide an alternative device and method for characterizing nanoparticle carriers or nanoparticle carrier systems, or to provide an device and method for characterizing nanoparticle carriers or nanoparticle carrier systems that overcomes the inconveniences of the aforementioned known devices and methods.

[0119] One object of this disclosure is to provide a nanoparticle carrier analyzer or a nanoparticle carrier measurement / characterization device.

[0120] One object of this disclosure is to provide a nanoparticle analyzer or nanoparticle measurement / characterization device for determining, for example, - The effective payload or effective payload concentration of the nanoparticle carrier, and / or - The effective load of the nanoparticle carrier versus the carrier content / concentration ratio, and / or - The composition of the nanoparticle carrier, including the ratio or fraction content of the carrier components, and / or - The concentration of nanoparticle carriers in the solution or formulation.

[0121] One object of this disclosure is to provide an analytical or measurement / characterization method for nanoparticle carriers.

[0122] One object of this disclosure is to provide an analytical or measurement / characterization method for nanoparticle carriers, for determining, for example... - The effective payload or effective payload concentration of the nanoparticle carrier, and / or - The effective load of the nanoparticle carrier versus the carrier content / concentration ratio, and / or - The composition of the nanoparticle carrier, including the ratio or fraction content of the carrier components, and / or - The concentration of nanoparticle carriers in the solution or formulation.

[0123] Embodiments of the present invention provide a nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device, comprising: An integrating cavity, including one or more reflective inner walls, is configured to receive a cuvette or sample holder within the integrating cavity, the cuvette or sample holder being configured to contain a liquid sample or solution comprising at least one or more nanoparticle carriers. The integrating cavity includes at least one optical inlet port and at least one optical outlet port, wherein one or more optical inlet ports are configured to receive light from at least one light source, and one or more optical outlet ports are configured to deliver light to a detector or spectrometer. The lipid-based drug delivery carrier analyzer or lipid-based drug delivery carrier measurement / characterization device is configured to operate in diffuse reflection mode, in which light from the light source follows an optical path from one or more inlet ports into the integrating cavity, is incident on the reflective inner wall of the integrating cavity, and is diffusely reflected within the integrating cavity, such that the light from the light source illuminates the liquid sample before being transmitted through one or more optical outlet ports and received by a detector or spectrometer for optical wavelength analysis to provide properties such as absorption spectra of at least one or more nanoparticle carriers contained in the liquid sample or solution.

[0124] In one specific embodiment, the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device is a lipid-based nanoparticle carrier analyzer or lipid-based nanoparticle carrier measurement / characterization device. Lipid-based nanoparticle carriers may include or consist of (e.g.) (solid) lipid nanoparticles and / or liposomes.

[0125] Embodiments of the present invention relate to a method for the analysis or measurement / characterization of nanoparticle carriers, comprising: - Provide a nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device, or provide a device configured to measure or determine at least one scattering-corrected absorption measurement result or scattering-corrected absorption spectrum; and / or - Acquire / receive at least one or more scatter-corrected absorption measurements / data or scatter-corrected absorption spectroscopy measurements / data for a liquid sample or solution containing multiple nanoparticle carriers.

[0126] In one specific embodiment, the nanoparticle carrier analysis or measurement / characterization method is a lipid-based nanoparticle carrier analysis or measurement / characterization method. Lipid-based nanoparticle carriers may include or consist of (e.g.) (solid) lipid nanoparticles and / or liposomes.

[0127] This invention provides alternative devices and methods to known devices and methods. This invention provides a device and method that overcomes the inconveniences of the previously mentioned known devices and methods.

[0128] Furthermore, this invention overcomes the shortcomings of the traditional UV-Vis method by employing an integrating cavity-based spectrophotometer for absorption measurement. Using an integrating cavity-based device eliminates the influence of scattering on the measured absorption spectrum, thereby generating a "pure absorption" spectrum that more accurately represents the characteristics of the nanoparticle carrier.

[0129] This invention guarantees prior scattering correction and absolute quantification, for example, using a quantification method similar to that of Beer-Lambert.

[0130] The apparatus and method of this invention ensure unique scattering correction, allowing for accurate composition measurements. The determination of the payload concentration is independent of particle size / scattering.

[0131] The apparatus and method of the present invention eliminate unwanted scattering contributions and ensure accurate or more accurate measurements of the concentration or characterization of nanoparticle carriers (e.g., drug delivery nanoparticle carriers).

[0132] The apparatus and method of the present invention advantageously ensure rapid and accurate measurement of the concentration of a payload (e.g., a drug), wherein the measurement requires only simple dilution. The apparatus and method of the present invention advantageously ensure rapid and accurate measurement of the concentration ratio of the payload to the carrier (e.g., a drug to a lipid).

[0133] Integrating cavities have never been used or suggested as a way to characterize nanoparticle carriers or lipid-based drug delivery carriers before.

[0134] The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device of the present invention may, for example, be, include, or consist of the spectrometer apparatus disclosed in International Patent Application WO2018070882, the entire contents of which are incorporated herein by reference and attached to this application. This apparatus is available from MARAMA LABS under the trade name CloudSpec™.

[0135] In some embodiments, the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device may differ from the apparatus described in WO2018070882 in several respects. For example, the analyzer / device described herein may not include the input optical path modulator 13 and / or the output optical path modulator 13B. Furthermore, the integrating cavity may only include the input port P2 and the output port P4 for absorption measurement.

[0136] Measuring the absorption of a scattering sample using an integrating sphere physically / optically eliminates the contribution of light scattering to the absorption measurement; therefore: • It is much more effective in obtaining true absorbance measurements compared to background subtraction methods.

[0137] • No scattering spectrum measurement or calculation (in other techniques, this is to subtract the appropriate scattering spectrum) is required to obtain scattering-corrected absorption. In particular, it is independent of particle size, particle size distribution (Pdi), or the refractive index of the material.

[0138] This method opens up many valuable applications that go beyond existing technologies, as described below.

[0139] A. Rapid payload measurement CloudSpec eliminates light scattering interference in NPV absorption measurements by using an integrating sphere, thus enabling accurate measurement of the target component's concentration. The advantages of CloudSpec are: • Rapid measurement of payload concentration – requires only simple sample dilution. • Measure the concentration ratio of payload to carrier (e.g., payload to lipid). • Measure the effective payload concentration in the scattering sample, which is much lower than that of the background subtraction method. • It can characterize NPV itself, for example, by composition or concentration.

[0140] Most NPVs scatter in the ultraviolet region and scatter little in the visible region, making scattering correction difficult or impossible.

[0141] As described in WO2018070882, the CloudSpec device can simultaneously measure two spectra of a liquid sample; the first is the "extinction spectrum," where the sample is measured under transmission geometry, and the decrease in light intensity is a result of sample absorption and scattering. This is equivalent to the spectrum produced by a conventional UV-Vis spectrophotometer. The second is the "absorption spectrum," where the sample is measured under integrating cavity geometry, and the decrease in light intensity is solely a result of sample absorption.

[0142] Exemplary measurements were performed on liquid samples of nanoparticle carriers contained in liquid using the CloudSpec device. The results clearly demonstrate that the CloudSpec device and / or the integrating cavity arrangement can provide true and useful absorption spectra of nanoparticle carrier samples, namely scatter-corrected absorption, which can be used to characterize multiple features of the nanoparticle carriers.

[0143] Some results of exemplary measurements of extinction and absorption performed on nanoparticle supports, such as Figure 12 As shown in (a) and 12(b), the nanoparticle carrier comprises a particle / carrier component consisting of (solid) lipid nanoparticles and a payload component, which is either (i) absent, (ii) composed of mRNA, or (iii) composed of siRNA.

[0144] The lipid nanoparticles used in the measurements typically contain three to four constituent carrier elements, such as cholesterol, a stable phospholipid, and a PEGylated lipid. The average size / diameter (measured by DLS) of the aforementioned lipid-based nanoparticle carriers are (i) 56.4 nm, (ii) 96.9 nm, and (iii) 76 nm, respectively.

[0145] The liquid sample is provided in the cuvette positioned within the integrating chamber, as mentioned earlier.

[0146] The sample was diluted 20-fold in Tris buffer and measured with distilled water as a blank. No stirring was performed during the measurement.

[0147] from Figure 12 (b) It can be seen that a strong absorption characteristic corresponding to the absorption of the payload (mRNA or siRNA) was obtained near 260 nm. The contribution of nanoparticle carriers lacking the payload to the absorption at this characteristic wavelength is negligible, thus allowing the absorption measured at this wavelength to be used for characterization, for example, the payload concentration.

[0148] Clearly, this method and principle can be extended and applied to characterize various features of nanoparticle carriers.

[0149] Figure 12The extinction measurement results in (a) confirm that the aforementioned correction method (i.e., subtracting / removing the scattering contribution of the unloaded nanoparticle carrier from the extinction measurement of the nanoparticle carrier with effective load in an attempt to obtain an accurate measurement) cannot provide sufficient correction, nor can it obtain accurate results. Figure 12 The absorption values ​​presented in (b) are the accurate measurements.

[0150] like Figure 12 As shown in (a), scattering masks the uptake of lipids and RNA, resulting in no signal at OD600. Figure 12 (b) shows that obtaining true lipid and RNA uptake makes it possible to measure absolute and relative concentrations.

[0151] The methods and principles disclosed herein can characterize various features of nanoparticle carriers, such as the effective payload capacity in NPV mentioned above.

[0152] As mentioned earlier, measuring the payload capacity in NPVs is difficult and is typically accomplished using inconvenient and resource-intensive techniques such as FFF-MALS and HPLC. UV / Vis spectroscopy can theoretically be used to quantify the payload capacity in NPVs, for example, by using the A260 peak, but particle scattering introduces artifacts whose magnitude varies across NPV systems.

[0153] NPVs both absorb and scatter light in the ultraviolet region due to factors such as refractive index, particle size, particle size distribution, and composition, which can introduce significant errors when quantifying components using light absorption. Since these factors vary from formulation to manufacturing process, it is neither convenient nor possible to construct simple corrections independent of other input data (e.g., particle size or concentration). CloudSpec enables prior scattering correction and absolute quantification using methods similar to Beer-Lambert quantification.

[0154] NPV varies depending on the formulation and typically has 3 to 4 constituent carrier elements, such as cholesterol, a stable phospholipid, a PEGylated lipid, etc.

[0155] The apparatus and methods disclosed herein can be advantageously used, for example, as follows: 1. The loading of the NPV formulation was quantified using scatter-corrected absorption, using the extinction coefficient of the loading. 2. Use scatter-corrected absorption to quantify the loading of the payload and / or carrier (e.g., lipid) portion in the NPV formulation, using the extinction coefficients of the payload and carrier (lipid) portions (combined or separate). 3. Use scatter-corrected absorption to quantify the relative fraction of the carrier (e.g., lipid) components, using the extinction coefficients of the individual carrier (lipid) constituent elements. 4. All of the above relate to various lipid-based carrier payloads, such as liposomes.

[0156] Another exemplary and valuable application that transcends existing technologies is: B. Rapid particle concentration measurement NPVs tend to absorb light in the ultraviolet region, but they also scatter light significantly. CloudSpec eliminates scattering, making Beer-Lambert concentration measurements possible. The advantages of CloudSpec are: • Use scattering-corrected absorption to measure particle concentration by mass.

[0157] • Measure particle concentration using externally measured size information.

[0158] • Use extinction (scattering) to approximate particle size parameters.

[0159] In other words, the methods and principles of this disclosure can characterize the features of nanoparticle carriers, namely the particle concentration of NPV.

[0160] Measuring the concentration of NPV in solution is difficult because NPV is small and discrete in buffer solutions, so the extinction coefficient varies with particle number and size. UV / Vis spectroscopy can theoretically be used to measure particle concentration in NPV, for example, by using the absorption of the payload or carrier (lipid) portion, but particle scattering causes artifacts whose size varies between NPV systems, and particle size may not be approximated by scattering-corrected absorption (SCA).

[0161] NPVs both absorb and scatter light in the ultraviolet region due to factors such as refractive index, particle size, particle size distribution, and composition, which can introduce significant errors when quantifying using light absorption. Since these factors vary depending on the formulation and manufacturing process, measuring particle concentration is neither convenient nor feasible. CloudSpec enables prior scattering correction and absolute quantification using methods similar to Beer-Lambert quantification.

[0162] If the particle size is known, for example, obtained from DLS measurements, the particle concentration can be measured using SCA.

[0163] Scattering spectroscopy and Mie theory can be used to approximate dimensions, thus eliminating the need for other measurements (such as DLS).

[0164] Therefore, the apparatus and method of this disclosure can be advantageously used, for example, as follows: 1. Particle concentration at NPV in formulations is quantified using scatter-corrected absorption, employing the extinction coefficient of the payload and particle size measured by techniques such as DLS and PTA. 2. Use scattering spectroscopy to quantify the particle concentration of NPV in the formulation, using calculated scattering spectra (from the Michaelis theory) and particle size measured by techniques such as DLS and PTA.

[0165] The above and other objects, features, and ways of achieving them will become more apparent, and the invention itself will be best understood by studying the following description. The foregoing general description and the following detailed description are used to explain the inventive features of this disclosure.

[0166] Exemplary and non-limiting embodiments of the present invention are now provided.

[0167] According to embodiments, the exemplary nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device of this disclosure may include, be based on or comprise the apparatus described in International Patent Application WO2018070882, the entire contents of which are incorporated herein by reference and are included as an appendix to this application.

[0168] Nanoparticle carrier analyzers or nanoparticle carrier measurement / characterization devices are, for example: - Nanoparticle carrier effective loading capacity or effective loading concentration analyzer or measurement / characterization equipment, and / or - Nanoparticle carrier effective load to carrier content / concentration ratio analyzer or measurement / characterization equipment, and / or - Nanoparticle carrier ratio or fractional carrier component content analyzer or measurement / characterization equipment, and / or - Nanoparticle carrier solution concentration analyzer or measurement / characterization equipment.

[0169] Nanoparticle carrier analyzers or nanoparticle carrier measurement / characterization devices include, for example: An integrating cavity, including one or more reflective inner walls, is configured to receive a cuvette, the cuvette being configured to hold a liquid sample within the integrating cavity. The integrating cavity includes at least one optical inlet port and at least one optical outlet port, wherein one or more optical inlet ports are configured to receive light from at least one light source, and one or more optical outlet ports are configured to transmit light to a spectrometer; The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device is configured to operate in diffuse reflection mode. In this mode, light from the light source enters the integrating cavity from one or more inlet ports, is incident on the reflective inner wall of the integrating cavity, and is diffusely reflected within the integrating cavity. This allows the light from the light source to irradiate the liquid sample before being transmitted through one or more light outlet ports and received by the spectrometer for wavelength analysis to provide the absorption spectrum of the liquid sample.

[0170] Specific embodiments of the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device are described in International Patent Application WO2018070882 and are also mentioned in the claims.

[0171] In one specific embodiment, the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device is, for example, a lipid-based nanoparticle carrier analyzer or a lipid-based nanoparticle carrier measurement / characterization device.

[0172] A nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device is, for example, a lipid-based drug delivery carrier analyzer or lipid-based drug delivery carrier measurement / characterization device. The lipid-based drug delivery carrier analyzer or lipid-based drug delivery measurement / characterization device is used (or configured to) determine, for example, the drug loading of a lipid-based drug delivery carrier, or determine the ratio of drug loading to lipid content / concentration of a lipid-based drug delivery carrier, or determine the lipid composition ratio of a lipid-based drug delivery carrier.

[0173] A nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device is, for example, a lipid-based nanoparticle analyzer or lipid-based nanoparticle measurement / characterization device, used to determine the concentration of lipid-based nanoparticles in a solution or formulation.

[0174] Lipid-based nanoparticle carriers may include or consist of (e.g.) (solid) lipid nanoparticles and / or liposomes, or lipid emulsions.

[0175] Nanoparticle carriers may, for example, include or consist of lipid-based nanoparticles as described above. Nanoparticle carriers may alternatively include or consist of polymer nanoparticles (e.g., polymer nanocapsules or nanospheres), polymer vesicles, dendritic polymers, or polymer micelles.

[0176] Nanoparticle carriers may alternatively include or consist of inorganic nanoparticles (e.g., silica nanoparticles, quantum dots, iron-based or iron oxide nanoparticles, or gold nanoparticles).

[0177] Nanoparticle carriers may alternatively include or consist of exosomes, microvesicles, lentiviruses, adeno-associated viruses (AAVs), or adenoviruses.

[0178] The particles or carrier components of the nanoparticle carrier may be made of, for example, various materials, including but not limited to: • Lipid preparations or lipid compositions, • Polymer formulations or polymer compositions, • Liposome formulations or liposome compositions, Other nanoparticle formulations or nanoparticle compositions; The payload portion or component of the nanoparticle carrier may, for example, be made of or contain the following: mRNA, ·siRNA, DNA Other therapeutic payloads or cargo, • Active agricultural ingredients • Active pharmaceutical ingredients.

[0179] This disclosure also relates to, for example, an analytical method or measurement / characterization method for nanoparticle carriers.

[0180] This disclosure also relates to an analytical or measurement / characterization method for nanoparticle carriers, for example, for: - Determine the effective loading amount or effective loading concentration of the nanoparticle carrier, and / or - Determine the effective load of the nanoparticle carrier as a ratio of carrier content / concentration, and / or - Determine the composition of the nanoparticle carrier, including the ratio or fractional content of the carrier components, and / or - Determine the concentration of nanoparticle carriers in the solution or formulation.

[0181] The analytical or measurement / characterization methods can be performed using the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization equipment described above.

[0182] The device can generate two spectra for a liquid sample, which can be measured simultaneously. The first is the "extinction spectrum," where the sample is measured under transmission geometry, and the decrease in light intensity is a result of sample absorption and scattering. This is equivalent to the spectrum produced by a conventional UV-Vis spectrophotometer. The second is the "absorption spectrum," where the sample is measured under integrating cavity geometry, and the decrease in light intensity is solely a result of sample absorption.

[0183] The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device may include, for example, at least one processor or computing device; and at least one memory or storage device, including a computer program, the computer program including instructions that, when executed by the processor or computing device, cause the analyzer or measurement / characterization device to perform all or part of the steps of the analytical method or measurement / characterization method, or to perform the method described in any of the following claims.

[0184] This disclosure also relates to a computer program comprising instructions that, when executed by a computer, processor, or computing device, cause the computer, processor, or computing device to perform an analysis method or a measurement / characterization method. This disclosure also relates to a computer-readable data carrier on which a computer program is stored.

[0185] Further details of non-limiting and exemplary embodiments of the analytical or measurement / characterization methods disclosed herein are now provided, including methods and measurements of payload capacity (NPV) and rapid particle concentration.

[0186] AI uses the extinction coefficient of the payload to quantify the payload loading in NPV formulations. To obtain the effective payload capacity of an NPV sample using the extinction coefficient of the effective payload: 1. Dilute the sample in an appropriate buffer solution as needed to bring the absorbance within the operating range of the integrating sphere spectrophotometer.

[0187] 2. The measurement buffer solution is used as a "blank".

[0188] 3. Using buffer solution as a blank, measure the scattering-corrected absorption of the NPV sample.

[0189] 4. Record the scattering-corrected absorbance of the payload at the target wavelength (e.g., 260 nm for mRNA payload).

[0190] 5. To obtain the extinction coefficient of the payload, it can be determined directly by measuring a known amount of payload or the concentration of a series of pure payload solutions in a UV-Vis instrument, or by looking up its value in the literature.

[0191] 6. Based on the NPV and payload properties, there are two methods to determine the payload capacity: a. Method A (Direct Beer-Lambert Law): If the payload and NPV do not absorb in the same wavelength region: i. Calculate the concentration of the effective payload in the NPV sample using the Beer-Lambert law: c payload = A(λ) SC / ε D (λ), where c payload It represents the effective payload concentration, A(λ). SC The measured scattering correction absorption of the NPV sample at the target wavelength (corrected for any dilution factor applied in step 1), where λ is the wavelength and ε is the scattering correction absorption. D (λ) is the molar extinction coefficient of the payload at the target wavelength.

[0192] b. Method B: If the payload and NPV do indeed absorb in the same wavelength region, use a spectral deconvolution method (e.g., multivariate curve resolution) to determine the separate scattering-corrected absorption spectra of the payload and NPV respectively, and repeat steps 6(a)(i) and 6(i) using the absorption values ​​in the separate payload spectra.

[0193] 7. If 100% of the payload is encapsulated in the NPV, the determined load concentration is equal to the payload capacity. If the payload encapsulation rate is less than 100%, the load value should be scaled accordingly.

[0194] A more detailed example is as follows: The generally accepted extinction coefficient for RNA at 260 nm is 0.025 μg / ml. -1 cm -1 In other words, an absorbance (optical density, OD, in a 1 cm cuvette) of 1 at 260 nm corresponds to a concentration of 40 μg / ml. Assuming no other components show significant absorption at 260 nm, the mRNA concentration can be extracted from the absorbance A_260 at 260 nm, using the formula: c mRNA = A 260 * 40 μg / ml If dilution has been performed, the above loading corresponds to the diluted sample; the original loading can be obtained by multiplying by the dilution factor.

[0195] For example, Figure 12 (a) and 12(b) are the absorption spectra (integrating sphere mode, i.e., scattering-corrected absorption) of the LNPs with and without payloads, measured in Cloudspec. For mRNA NPV, we see A_260 ≈ 0.044. Considering a 20-fold dilution, this corresponds to the payload: c mRNA = 0.044 * 40 * 20 = 35.2 μg / ml The spectrum of empty NPVs clearly shows that they do not contribute to A260. This means that the payload concentration can be directly quantified from the A260 of the CS scattering-corrected absorption spectrum.

[0196] A.II. Quantification of payload and / or lipid fractions in NPV formulations using extinction coefficients of payload and lipid fractions. Partial load capacity To obtain the payload capacity and / or lipid fraction of an NPV sample using the extinction coefficients of the payload and lipid fraction: 1. Dilute the sample in an appropriate buffer solution as needed to bring the absorbance within the operating range of the integrating sphere spectrophotometer.

[0197] 2. The measurement buffer solution is used as a "blank".

[0198] 3. Using buffer solution as a blank, measure the scattering-corrected absorption of the NPV sample.

[0199] 4. Record the scattering-corrected absorbance of the payload at the target wavelength (e.g., 260 nm for mRNA) and NPV, and multiply the measured absorbance by the dilution factor used in step 1.

[0200] 5. To obtain the extinction coefficient of the payload, it can be determined directly by measuring a known amount of payload or the concentration of a series of pure payload solutions in a UV-Vis instrument, or by looking up its value in the literature.

[0201] 6. To obtain the extinction coefficient of the NPV fraction, it can be determined directly by measuring the concentration of a known amount of NPV fraction or a series of pure NPV fraction solutions in a UV-Vis instrument, or by looking up its value in the literature.

[0202] 7. Based on the properties of NPV and payload, there are two methods to determine the payload and NPV concentration: a. Method A (Direct Beer-Lambert Law): If the payload and NPV do not absorb in the same wavelength region: i. Calculate the concentration of the effective payload in the NPV sample using the Beer-Lambert law: c payload = A(λ) SC / ε D (λ), where c payload It represents the effective payload concentration, A(λ). SC The measured scattering correction absorption of the NPV sample at the target wavelength (corrected for any dilution factor applied in step 1), where λ is the wavelength and ε is the scattering correction absorption. D (λ) is the molar extinction coefficient of the payload at the target wavelength.

[0203] ii. Calculate the NPV concentration using Beer-Lambert's law: c NPV = A(λ) SC / ε NPV (λ), where c NPV This is the NPV partial concentration, A(λ). SC The measured scattering correction absorption of the NPV sample at the target wavelength (corrected for any dilution factor applied in step 1), where λ is the wavelength and ε is the scattering correction absorption. NPV (λ) is the molar extinction coefficient of the NPV portion at the target wavelength.

[0204] b. Method B: If the payload and NPV do indeed absorb in the same wavelength region, use a spectral deconvolution method (e.g., multivariate curve resolution) to determine the separate scattering-corrected absorption spectra of the payload and NPV respectively, and repeat steps 7(a)(i) and 7(a)(ii) using the absorbance values ​​in the separate spectra.

[0205] 8. If 100% of the payload is encapsulated in the NPV, the determined load concentration is equal to the payload capacity. If the payload encapsulation rate is less than 100%, the load value should be scaled accordingly.

[0206] 9. The ratio of payload to NPV can be calculated by adjusting c. payload Divide by c NPV To calculate.

[0207] A.III. Quantification of the relative lipid fraction in NPV formulations using the extinction coefficients of each lipid component. To obtain the relative lipid component fraction of the NPV sample using the extinction coefficients of each lipid component: 1. Dilute the sample in an appropriate buffer solution as needed to bring the absorbance within the operating range of the integrating sphere spectrophotometer.

[0208] 2. The measurement buffer solution is used as a "blank".

[0209] 3. Using buffer solution as a blank, measure the scattering-corrected absorption of the NPV sample.

[0210] 4. Record the scattering-corrected absorbance of each lipid fraction at its respective target wavelength.

[0211] 5. To obtain the extinction coefficient of the lipid fraction, it can be determined directly by measuring the concentration of a known amount of the lipid fraction or a series of pure lipid fraction solutions in a UV-Vis instrument, or by looking up its value in the literature.

[0212] 6. Calculate the concentration of each lipid fraction in the sample using Beer-Lambert's law: c f = A(λ) SC / ε f (λ), where c f It represents the component concentration, A(λ). SC The measured scattering correction absorption of the NPV sample at the target wavelength (corrected for any dilution factor applied in step 1), where λ is the wavelength and ε is the scattering correction absorption. f (λ) is the molar extinction coefficient of the component at the target wavelength.

[0213] BI uses the extinction coefficient of the payload and particle size to quantify the particle concentration of NPV in the formulation. To obtain the particle concentration of an NPV sample using the extinction coefficient of the payload, the extinction coefficient of the lipid fraction, and particle size information: 1. Dilute the sample in an appropriate buffer solution as needed to bring the absorbance within the operating range of the integrating sphere spectrophotometer.

[0214] 2. The measurement buffer solution is used as a "blank".

[0215] 3. Using buffer solution as a blank, measure the scattering-corrected absorption of the NPV sample.

[0216] 4. Record the scattering-corrected absorbance values ​​of the lipid fraction and the payload at their respective target wavelengths.

[0217] 5. To obtain the extinction coefficient of the lipid fraction, it can be determined directly by measuring the concentration of a known amount of lipid fraction or a series of pure lipid fraction solutions in a UV-Vis instrument, or by looking up its value in the literature.

[0218] 6. To obtain the extinction coefficient of the payload, it can be determined directly by measuring a known amount of payload or the concentration of a series of pure payload solutions in a UV-Vis instrument, or by looking up its value in the literature.

[0219] 7. Based on the properties of NPV and payload, there are two methods to determine the payload and NPV concentration: a. Method A (Direct Beer-Lambert Law): If the payload and NPV do not absorb in the same wavelength region: i. Calculate the concentration of the effective payload in the NPV sample using the Beer-Lambert law: c payload = A(λ) SC / ε D (λ), where c payload It represents the effective payload concentration, A(λ). SC The measured scattering correction absorption of the NPV sample at the target wavelength (corrected for any dilution factor applied in step 1), where λ is the wavelength and ε is the scattering correction absorption. D (λ) is the molar extinction coefficient of the payload at the target wavelength.

[0220] ii. Calculate the concentration of the lipid fraction using Beer-Lambert's law: c NPV = A(λ) SC / ε NPV (λ), where c NPV This refers to the lipid fraction concentration, A(λ). SC The measured scattering correction absorption of the NPV sample at the target wavelength (corrected for any dilution factor applied in step 1), where λ is the wavelength and ε is the scattering correction absorption. NPV (λ) is the molar extinction coefficient of the lipid fraction at the target wavelength.

[0221] b. Method B: If the payload and lipid fractions do indeed absorb in the same wavelength region, use a spectral deconvolution method (e.g., multivariate curve resolution) to determine the separate scattering-corrected absorption spectra of the payload and lipids, and repeat steps 7(a)(i) and 7(a)(ii) using the absorbance values ​​in the separate spectra.

[0222] 8. Determine particle size using external methods (e.g., dynamic light scattering, particle tracking analysis, or other appropriate techniques).

[0223] 9. For example, assuming the lipid portion of each particle is known, calculate the number of lipid molecules in each particle based on the size determined in step 8.

[0224] 10. Calculate, for example, the particle concentration c. P [Number of particles per milliliter] = c NPV / N P , where N P It represents the lipid fraction of each particle, expressed in moles per particle.

[0225] B.II To obtain the particle concentration (by particle size) of the NPV sample using particle scattering and particle size information: 1. Dilute the sample in an appropriate buffer solution as needed to bring the absorbance within the operating range of the integrating sphere spectrophotometer.

[0226] 2. The measurement buffer solution is used as a "blank".

[0227] 3. Using buffer solution as a blank, measure the scattering-corrected absorption spectrum of the NPV sample.

[0228] 4. Using buffer solution as a blank, measure the extinction spectrum of the NPV sample.

[0229] 5. The scattering spectrum of the particles is calculated by subtracting the scattering correction absorption from the extinction spectrum.

[0230] 6. Determine particle size using external methods (e.g., dynamic light scattering, particle tracking analysis, or other appropriate techniques).

[0231] 7. Calculate the wavelength-dependent scattering cross section σ of NPV using Mie theory. scat The particle size and wavelength-dependent dielectric function of the particle composition obtained in step 6 are used as inputs for Mie theory calculations.

[0232] 8. Through OD theo = σ scat * c P Calculate the theoretical scattering spectrum (optical density OD) of the particle, where c P It is the particle concentration [mol / L].

[0233] 9. By adjusting c P Obtain OD theo The best fit between the measured scattering spectrum and the NPV particle concentration is used to determine the NPV particle concentration.

[0234] 10. Through c P [Number of particles per milliliter] = cP [moles / liter] * N * 0.001 will c P [moles / liter] converted to c P [Number of particles per milliliter], where N = Avogadro's constant.

[0235] The implementations described herein are not intended to limit the scope of this disclosure, but merely to provide an illustration of possible implementations.

[0236] While the invention has been disclosed with reference to certain preferred embodiments, numerous modifications, alterations, and changes, as well as equivalents thereof, can be made to the said embodiments without departing from the scope of the invention as defined in the appended claims. Therefore, the invention is intended to be limited to the said embodiments and should be given the broadest reasonable interpretation based on the language of the appended claims. Features of any of the foregoing embodiments may be included in any other embodiments described herein.

[0237] Those skilled in the art will readily understand that various modifications and alterations can be made to the methods and apparatus described above. These modifications can be made without departing from the scope of the appended claims. For example, the invention is not limited to measuring lipid nanoparticles carrying RNA, but is also applicable to other delivery carriers carrying other payloads. Different wavelengths and integrating cavity shapes can also be used.

[0238] Embodiments of the present invention are set forth in the following numbered clauses.

[0239] A1. Nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization equipment, including: An integrating cavity, including one or more reflective inner walls, is configured to receive a cuvette within the integrating cavity, the cuvette being configured to contain a liquid sample or solution comprising at least one or more nanoparticle carriers. The integrating cavity includes at least one optical inlet port and at least one optical outlet port, wherein one or more optical inlet ports are configured to receive light from at least one light source, and one or more optical outlet ports are configured to deliver light to a spectrometer; The lipid-based drug delivery carrier analyzer or lipid-based drug delivery carrier measurement / characterization device is configured to operate in diffuse reflection mode, in which light from the light source follows an optical path from one or more inlet ports into the integrating cavity, is incident on the reflective inner wall of the integrating cavity, and is diffusely reflected within the integrating cavity, such that the light from the light source irradiates the liquid sample before being transmitted through one or more optical outlet ports and received by a spectrometer for optical wavelength analysis to provide the absorption spectrum of at least one or more nanoparticle carriers contained in the liquid sample or solution.

[0240] A2. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device as described in Clause A1, wherein the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device is used for or configured to: Determine the effective loading amount or effective loading concentration of the nanoparticle carrier, and / or Determine the effective load of the nanoparticle carrier and the carrier content / concentration ratio, and / or Determine the composition of the nanoparticle carrier, including the ratio or fractional content of the carrier components, and / or Determine the concentration of nanoparticle carriers in the solution or formulation.

[0241] A3. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device described in any of the preceding clauses, wherein the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device is: Nanoparticle carrier effective loading capacity or effective loading concentration analyzer or measurement / characterization equipment, and / or Nanoparticle carrier effective load to carrier content / concentration ratio analyzer or measurement / characterization equipment, and / or Nanoparticle carrier ratio or fractional carrier component content analyzer or measurement / characterization equipment, and / or Nanoparticle carrier solution concentration analyzer or measurement / characterization equipment.

[0242] A4. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, wherein the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device is, for example, a lipid-based nanoparticle carrier analyzer or a lipid-based nanoparticle carrier measurement / characterization device.

[0243] A5. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, wherein the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device is a lipid-based drug delivery carrier analyzer or a lipid-based drug delivery carrier measurement / characterization device.

[0244] A6. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device as described in the preceding clause, wherein the lipid-based drug delivery carrier analyzer or lipid-based drug delivery measurement / characterization device is used or configured to determine the drug loading of the lipid-based drug delivery carrier, or to determine the ratio of drug loading to lipid content / concentration of the lipid-based drug delivery carrier, or to determine the lipid composition ratio of the lipid-based drug delivery carrier.

[0245] A7. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, wherein the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device is a lipid-based nanoparticle analyzer or lipid-based nanoparticle measurement / characterization device, used or configured to determine the concentration of lipid-based nanoparticles in a solution or formulation, or used or configured to determine the concentration of lipid-based drug delivery carriers in a solution or formulation.

[0246] A8. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, wherein the lipid-based nanoparticle carrier comprises or is composed of (solid) lipid nanoparticles and / or liposomes.

[0247] A9. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses further includes an optical path modulator configured to selectively modulate the optical path through the integrating cavity, thereby providing at least two different optical paths; When the optical path modulator is in the first configuration, the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device is in transmission mode. In this mode, the light from the light source follows a first optical path from one or more optical inlet ports to the liquid sample, so that the light from the light source directly irradiates the liquid sample before the light transmitted by the sample is transmitted through one or more optical outlet ports and received by the spectrometer for wavelength analysis to provide the extinction spectrum of the liquid sample; and When the optical path adjuster is in the second configuration, the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device is in diffuse reflection mode. In this mode, light from the light source follows a second optical path into the integrating cavity from one or more inlet ports, is incident on the reflective inner wall of the integrating cavity, and is diffusely reflected within the integrating cavity. This allows the light from the light source to irradiate the liquid sample before being transmitted through one or more optical outlet ports and received by the spectrometer for optical wavelength analysis, thereby providing the absorption spectrum of the liquid or liquid matrix contained in the liquid sample.

[0248] A10. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, arranged such that light is transmitted: a. Directly from the inlet port to the wall of the integrating cavity; and / or b. Irradiate directly from the inlet port, through the sample, and then onto the wall of the integration chamber.

[0249] A11. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, wherein the inlet port used in the first configuration is directly opposite the outlet port used in the first configuration, such that when in the first configuration, the first optical path extends directly through the integrating cavity.

[0250] A12. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device described in any of the preceding clauses also includes a light source.

[0251] A13. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses further includes a controller, or a controller configured to control an optical path modulator to selectively adjust the optical path through the device.

[0252] A14. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device as described in Clause A13, wherein the controller is an integral part of the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device and communicates directly with the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device.

[0253] A15. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device as described in Clause A13, wherein the controller is located remotely from the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device and is configured to wirelessly communicate with the transceiver of the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device.

[0254] A16. A nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any one of clauses A13 to A15, wherein the controller is configured to control the spectrometer, in particular to process the light received by the spectrometer for optical wavelength analysis, thereby providing the extinction and / or absorption spectra of the liquid sample contained in the cuvette.

[0255] A17. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device described in any of the preceding clauses also includes a spectrometer.

[0256] A18. A nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any one of clauses A13 to A17, wherein the controller is configured to control one or more of the following: a. Switch between the first and second configurations; b. Obtain the spectrum from the integrating cavity; c. Select operating conditions; d. Display the spectrum on the display of the device or controller, or on a display that communicates with the device or controller; e. To store data in the memory of the device or controller, or in a memory that communicates with the device or controller; f. A user interface for a device or controller, or a user interface that communicates with a device or controller, interacts with the device, and allows the user to control the position of the optical path adjustment mechanism.

[0257] A19. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, wherein the optical path adjuster includes at least one movable optical element configured to manipulate light incident on the optical element from a light source, and the optical path adjuster is configured to adjust the movable optical element to selectively provide a first optical path and a second optical path.

[0258] A20. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to clause A19, wherein the optical element can be adjusted from a first position to a second position by moving the optical element relative to the integrating cavity, in the first position, light propagates along a first optical path, and in the second position, light propagates along a second optical path.

[0259] A21. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to clause A20, wherein the integrating cavity includes orthogonal longitudinal, vertical, and transverse axes, and one or more positional characteristics of the optical elements can be adjusted relative to one or more axes: a. Vertical position; b. Vertical position; c. Lateral position; d. Direction; e. Inclination.

[0260] A22. A nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any one of clauses A19 to A21, wherein a plurality of movable optical elements are provided.

[0261] A23. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device as described in clause A19, wherein the movable optical element is selected from: a. Prism; b. Lens; c. Reflector; d. Diffraction grating; e. Fiber optic cable; f. Light source.

[0262] A24. A nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any one of clauses A19 to A23, wherein the optical path adjuster includes at least one fixed optical element that is not adjustable relative to the integrating cavity.

[0263] A25. A nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device as described in clause A24, wherein a fixed optical element is configured to manipulate light from a light source before the light reaches the light inlet port.

[0264] A26. A nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device as described in Clause A24 or Clause A25, wherein a fixed optical element is configured to manipulate light from a light exit port.

[0265] A27. A nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any one of clauses A24 to A26, wherein the fixed optical element is selected from: a. Prism; b. Lens; c. Reflector; d. Diffraction grating; e. Fiber optic cable; f. Light source.

[0266] A28. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, wherein the optical path modulator includes at least one electronic controller for selectively operating one or more light sources to selectively provide a first optical path and a second optical path.

[0267] A29. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device as described in Clause A28 includes at least a first light source and a second light source, and the controller is configured to independently control each light source.

[0268] A30. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, wherein the optical path modulator is located between the light source and the optical entrance port.

[0269] A31. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, wherein the optical path adjuster is located between the spectrometer and the optical exit port.

[0270] A32. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, wherein multiple optical path adjustment mechanisms are provided.

[0271] A33. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, wherein a plurality of optical inlet ports are provided, and an optical path modulator is configured to provide a first optical path by guiding light from a light source through a first optical inlet port and to provide a second optical path by guiding light from a light source through a second optical inlet port.

[0272] A34. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, wherein a plurality of optical exit ports are provided, a first optical path guides light from the integrating cavity through the first optical exit port, and a second optical path guides light from the integrating cavity through the second optical exit port.

[0273] A35. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, wherein the integrating chamber comprises any one of the following: a. Diffuse reflection spherical integrating cavity; b. Cylindrical cavity; c. Rectangular or square cavity.

[0274] A36. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, wherein the integrating cavity includes an internal coating configured to provide one or more of the following: a. Specular reflection; b. Diffuse reflection; c. Reflectance in the ultraviolet spectrum; d. Reflection in the visible spectrum; e. Reflection in the infrared spectrum.

[0275] A37. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, wherein the light source includes one or more of the following: a. Quartz halogen light source; b. LED; c. Laser; d. Any multi-color light source.

[0276] A38. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, wherein the cuvette has one or more shapes: a. square; b. Plate-shaped; c. Cylindrical; d. Sphere-shaped.

[0277] A39. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device described in any of the preceding clauses is configured to perform UV-VIS spectroscopy measurements.

[0278] A40. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses further includes a sample holder configured to hold a cuvette containing a liquid sample within the integration chamber.

[0279] A41. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, wherein the light source includes a first LED light source and a second LED light source, and the optical path adjuster includes a controller configured to control the first LED light source and the second LED light source such that, in a first configuration, the first LED light source is controlled to provide light in a first optical path, and in a second configuration, the second LED light source is controlled to provide light in a second optical path.

[0280] A42. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device as described in Clause A40, wherein light from each LED light source is transmitted to the integrating cavity via a corresponding fiber optic cable.

[0281] A43. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device as described in clauses A41 or A42, wherein each LED light source transmits light to a corresponding light inlet port.

[0282] A44. A nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any one of clauses A41 to A43, wherein each optical path transmits light through a corresponding optical exit port.

[0283] A45. A nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any one of clauses A41 to A44, wherein a first LED light source is associated with a collimating lens located between the first LED light source and a light entrance port associated with the LED light source.

[0284] A46. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any one of clauses A41 to A45 further includes a first exit port and a second exit port, and a beam splitter configured to selectively allow light from the first exit port and the second exit port to be transmitted to the spectrometer.

[0285] A47. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, wherein the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device is: (Solid) lipid nanoparticle drug delivery carrier drug loading analyzer or (solid) lipid nanoparticle drug delivery carrier drug loading measurement / characterization equipment; or (Solid) lipid nanoparticle drug delivery carrier drug loading capacity and lipid content / concentration ratio analyzer or (solid) lipid nanoparticle drug delivery carrier drug loading capacity and lipid content / concentration ratio measurement / characterization equipment; or (Solid) lipid nanoparticle drug delivery carrier lipid component ratio analyzer or (solid) lipid nanoparticle drug delivery carrier lipid component ratio measurement / characterization device.

[0286] A48. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, wherein the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device is a liposome drug delivery carrier analyzer or measurement / characterization device.

[0287] A49. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device as described in the preceding clause, wherein the nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device is: Liposome nanoparticle drug delivery carrier drug loading analyzer or liposome nanoparticle drug delivery carrier drug loading measurement / characterization equipment; or Liposome nanoparticle drug delivery carrier drug loading and lipid content / concentration ratio analyzer or liposome nanoparticle drug delivery carrier drug loading and lipid content / concentration ratio measurement / characterization equipment; or Liposome nanoparticle drug delivery carrier lipid component ratio analyzer or liposome nanoparticle drug delivery carrier lipid component ratio measurement / characterization equipment.

[0288] B50. Methods for analyzing or measuring / characterizing nanoparticle carriers, including: - Provide a nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device as described in any of the foregoing clauses, or provide a device configured to measure or determine at least one scattering-corrected absorption measurement result or scattering-corrected absorption spectrum; and / or - Acquire / receive at least one or more scatter-corrected absorption measurements / data or scatter-corrected absorption spectroscopy measurements / data for a liquid sample or solution containing multiple nanoparticle carriers.

[0289] B51. The method according to the preceding clause further includes using at least one scattering-corrected absorption measurement / data or scattering-corrected absorption spectral measurement / data, and utilizing the extinction coefficient of the nanoparticle carrier's effective load at the target absorption wavelength to determine the effective load of the nanoparticle carrier in the liquid sample or solution.

[0290] B52. The method described in the preceding clause, wherein the effective loading concentration of the nanoparticle carrier in the liquid sample or solution is determined using the Beer-Lambert law, as follows: c payload = A(λ) SC / ε D (λ), Where c payloadIt represents the effective payload concentration, A(λ). SC This is the scattering-corrected absorption of the nanoparticle-carried liquid sample at the target absorption wavelength (ultimately corrected for any dilution factor of the application), where λ is the wavelength and ε is the scattering-corrected absorption. D (λ) is the molar extinction coefficient of the payload at the target absorption wavelength.

[0291] B53. The method described in accordance with the preceding clause B51 or B52 further includes: if the payload and the carrier component / part absorb in the same wavelength region, applying a spectral deconvolution method to determine the separate scattering-corrected absorption or absorption spectra of the payload and the carrier component / part.

[0292] B54. The method described in accordance with clauses B51 or B52 further includes applying a scaling value to the determined load concentration based on the encapsulation level or amount of payload within the nanoparticle carrier.

[0293] B55. The method according to clause B50 further includes using at least one scatter-corrected absorption measurement / data or scatter-corrected absorption spectral measurement / data, and using the extinction coefficient of the effective load of the nanoparticle carrier and the extinction coefficient of the carrier component of the nanoparticle carrier at the target absorption wavelength to determine the ratio of the effective load of the nanoparticle carrier to the carrier component / part of the liquid sample or solution.

[0294] B56. The method described in the preceding clause, wherein the effective load concentration of the nanoparticle carrier in the liquid sample or solution is determined using the Beer-Lambert law, as follows: c payload = A(λ) SC / ε D (λ), Where c payload It represents the effective payload concentration, A(λ). SC This is the scattering-corrected absorption of the nanoparticle-carried liquid sample at the target absorption wavelength (ultimately corrected for any dilution factor of the application), where λ is the wavelength and ε is the scattering-corrected absorption. D (λ) is the molar extinction coefficient of the payload at the target absorption wavelength; and The Beer-Lambert law is used to determine the carrier component / partial concentration of the nanoparticle carrier in the liquid sample or solution, as shown below: c NPV = A(λ) SC / ε NPV (λ), Where c NPV A(λ) represents the concentration of the carrier component / partial concentration of the nanoparticle carrier. SCThis is the scattering-corrected absorption of the nanoparticle-carried liquid sample at the target absorption wavelength (ultimately corrected for any dilution factor of the application), where λ is the wavelength and ε is the scattering-corrected absorption. NPV (λ) is the molar extinction coefficient of the carrier component / part of the nanoparticle carrier at the target absorption wavelength.

[0295] B57. The method described in accordance with the preceding clause B55 or B56 further comprises: if the payload and the carrier component / part absorb in the same wavelength region, applying a spectral deconvolution method to determine the separate scattering-corrected absorption or absorption spectra of the payload and the carrier component / part.

[0296] B58. The method described in accordance with clauses B55 or B56 further includes applying a scaling value to the determined load concentration based on the encapsulation level or amount of payload within the nanoparticle carrier.

[0297] B59. Further, by means of the method described in Clause B55 or B56, through c payload Divide by c NPV To determine the effective load of a nanoparticle carrier for a liquid sample or solution relative to the amount of carrier components / parts.

[0298] B60. The method according to clause B50 further includes using at least one scattering-corrected absorption measurement / data or scattering-corrected absorption spectral measurement / data, and using the extinction coefficients of the constituent carrier elements of the carrier component / part of the nanoparticle carrier to determine the molar ratio of the constituent carrier elements of the carrier component / part of the nanoparticle carrier.

[0299] B61. The method described in the preceding clause, wherein the concentration of each constituent element of the carrier component / part of the nanoparticle carrier is determined using the Beer-Lambert law, as follows: c f = A(λ) SC / ε f (λ), Where c f It represents the concentration of the constituent elements of the carrier, A(λ). SC This is the scattering-corrected absorption of the nanoparticle-carried liquid sample at the target absorption wavelength (ultimately corrected for any dilution factor of the application), where λ is the wavelength and ε is the scattering-corrected absorption. f (λ) is the molar extinction coefficient of the carrier element at the target absorption wavelength.

[0300] B62. The method according to clause B50 further includes determining the nanoparticle carrier concentration in a liquid sample or solution using at least one scattering-corrected absorption measurement / data or scattering-corrected absorption spectroscopy measurement / data and the measured or determined nanoparticle carrier size.

[0301] B63. The method described in the preceding clause, wherein the effective load concentration of the nanoparticle carrier in the liquid sample or solution is determined using the Beer-Lambert law, as follows: c payload = A(λ) SC / ε D (λ), Where c payload It represents the effective payload concentration, A(λ). SC This is the scattering-corrected absorption of the nanoparticle-carried liquid sample at the target absorption wavelength (ultimately corrected for any dilution factor of the application), where λ is the wavelength and ε is the scattering-corrected absorption. D (λ) is the molar extinction coefficient of the payload at the target absorption wavelength; and The Beer-Lambert law is used to determine the carrier component / partial concentration in liquid samples or solutions of nanoparticle carriers, as shown below: c NPV = A(λ) SC / ε NPV (λ), Where c NPV This represents the concentration of the carrier component / partial concentration, A(λ). SC This is the scattering-corrected absorption of the nanoparticle-carried liquid sample at the target absorption wavelength (ultimately corrected for any dilution factor of the application), where λ is the wavelength and ε is the scattering-corrected absorption. NPV (λ) is the molar extinction coefficient of the carrier component / part of the nanoparticle carrier at the target absorption wavelength.

[0302] B64. The method according to the preceding clause B62 or B63 further comprises: if the payload and the carrier component / part absorb in the same wavelength region, applying a spectral deconvolution method to determine the separate scattering-corrected absorption or absorption spectra of the payload and the carrier component / part.

[0303] B65. The method according to clauses B62 or B63 further includes providing constituent elemental values ​​of the carrier component / part of the nanoparticle carrier and using the measured or determined nanoparticle carrier size to determine the number of molecules in the carrier component / part of the nanoparticle carrier.

[0304] B66. The method according to any one of the preceding clauses B62 to B65, further according to cP = c NPV / N P Determine particle concentration c P [Number of particles per milliliter], of which N P It represents the lipid fraction of each particle, expressed in moles per particle.

[0305] B67. The method according to clause B50 further includes determining the nanoparticle carrier concentration in a liquid sample or solution using at least one scattering spectrum or scattering spectrum data and the measured or determined nanoparticle carrier size.

[0306] B68. The method described pursuant to Clause B57 also includes Acquire / receive at least one or more extinction measurement results / data or extinction spectral measurement results / data for a liquid sample or solution containing multiple nanoparticle carriers; Using (i) at least one scattering-corrected absorption measurement result / data or scattering-corrected absorption spectral measurement result / data and (ii) at least one extinction measurement result / data or extinction spectral measurement result / data determined, determine at least one scattering spectrum or scattering spectral data; The wavelength-dependent dielectric function of the nanoparticle carrier, based on the measured or determined nanoparticle carrier size and particle composition, is used to determine the wavelength-dependent scattering cross section σ of the nanoparticle carrier. scat ; According to OD theo = σ scat * c P Determine the scattering spectrum (optical density OD) of the particles, where c P It is the particle concentration [mol / L].

[0307] B69. The method described under the preceding clause also includes Through c P [Number of particles per milliliter] = c P [moles / liter] * N * 0.001 will c P [moles / liter] converted to c P [Number of particles per milliliter], where N = Avogadro's constant.

[0308] B70. The method according to any of the preceding clauses, wherein the nanoparticle carrier is a lipid-based nanoparticle carrier, and the carrier component / part of the nanoparticle carrier comprises at least one lipid, and the constituent carrier element comprises at least one lipid.

[0309] B71. The method according to the preceding clause, wherein the payload of the lipid-based nanoparticle carrier comprises oligonucleotides, such as mRNA, siRNA, or DNA.

[0310] B72. The method according to any of the preceding clauses, wherein the nanoparticle carrier is a lipid-based drug delivery carrier.

[0311] B73. The method according to any of the preceding clauses, wherein the nanoparticle carrier comprises or is composed of (solid) lipid nanoparticles and / or liposomes.

[0312] C74. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any one of the clauses A1 to A49 further includes at least one processor or computing device; and at least one memory or storage device including a computer program, the computer program including instructions that, when executed by the processor or computing device, cause the analyzer or measurement / characterization device to perform the steps of the method according to any one of the clauses B50 to B73.

[0313] C75. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device as described in the preceding clause, wherein the controller includes at least one processor or computing device; and at least one memory or storage device including a computer program.

[0314] D76. A computer program comprising instructions that, when executed by a computer, processor, or computing device, cause the computer, processor, or computing device to perform the method according to any one of clauses B50 to B73.

[0315] E77. A computer-readable data carrier having a computer program stored thereon as described in the preceding clause.

[0316] F78. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, wherein the nanoparticle carrier includes polymer nanoparticles or inorganic nanoparticles.

[0317] F79. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device as described in the preceding clause, wherein the polymer nanoparticles include polymer nanocapsules or nanospheres, polymer vesicles, dendritic polymers or polymer micelles.

[0318] F80. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device as described in the preceding clause B78, wherein the inorganic nanoparticles include silica nanoparticles, quantum dots, iron-based or iron oxide nanoparticles or gold nanoparticles.

[0319] F81. A nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any of the preceding clauses, wherein the nanoparticle carriers include exosomes, microvesicles, lentiviruses, adeno-associated viruses (AAVs) or adenoviruses.

[0320] G82. The method for analyzing or measuring / characterizing nanoparticle carriers according to any of the preceding clauses, wherein the nanoparticle carriers include polymer nanoparticles or inorganic nanoparticles.

[0321] G83. The nanoparticle carrier analysis or measurement / characterization method according to the preceding clause, wherein the polymer nanoparticles include polymer nanocapsules or polymer nanospheres, or polymer vesicles, or dendritic polymers, or polymer micelles.

[0322] G84. The method for analyzing or measuring / characterizing nanoparticle carriers according to the preceding clause G82, wherein the inorganic nanoparticles include silica nanoparticles, quantum dots, iron-based or iron oxide nanoparticles or gold nanoparticles.

[0323] G85. The analytical or measurement / characterization method for nanoparticle carriers according to any of the preceding clauses, wherein the nanoparticle carriers include exosomes, microvesicles, lentiviruses, adeno-associated viruses (AAVs), or adenoviruses.

[0324] appendix The contents of international patent application PCT / NZ2017 / 050131 have been published as WO2018 / 070882 A1. Spectrometer apparatus for measuring the spectrum of liquid samples using an integrating cavity. Technical Field

[0325] The present invention relates to a spectrometer apparatus for measuring the spectrum of a liquid sample using an integrating cavity, and in some embodiments, the present invention relates to a UV-vis spectrometer apparatus for measuring turbid liquids. Background Technology

[0326] Standard UV-VIS spectroscopy is performed by passing a light source through a sample and measuring the transmitted light at different wavelengths. The sample is typically a liquid contained in a square tank, positioned so that the tank surface is perpendicular to the light beam. The transmitted light is then converted into an absorption spectrum, which provides a measure of the sample's absorbance at each wavelength used. Absorbance can be used as a measure of the concentration of dissolved species (absorbance is proportional to concentration, known as the Beer-Lambert law) or to determine the chemical composition of the solution based on the absorption peak of the species at a known wavelength.

[0327] UV-VIS spectrometers are standard instruments in analytical chemistry and can be used for both quantitative and qualitative analysis of liquids. A UV-VIS spectrometer measures the spectrum transmitted directly from the sample and determines the absorption spectrum based on the assumption that only light loss occurs due to absorption within the sample. This results in the generally required clarity of the sample liquid in a UV-VIS spectrometer.

[0328] In the more general case, including turbid liquids, light is lost due to scattering by the sample, and the UV-VIS spectrometer will measure the extinction spectrum instead of the absorption spectrum. In short: Extinction = Scattering + Absorption.

[0329] The intensity of scattered light is generally wavelength-dependent, resulting in a scattering spectrum. In a UV-VIS spectrometer, the absorption and scattering spectra are superimposed and cannot be deconstructed without individual knowledge of either component spectrum. In strongly scattering liquids (e.g., milk, paint, blood, wine), even if the scattering spectrum is known, the light reaching the detector is reduced to such an extent that the absorption spectral component is almost indistinguishable from the measured extinction spectrum. Standard UV-VIS therefore has very limited general applicability to scattering / turbid samples and, if used, still requires sample pretreatment (e.g., filtration, centrifugation, or other methods to remove scattering species). Sample dilution is generally unhelpful, as it reduces both scattering and absorption of the sample in the same proportion.

[0330] In summary, there are large numbers of samples where UV-VIS is ineffective or requires time-consuming processing to allow analysis of turbid solutions. Furthermore, it may be impossible to distinguish the relative contributions of scattering and absorption using standard UV-VIS spectroscopy.

[0331] Purpose of the invention Therefore, the object of the present invention is to provide a spectrometer device that overcomes or at least improves one or more disadvantages of the prior art or, alternatively, provides the public with at least a useful option.

[0332] Other objects of the present invention will become apparent from the following description. Summary of the Invention

[0333] Therefore, in one aspect, the present invention can be broadly described as a spectrometer apparatus for measuring the spectrum of a liquid sample, the apparatus comprising: An integrating cavity, the integrating cavity including one or more reflective inner walls, and the integrating cavity being configured to house a liquid tank containing a liquid sample within the integrating cavity. The integrating cavity includes at least one optical inlet port and at least one optical outlet port, wherein each optical inlet port is configured to receive light from a light source and each optical outlet port is configured to transmit light to a spectrometer. The device further includes an optical path adjuster configured to selectively adjust the optical path through the integrating cavity, such that at least two different optical paths are provided; wherein When the optical path modulator is in the first configuration, the device is in transmission mode, in which light from the light source follows a first optical path from the light inlet port or one thereof to the liquid sample, such that the light from the light source directly illuminates the liquid sample before the light transmitted by the sample is transmitted through the light outlet port or one thereof and received by the spectrometer for wavelength analysis to provide the extinction spectrum of the liquid sample; and When the optical path adjuster is in the second configuration, the device is in diffuse reflection mode, in which light from the light source follows a second optical path from the inlet port or one of them into the integrating cavity, is incident on one or more reflective inner walls of the integrating cavity and undergoes diffuse reflection within the integrating cavity, such that the light from the light source illuminates the liquid sample before being transmitted through the light outlet port or one of them and received by the spectrometer for wavelength analysis to provide the absorption spectrum of the liquid sample contained in the liquid sample.

[0334] This spectrometer device can be used in particular to obtain the spectrum as the absorption and extinction spectra of the sample, thereby generating the absorption and extinction spectra defined for a given path length through the sample using a suitable calibration procedure implemented by one or more electronic data processors.

[0335] By providing a device that can be used in each of the above configurations, quantitative spectra can be obtained, wherein the path length of light through the sample is well defined in each configuration, so that the data obtained in each configuration are relevant.

[0336] The device can be configured such that, in the second configuration, light from the second optical path is transmitted as follows: a) Directly from the inlet port to one or more walls of the integrating cavity; and / or b) Directly from the inlet port to the sample and subsequently to one or more walls of the integrating cavity via the sample.

[0337] Therefore, in the second configuration, the second optical path can first be transmitted through the sample from the inlet port or directly to the one or more cavity walls. With any variation, the device is configured such that the outlet port used in the second configuration does not look towards the inlet port. In other words, the outlet port used in the second configuration "faces" the wall of the integrating cavity. The outlet port may, for example, be at 90° to the inlet port or at any other location on the integrating cavity. The relative positions of the inlet and outlet ports used in the second configuration prevent the spectrometer from collecting incident light or light directly transmitted from the sample.

[0338] Preferably, in the first configuration, the inlet port and the outlet port are directly opposite each other, such that the first optical path extends directly across the integrating cavity.

[0339] In another aspect of the invention, a spectrometer apparatus for measuring the spectrum of a liquid sample is provided, specifically wherein the obtained spectrum is the absorption and extinction spectrum of the sample, and the apparatus includes: An integrating cavity, the integrating cavity including one or more reflective inner walls, and the integrating cavity being configured to house a liquid tank containing a liquid sample within the integrating cavity. The integrating cavity includes at least one optical inlet port and at least one optical outlet port, wherein the optical inlet port is configured to receive light from a light source and the optical outlet port is configured to transmit light to a spectrometer; The device further includes an optical path adjuster configured to selectively adjust the optical path through the integrating cavity, such that at least two different optical paths are provided; wherein When the optical path modulator is in the first configuration, the device is in transmission mode, in which light from the light source follows a first optical path from the light inlet port to the liquid sample, such that the light from the light source directly illuminates the liquid sample before the light transmitted by the sample is collected via the light outlet port, which is positioned directly opposite the inlet port, and received by the spectrometer for wavelength analysis to provide the extinction spectrum of the liquid sample; and When the optical path adjuster is in the second configuration, the device is in diffuse reflection mode, in which light from the light source follows a second optical path from the inlet port into the integrating cavity and is incident on one or more reflective inner walls of the integrating cavity or directly onto the liquid sample; wherein light transmitted and / or scattered by the sample is transmitted through the outlet port, and the device is configured such that light directly transmitted and / or reflected by the sample is reflected by one or more inner walls of the cavity before being transmitted through the outlet port and received by the spectrometer for wavelength analysis to provide the absorption spectrum of the liquid sample contained in the liquid sample.

[0340] Preferably, a suitable calibration procedure is used to generate an absorbance and extinction spectrum defined for a given path length through the sample.

[0341] A preferred embodiment of the second configuration is to position the outlet port such that it faces the area of ​​the cavity wall from which the light from the inlet port does not directly illuminate.

[0342] In both configurations, the apparatus, used with a suitable calibration procedure, generates the extinction and absorption spectra of the liquid sample, wherein in both configurations the path length through the sample is well defined such that the obtained spectra give the wavelength-dependent extinction and absorption coefficients of the sample, respectively, across the wavelength range of the light illuminating the sample.

[0343] The device may include one or more integral light sources, or the light source may be configured to be connected to one or more individual light sources.

[0344] The device may also include an integral or remote controller configured to control the optical path modulator to selectively adjust the path of light passing through the device.

[0345] The controller is preferably configured to control the spectrometer, and particularly to process the light received by the spectrometer for wavelength analysis to provide the extinction and / or absorption spectra of the liquid sample contained in the tank. The spectrometer may be integrated with the device.

[0346] The one or more controllers may be configured to control one or more of the following: a) Switch between the first configuration and the second configuration; b) Obtain the spectrum from the integrating cavity; c) Select operating conditions; d) Displaying the spectrum on the display of the device or the display of the controller or a display communicating with the device or controller; e) Store the data in the memory of the device or the memory of the controller or in communication with the device or controller; f) The user interface of the device or the user interface of the controller, or the user interface that communicates with the device or the controller, interacts with the device and allows the user to control the position of the optical path adjustment mechanism.

[0347] The optical path adjuster may include at least one movable optical element configured to manipulate light incident on the optical element from the light source, and the optical path adjuster is configured to adjust the movable optical element to selectively provide the first optical path and the second optical path.

[0348] The optical element can be adjusted by moving it relative to the integrating cavity from a first position where the light travels along the first optical path and a second position where the light travels along the second optical path.

[0349] The integrating cavity includes orthogonal longitudinal, vertical, and transverse axes, and any one or more of the following positional characteristics of the optical element can be adjusted relative to any one or more of these axes: a) Vertical position; b) Vertical position; c) Lateral position; d) Orientation; e) Inclination angle.

[0350] Multiple movable optical elements can be provided.

[0351] The movable optical element is preferably selected from any one or a combination of the following: Prism; lens; Reflector; Diffraction grating; Fiber optic cable; The light source; shutter.

[0352] The optical path adjuster may additionally or alternatively include at least one fixed optical element, which is non-adjustable relative to the integrating cavity. The fixed optical element may be configured to manipulate the light from the light source before the light inlet port. The fixed optical element may also be configured to manipulate the light from the light outlet port.

[0353] The fixed optical element may be selected from any one or a combination of the following: a) Prism; b) Lens; c) Reflector; d) Diffraction grating; e) Fiber optic cable; f) The light source.

[0354] The optical path modulator may include at least one electronic controller operable to selectively operate one or more light sources to selectively provide the first optical path and the second optical path.

[0355] The device may include at least a first light source and a second light source, and the controller is configured to independently control each light source. The light sources can be switched on and off in a flashing or sequential manner, wherein in configuration one the first light source is switched on, and in configuration two the second light source is switched on while initially switched off. The light sources can be controlled such that two or all light sources can be switched off to obtain a dark spectrum.

[0356] The optical path modulator can be positioned: a) Between the light source and the light inlet port, and / or b) Between the spectrometer and the light output port.

[0357] Multiple optical path adjustment mechanisms can be provided.

[0358] Multiple optical inlet ports can be provided, and the optical path modulator is configured to provide a first optical path by guiding light from the light source through a first optical inlet port, and to provide a second optical path by guiding light from the light source through a second optical inlet port.

[0359] Multiple optical exit ports can be provided. The first optical path guides the light from the integrating cavity through the first optical exit port, and the second optical path guides the light from the integrating cavity through the second optical exit port.

[0360] The integrating cavity may include any one of the following: a) Diffuse reflection spherical integrating cavity; b) Cylindrical cavity; c) Cubic or square cavity.

[0361] It should be understood that the integrating cavity can be any other shape or combination of shapes.

[0362] The integrating cavity may include an internal coating configured to provide any one or more of the following: a) Specular reflection; b) Diffuse reflection; c) Reflection in the ultraviolet spectrum; d) Reflection in the visible light spectrum; e) Reflection in the infrared spectrum.

[0363] The light source may include any one or more of the following: a) Quartz halogen source; b) LED; c) Laser; d) Any multicolor light source.

[0364] The shape of the liquid tank can be: a) Square; b) plate-shaped; c) Cylindrical; d) Spherical.

[0365] The device may be a UV-VIS spectrometer.

[0366] The apparatus may also include a sample holder configured to hold a liquid tank containing a liquid sample within the integrating chamber.

[0367] The light source may include a first LED light source and a second LED light source, and the light path adjuster includes a controller configured to control the first LED light source and the second LED light source such that, in the first configuration, the first LED light source is controlled to provide light in the first light path, and in the second configuration, the second LED light source is controlled to provide light in the second light path.

[0368] Light from each LED light source can be transmitted to the integrating cavity via corresponding optical fibers and cables. Each LED light source can transmit light to a corresponding optical inlet port. Each optical path transmits light through a corresponding optical outlet port.

[0369] The first LED light source may be associated with a collimating lens, which is positioned between the first LED light source and the light inlet port associated with the LED light source.

[0370] The device may further include a first exit port and a second exit port, as well as a beam splitter configured to selectively allow light from the first exit port and the second exit port to be transmitted into the spectrometer.

[0371] According to another aspect of the present invention, a spectrometer apparatus for measuring the spectrum of a liquid sample is provided, the apparatus comprising: An integrating cavity, the integrating cavity including one or more reflective inner walls, and configured to house a liquid tank containing a liquid sample within the integrating cavity. The integrating cavity includes a first optical inlet port and a second optical inlet port. The first optical inlet port is configured to receive light from a first LED light source and the second optical inlet port is configured to receive light from a second LED light source. At least one optical outlet port is provided and configured to transmit light to a spectrometer. The device further includes an optical path adjuster configured to selectively adjust the optical path through the integrating cavity, such that at least two different optical paths are provided; wherein When the optical path modulator is in the first configuration, the device is in transmission mode, in which light from the first LED light source follows a first optical path from the first light inlet port to the liquid sample, such that the light from the first LED light source directly illuminates the liquid sample before the light transmitted by the sample is transmitted through the light outlet port and received by the spectrometer for wavelength analysis to provide the extinction spectrum of the liquid sample; and When the optical path adjuster is in the second configuration, the device is in diffuse reflection mode, in which light from the second LED light source follows a second optical path from the second inlet port into the integrating cavity, is incident on one or more reflective inner walls of the integrating cavity and diffusely reflected within the integrating cavity, such that the light from the light source illuminates the liquid sample before being transmitted through the one or another optical outlet port and received by the spectrometer for wavelength analysis to provide the absorption spectrum of the liquid sample contained in the liquid sample.

[0372] The spectrometer apparatus can be configured to measure the spectra of liquid samples selected from any one or more of the following: a.water; b. Wine; c. Beverages; d. Edible liquid or partially liquid products.

[0373] According to another aspect of the present invention, a method for measuring the spectrum of a liquid sample using the apparatus described in any of the other aspects of the present invention is provided, comprising the following steps: a. Activate the light source; b. Control the optical path adjuster to be in either transmission mode or diffuse mode; and c. Wavelength analysis of the light transmitted through the light exit port via a spectrometer is performed to provide the absorption spectrum of the liquid sample contained in the liquid tank. Attached Figure Description

[0374] Several embodiments of the invention will now be described by way of example with reference to the accompanying drawings, in which: Figure 1 This is a schematic diagram of exemplary components of a spectrometer apparatus according to the present invention; Figure 2 a and Figure 2 b is a schematic diagram of a first embodiment of the spectrometer device according to the present invention in the first and second configurations; Figure 3 a and Figure 3 b is a schematic diagram of a second embodiment of the spectrometer device according to the present invention in the first and second configurations; Figure 4 This is a schematic diagram of a third embodiment of a spectrometer apparatus according to the present invention, which also shows a first configuration and a second configuration of the apparatus; Figure 5 This is a schematic diagram of a fourth embodiment of a spectrometer apparatus according to the present invention, which also shows a first configuration and a second configuration of the apparatus; Figure 6 This is a schematic diagram of a fifth embodiment of a spectrometer apparatus according to the present invention, which also shows a first configuration and a second configuration of the apparatus; and Figure 7 This is a schematic diagram of a sixth embodiment of a spectrometer apparatus according to the present invention, which also shows a first configuration and a second configuration of the apparatus. Detailed Implementation

[0375] Throughout the specification, in different embodiments, the same reference numerals will be used to refer to the same features.

[0376] refer to Figure 1 A spectrometer apparatus 1 is provided for measuring the spectrum of a liquid sample. The spectrometer apparatus 1 is configured to measure a variety of optical properties of the liquid sample, including the wavelength-dependent extinction and absorption coefficients of the liquid.

[0377] The device 1 includes an integrating cavity 3, which includes a reflective inner wall 5. The device 1 is configured to hold a liquid tank 7 containing liquid within the integrating cavity 3. Light from a light source 9 is transmitted into the cavity 3 via different optical paths 15 and 17, which can be selectively adjusted via an optical path modulator 13. The optical path modulator 13 is used to transmit light into the cavity 3 along different paths through at least one inlet port P1 and P2, depending on the configuration of the optical path modulator 13.

[0378] The apparatus 1 also includes at least one optical output port P3, P4 configured to transmit light to the spectrometer 11. In some examples, an output optical path modulator 13B is provided to control the path of light from the integrating cavity 3 to the spectrometer 11.

[0379] In the first configuration, device 1 is in transmission mode, wherein the input path adjuster 13 is positioned such that light from light source 9 enters cavity 3 through inlet port P1 to directly irradiate the liquid contained in liquid tank 7, and the outlet light path adjuster 13B is configured such that light is collected through outlet port P3 and sent to spectrometer 11, so that a portion of the light from light source 9 is directly transmitted through the sample after irradiating the sample. In this configuration, the extinction spectrum of the sample is obtained.

[0380] In the second configuration, device 1 is in diffuse reflection mode, wherein the inlet optical path modulator 13 is positioned such that light entering cavity 3 from light source 9 through inlet port P2 can directly illuminate the liquid contained in liquid tank 7 or can be incident on cavity wall 5 and diffusely reflected within cavity 3 before interacting with the liquid sample. Furthermore, in this second configuration, outlet optical path modulator 13B is configured such that light transmitted and / or reflected by the sample and collected through outlet port P4 and sent to spectrometer 11 has undergone at least one reflection from cavity wall 5 before entering outlet port P4. In this configuration, the absorption spectrum of the sample is obtained without the influence of scattering by the liquid sample.

[0381] The means of switching between configuration modes is provided by one or more electronic controllers, which select the configuration of both the inlet optical path modulator 13 and the outlet optical path modulator 13B (if provided) to obtain the extinction spectrum or absorption spectrum of the liquid sample according to the selected configuration mode.

[0382] Apparatus 1 and the method used in apparatus 1 allow for the measurement of the extinction and absorption spectra of a liquid sample using a single apparatus without moving the liquid sample.

[0383] Now for reference Figure 2 a, Figure 2 b. A first embodiment of the spectrometer apparatus 1 for measuring the spectrum of a liquid sample includes an integrating cavity 3, which includes one or more reflective inner walls 5, and the spectrometer apparatus 1 is configured to hold a liquid tank 7 containing a liquid sample within the integrating cavity 3. The integrating cavity 3 includes at least one light inlet port P1, P2 and at least one light outlet port P3, P4, the light inlet port(s) P1, P2 being configured to receive light from a light source 9, and the light outlet port(s) P3, P4 being configured to transmit light to the spectrometer 11.

[0384] The device 1 also includes an optical path modulator 13, which is configured to selectively adjust the path of light passing through the integrating cavity 3, such that at least two different optical paths 15, 17 are provided.

[0385] When the optical path modulator 13 is in the first configuration, the device 1 is in transmission mode, in which light from the light source 9 follows a direct optical path 15 from one of the light inlet ports P1 to the liquid sample, such that the light from the light source 9 directly illuminates the liquid sample before being transmitted through one of the light outlet ports P3, P4 and received by the spectrometer 11 for wavelength analysis to provide the extinction spectrum of the liquid sample in the liquid tank 7.

[0386] When the optical path adjuster 13 is in the second configuration, the device 1 is in diffuse reflection mode, in which light from the light source 9 follows optical path 17 from one of the inlet ports P1, P2, or P2 into the integrating cavity 3, and: a) The light is directly incident on one or more reflective inner walls 5 of the integrating cavity 3 and diffusely reflected within the integrating cavity 3, so that the light from the light source 9 indirectly illuminates the liquid sample; or b) Directly incident (not shown) onto the liquid sample 7, such that light from the light source 9 directly illuminates the liquid sample, and the light transmitted and / or reflected by the sample undergoes diffuse reflection within the integrating cavity. The light is then transmitted through one of the light exit ports P3, P4 and is received by the spectrometer 11 for wavelength analysis to provide the absorption spectrum of the liquid sample contained in the liquid tank 7.

[0387] Apparatus 1 and the method used therein allow for the measurement of the extinction and absorption spectra of a liquid sample using a single device without moving the liquid sample. The method includes placing a liquid sample, which can be contained in a standard 1 cm square liquid tank 7, into an integrating cavity 3; and transmitting light to the sample in either a transmission or diffuse reflection configuration. In the first configuration, light transmitted from the sample is sent to the spectrometer 11 and an extinction spectrum is obtained, while in the second configuration, the light undergoes diffuse reflection within the cavity 3 and interacts with the sample such that no light scattered by the sample is lost. In the second configuration, the light may initially interact with the sample or be directly incident on the walls of the cavity. The spectrum collected by the spectrometer 11 in the second configuration can then be correlated with the absolute absorption spectrum using suitable calibration and modeling. Switching between measurement configurations is provided via one or more tunable optical elements L1-L5, M1-M4 configured to manipulate the light from the light source 9 before it enters the integrating cavity 3. Such an optical element may include one or more shutters and / or movable mirrors that control the optical path through the integrating cavity 3, and thus allow both the extinction spectrum and the absorption spectrum of the liquid to be obtained using a single device 1.

[0388] The device 1 suspends or supports the sample liquid tank 7 within the integrating cavity 3, which has a specific optical inlet / outlet port configuration (in combination with one or more optical elements) allowing two different optical paths to be provided between the light source 9 and the spectrometer 11 (and specifically the photodetector of or connected to the spectrometer) via the integrating cavity 3.

[0389] Technicians should be aware that the first and second optical paths through the integrating cavity 3 can be provided in several different ways and by changing at least one or more of the following: a. The number and / or location of the entry ports; b. The number and / or location of exit ports; c. The number and / or location and / or type of movable optical elements; d. The number and / or location and / or type of any auxiliary fixed optical elements that may be used; e. The relative position of the integrating cavity with respect to the light source and / or spectrometer.

[0390] In practice, the use of device 1 provides one or more of the following advantages: This method is used to perform standard UV-VIS measurements in any other commercially available device with a standard liquid tank.

[0391] The ability to switch to absorption mode to remove any scattering effects.

[0392] From the user's perspective, both cancellation spectrum and absorption spectrum are immediately provided.

[0393] Absorption and extinction spectra are measured in a single instrument without user intervention.

[0394] It allows for easy sample replacement via the cavity port, similar to the replacement in a standard UV-VIS instrument. Provides means for determining the absolute absorbance of turbid / scattering media. Different entry ports refer to Figure 2 a and Figure 2In the first example of b, light travels along a first optical path 15 from the light source 9 through one of the two optical inlet ports P and P2 into the integrating cavity 3. When the device 1 is in the first configuration, light enters through the first optical inlet port P1 and is directly incident on the liquid sample in the liquid tank 7. The light transmitted from the liquid sample is collected via the first optical outlet port P3 and processed in the same manner as a standard UV-VIS measurement, which is performed by measuring the wavelength-dependent extinction spectrum of the sample, which determines the wavelength-dependent extinction coefficient of the sample.

[0395] In the second configuration, light from the light source is transmitted through P2 along the second optical path 17 and first directly incident on the reflective wall 5 of cavity 3. The surface of the wall 5 of cavity 3 is approximately a perfect diffuse reflector (Lambertian surface). The incident light thus diffuses diffusely within cavity 3 and illuminates and interacts with the sample. The light can be absorbed by the sample, but the light scattered by the sample is still part of the diffuse illumination present in cavity 3.

[0396] In the second configuration, light is then collected via a second optical exit port P4, which is specifically positioned such that as much light as possible transmitted or reflected directly from the sample does not enter the exit port P4 before being reflected from the cavity wall 5 and processed by the spectrometer 11. This allows for the determination of the sample's true absorption spectrum without spectral loss due to scattering. Switching between extinction and absorption modes is accomplished via an optical path adjuster without requiring changes to the sample position or any other optics of the apparatus.

[0397] The optical path modulator 13 thus modulates the light received from the light source 9 by the integrating cavity 3 to provide a first optical path 15 and a second optical path 17, in which the light directly incident on the liquid sample without incident on the wall 5 of the cavity 3, and in the second optical path 17, the light directly incident on the wall 5 of the cavity 3 without incident on the liquid sample.

[0398] exist Figure 1 a, Figure 1 In example b, the optical path adjuster 13 includes optical elements in the form of two laterally spaced, angled inlet mirror groups M1 and M2 between the light source 9 and the cavity 3, and a pair of corresponding laterally spaced, angled outlet mirror groups M3 and M4 between the cavity 3 and the spectrometer 11. In this example, the cavity 3 includes two laterally spaced light inlet ports P1 and P2, and two laterally spaced light outlet ports P3 and P4. In this example, a plurality of lenses L1-L5 are provided at different positions along the first optical path 15 and the second optical path 17. The optical path adjuster also includes a movable shutter S1 configured to open and close the first outlet port P3.

[0399] An inlet mirror M1 and an outlet mirror M4 are movable along the transverse axis of cavity 3; however, the second inlet mirror M2 and the second outlet mirror M3 are fixed and immovable. In the first configuration, the two mirror groups are positioned so as not to obstruct the conceptual path (from the light source 9, to the first inlet port P1, to the liquid sample, and then to the first outlet port P3). In this position, light from the light source 9 is transmitted along the direct optical path 15 and directly incident on the liquid sample.

[0400] In parallel, when the reflector M1 leaves the first optical path 15, the shutter S1 opens simultaneously, allowing the light transmitted by the sample to leave the cavity 3 from the extinction light exit port P3. The movable exit reflector M3 is also simultaneously positioned outside the first optical path 15, so that the light leaving P3 can be directly focused onto the spectrometer 11 via the lens L5.

[0401] In configuration 2, a movable inlet reflector M1 is placed in the optical path between the light source 9 and the first inlet port P1. The reflector M1 is positioned at 45° to the optical path, guiding the light to a fixed reflector M2. The fixed reflector M2 thus allows the light to be focused by the focusing lens L2 into the absorption inlet port P2. In this configuration, the light is directly incident on the inner wall 5 of the cavity 3 and undergoes diffuse reflection within the cavity 3. The light within the cavity 3 is then collected by the lens L4 via the outlet port P4 and sent to the spectrometer. The light is prevented from leaving the cavity 3 via the first outlet port P3 because this has been closed by the movable shutter S1.

[0402] In practice, the use of device 1 provides one or more of the advantages stated above.

[0403] Device 1 may include, or communicate with, an electronic controller / software configured to perform measurements (i.e., reference and sample measurements, acquisition time, integration time) and display of the obtained extinction, absorption, and scattering spectra.

[0404] Same entry port Now for reference Figure 3 a and Figure 3 b. A second embodiment of device 1 is provided with the same features given the same reference numerals. In this example, device 1 and Figure 2 The device is similar, but provides a single optical entry port P1. The optical path adjuster 13 includes a combined pinhole lens system comprising a pinhole PN1 positioned between the light source 9 and the entry port P1, a focusing lens L2, and a movable shutter S1 positioned after the exit ports P3 and P4. Figure 3In the first configuration shown in b, the optical path adjuster 13 is configured such that the pinhole PN1 is aligned with the incoming optical path and the light entering the inlet port P1 is substantially collimated, and at this position, the light from the light source 9 is transmitted along the direct optical path 15 and directly incident on the liquid sample. In parallel, when the pinhole PN1 is in the optical path, the shutter S2 is simultaneously closed, thereby allowing the light transmitted by the sample to exit the cavity 3 from the extinction light exit port P3. The movable exit mirror M3 is also simultaneously positioned outside the first optical path 15, such that the light exiting P3 can be directly focused onto the spectrometer 11 via the lens L5.

[0405] In configuration 2, the optical path adjuster 13 is positioned such that the input pinhole PN1 is outside the optical path and the focusing lens L2 is inside the optical path, and the incident light from the light source 9 is focused onto the inlet port P1, allowing the light to be transmitted to the sample along the direct optical path. However, because it has been focused to a point at the inlet port, the light is diverged, causing the light to illuminate the entire lateral width of the sample reservoir. In parallel, when the focusing lens L2 is inside the optical path, the shutter S2 simultaneously opens, thereby covering the outlet port P3, where the movable mirror M4 is positioned at 45° to the optical path. In this configuration, the light scattered, transmitted, and reflected by the sample undergoes diffuse reflection within the cavity 3, which then allows the diffusely reflected light to exit the cavity 3 from the absorption outlet port P4. This light is then collected via the outlet port P4 using the lens L4 and transmitted to the spectrometer via the mirror M3 and the movable mirror M4.

[0406] Shutter selection to avoid sample Now for reference Figure 4 A third embodiment of device 1 is provided with the same features given the same reference numerals. In this example, the movable inlet reflector M1 has been replaced by an inlet shutter S2 and a second fixed inlet reflector M1. The outlet reflectors M3 and M4 together can exit from... Figure 2 The position shown is laterally moved such that the exit mirror M3, angled at this position, is in the optical path of the exit port P3 to guide light from the first optical path 15 to the second exit mirror M4 and then to the spectrometer 11. The exit shutter S1 includes a shutter aperture aligned with the exit port P3 in this first configuration. The entrance shutter S2 includes a pair of laterally spaced shutter apertures. In the first configuration, the shutter S2 is positioned such that one of the shutter apertures is aligned with the entrance port P1, but the entrance port P2 is closed. Angled, fixed entrance mirrors M1 and M2 guide light to the entrance port P1.

[0407] In the second configuration, the inlet shutter S2 is moved laterally, causing the inlet port P1 to close and the inlet port P2 to align with one of the apertures of the inlet shutter S2, so that light from the light source 9 is directly transmitted into the inlet port P2. The outlet mirrors M3 and M4 are moved laterally, so that mirror M3 is no longer in the optical path between the outlet port P4 and the spectrometer 11.

[0408] Shutter selection through sample refer to Figure 5 The fourth embodiment of device 1 is provided with the same features as those given the same reference numerals. In this example, device 1 and Figure 4 The device is similar, but without an entrance mirror. The entrance shutter S2 is positioned adjacent to the entrance lens L6. Lateral adjustment of the position of the entrance shutter S2 aligns one or more shutter apertures with the entrance lens L6 and the light source. One entrance shutter aperture is relatively small, while the other is relatively large. By adjusting which aperture is aligned with the light source in conjunction with the lens L6, both optical paths 15 and 17 can be directly incident on the liquid sample. The first optical path passes through the sample and exits the cavity via the exit port P3, and when the exit shutter S1 closes the first exit port P3, the second optical path also passes through the liquid sample but is diffused to contact the wall 5 of the cavity 3 before exiting the cavity 3 via the second exit port P4.

[0409] Now for reference Figure 6 The fifth embodiment of device 1 is provided with the same features as those given the same reference numerals. In this example, device 1 and Figure 3 a and Figure 3 The device in embodiment b is similar, but provides manipulation of the optical path for two different configurations via an off-axis parabolic (OAP) mirror instead of a lens and a plane mirror. Furthermore, two entry ports P1 and P2 are provided in this embodiment. In this example, OAP M1 includes a mirror positioned between the light source 9 and the first entry port P1, with a centrally drilled hole parallel to the incident light path, while OAP M2 has no hole and redirects the light at an angle (60° in this example) between the light source 9 and the second entry port P2. The optical path adjuster includes two movable shutters S1 and S2 on the entry and exit sides of the integrating cavity 3, which are parallel and move according to their positions to block light entering and exiting simultaneously from ports P1 and P3 or simultaneously from ports P2 and P4.

[0410] In the first configuration, the optical path modulator is positioned such that light reflected and focused from OAP M2 is blocked from entering cavity 3 via the second inlet port P2, allowing only light passing through the hole in OAP M1 to enter cavity 3 via the first inlet port P1 and be transmitted along the direct optical path 15. This light is directly incident on the liquid sample 7. In parallel, on the outlet side of cavity 3, the shutter S2 of the optical path modulator is positioned such that the second outlet port P4 is closed and light diffusely reflected within cavity 3 does not reach spectrometer 11. In parallel, the first outlet port P3 is opened, allowing light transmitted from sample 7 to exit through the first optical outlet port P3, be transmitted through a hole drilled parallel to the optical path in OAP M3, and be directly focused onto spectrometer 11 via lens L5.

[0411] In the second configuration, the shutter S1 of the optical path modulator is positioned such that light passing through the aperture in OAP M1 is blocked from entering cavity 3 via inlet port P1. Therefore, the diverging light arriving at OAP M1 is collimated by OAP M1 and redirected 90° onto OAP M2, which is then focused from OAP M2 and redirected at 60° to a point at the second inlet port P2. The light entering cavity 3 then diverges, illuminating the entire lateral width of the sample reservoir while preventing any light from being directly transmitted to the first optical inlet port P1. In parallel, on the outlet side of cavity 3, the shutter S2 of the optical path modulator is positioned such that outlet port P3 is closed and light directly transmitted by sample 7 does not reach spectrometer 11. In parallel, outlet port P4 is opened, allowing light scattered, transmitted, and reflected by sample 7 to undergo diffuse reflection within cavity 3, after which it exits cavity 3 via the second outlet port P4. The diffused light is then collected by OAP M4, collimated by OAP M4 and redirected at 90° to OAP M3, and then redirected at 90° from OAP M3 and focused directly onto the spectrometer 11.

[0412] Now for reference Figure 7 A sixth embodiment of device 1 is provided with the same features given the same reference numerals. In this example, manipulation of the optical paths for the two different configurations is provided via a pair of fiber optic cables 21, 23, each of which is associated with a corresponding light source 25, 27 and with a corresponding entry port P1, P2. Each light source 25, 27 may include a corresponding LED light source 25, 27, which in this example includes a light modulator together with an associated LED electronic controller 29, thereby controlling the supply of light to entry port P1 or P2 by appropriately activating and deactivating the LED source 25, 27 by the controller 29.

[0413] In this example, fiber optic cable 21 supplies light directly to the first entry port P1. Fiber optic cable 23 supplies light to the second entry port P2 via collimating lens 30.

[0414] The exit mirror 32 and beam splitter 33 are disposed between the exit ports P3, P4 and the spectrometer 11 and are configured to selectively allow light from the first exit port P3 and the second exit port P4 to reach the spectrometer 11, depending on which configuration the device is operating in.

[0415] In the first configuration, device 1 is in transmission mode, in which the optical path modulator (i.e., controller 29) is controlled to provide light from LED source 25 to inlet port P1 via first optical fiber cable 21. The light entering cavity 3 through inlet port P1 directly irradiates the liquid contained in liquid tank 7, and the outlet optical path modulator (i.e., outlet reflector 31 and beam splitter 33) is configured such that the light collected through outlet port P3 and sent to spectrometer 11 includes a portion of the light from first LED source 25, which is directly transmitted through the sample after irradiating it. In this configuration, the extinction spectrum of the sample is obtained.

[0416] In the second configuration, device 1 is in diffuse reflection mode, where controller 29 controls the second LED source 27 to provide light to the second inlet port P2 via the second optical fiber cable 23. Light from the LED source 25 entering cavity 3 through inlet port P2 can directly illuminate the liquid contained in the liquid tank 7 or can be incident on the cavity wall 5 and diffusely reflected within cavity 3 before interacting with the liquid sample. In this second configuration, the outlet reflector 31 and / or beam splitter 33 are configured such that light transmitted and / or reflected by the sample and collected through the second outlet port P4 and sent to spectrometer 11 has undergone at least one reflection from the cavity wall 5 before entering outlet port P4. In this configuration, the absorption spectrum of the sample is obtained without the effect of scattering by the liquid sample.

[0417] A slightly simpler device can be produced by using independently controllable LED light sources (each of which feeds a specific inlet port P1, P2), which requires fewer separate movable and / or fixed optical elements to control the light entering the sphere 3 and allows the device to operate in both a first and a second configuration.

[0418] In this embodiment, the inlet port P2 is not parallel to the inlet port P1, so that light enters the cavity through the inlet port P2 at an angle tilted to the cavity's main axis. The position / angle of port P2 should be selected to minimize the chance that any Fresnel reflection from the liquid tank 7 when light hits the liquid tank 7 will exit through the transmission ports P3 and P4 during the first reflection. The angle of the light path through port P2 can be selected accordingly.

[0419] In device 1, movable and / or fixed optical elements can be selected from the following: a. Prism; b. Lens; c. Reflector; d. Diffraction grating; e. Fiber optic cable; f. Light source.

[0420] Exemplary Components Below is a non-limiting outline of exemplary components that can be used with some examples of device 1: • Light source 9: Tungsten halogen lamps purchased from ThorLabs that provide excitation light from 350 nm to 900 nm.

[0421] • Movable reflectors (M1, M4, in) Figure 1 and Figure 2 (In the example): A standard optical reflector mounted at 45° to the optical path, which can be shifted in and out of the beam path to select either a first or second configuration. Purchased from ThorLabs.

[0422] • Fixed reflectors (M2, M4, in) Figure 1 and Figure 2 (In the example): A standard optical reflector mounted at 45° to the optical path, which can be shifted in and out of the beam path to select either a first or second configuration. Purchased from ThorLabs.

[0423] • Transmission lens (L2, in) Figure 1 and Figure 2 (In the example): A standard convex lens with a defined focal length used in the second configuration to focus incoming light through the inlet port P2 onto the cavity wall 5 for absorption measurements. Purchased from ThorLabs.

[0424] • Integrating Cavity 3: A spherical integrating cavity with a 50 mm inner diameter and diffuse inner walls. The sphere has four ports (P1-P4) drilled in the walls for light transmission and collection, and a custom-drilled sample port at the north pole for suspending the liquid tank 7 at the center of cavity 3. Integrating Cavity 3 is purchased from Avian Technologies. The sphere geometry can be customized to suit the application used with Device 1. Cavity 3 can be non-spherical and may be cylindrical or cubic. The coating of the walls 5 can have different types of surface reflectivity, including specular and diffuse reflection or combinations thereof in the UV, visible, or infrared regions or combinations thereof.

[0425] • Sample Holder / Liquid Tank 7: The liquid tank 7 is held in the device 1 by a holder that clamps around the liquid tank 7 and also allows the liquid tank 7 to be suspended in a fixed position within the cavity 3. The following liquid tank geometries can be provided: standard (1 cm square), thin or plate-shaped (10 × 1 mm), cylindrical, spherical (combinations are also possible, such as a cylinder with a flat area). • USB Spectrometer 11: Analyzes the intensity of light leaving cavity 3 based on wavelength, thereby allowing the spectrum to be acquired and displayed on, for example, a computer screen. This can be a standalone device powered via a USB connection and interfaced to a controller in the form of a laptop / computer. Light detection can be performed using a standard spectrometer with dispersive optics or via CMOS, CCD, diode array, or scanning monochromator.

[0426] • Electronic Devices: The movable reflector is driven by a stepper motor and controlled by a programmable microcontroller with a stepper motor driver board. Both the microcontroller and the USB spectrometer are attached to a controller, such as a microcomputer within device 1. The microcomputer serves a dual purpose: i) it facilitates communication with the spectrometer 11 and the motor driver, and ii) it provides a web-based graphical user interface. This facilitates interaction with device 1 because it eliminates the need for users to install special software and for developers to maintain customized operating system-related software.

[0427] • Light sources: standard UV-VIS (i.e., halogen lamps, xenon lamps, deuterium lamps), any kind of LED, lasers, combinations of all of these; and any multicolor light source with an attached monochromator for wavelength selection.

[0428] • Transmission optics: Assemblies of standard optical components such as lenses, mirrors, shutters, diffraction gratings, optical fibers, or any combination thereof.

[0429] • Optical path switching: (one or more) motorized linear stages and / or (one or more) shutters.

[0430] Parameters / Variables There are many physical and geometric parameters / variables that are factors in the design and operation of device 1 as described above, including any one or more of the following: • Cavity surface reflectivity ρ It is the ratio of reflected light to incident light. For the cavity of device 1 to operate, the reflectivity must be close to one, meaning that wall 5 comprises a highly reflective material. Device 1 further requires a strongly diffuse (Lambertian) reflectivity.

[0431] • Port score fIt is the ratio of the surface area of ​​all cavity ports P1-P4 to the total surface area of ​​the wall 5 of cavity 3. Light rays randomly crossing cavity 3 therefore have a chance of escaping. f .

[0432] • Enhancer M This approximates the amount of light reflected from the diffuse cavity surface before being absorbed by the cavity walls or leaving through the ports. In the ideal case of an empty spherical cavity, we have... M = .

[0433] • Sample hit probability μ: a purely geometric factor, stating the probability that diffusely reflected light leaving the cavity surface interacts with the sample reservoir.

[0434] • Path length L L is the average length of the path that light travels within the sample volume. If M and i are large, then L is large.

[0435] Device Calibration / Measurement / Control Overview The following factors form the basis of device 1 in order to obtain error-free spectra: Related to absorption rate measurement: • The controller determines the absolute absorption cross-section of the sample inserted into the integrating chamber; this requires accurate calibration of the measurable intensity against known standards.

[0436] • Input port location for absorption rate: There are two options for the placement of this port: ○ i) Avoiding direct irradiation of the sample improves the reproducibility of the measurement because it is less sensitive to the exact geometrical replacement of the sample reservoir. The disadvantage of this method is that even for a fully absorbed sample, some light reaches the detector (determined by μ) without interacting with the sample, which limits the range of measurable optical densities.

[0437] ○ ii) Alternatively, all incident light can pass through the sample. This solves the problem of saturation absorptivity and allows measurement of strongly absorbing samples. In this case, the detection port needs to collect light from a portion of the cavity wall that does not receive light from direct or reflected illumination.

[0438] • Detection port location for absorbance: The field of view of the detection port must not intersect with the sample; instead, it should focus light only from the cavity surface. This minimizes the dependence of the measurement on the scattering properties of the sample.

[0439] • Geometry optimization settings: Average path length in the sample L The ratio of sample volume to cavity volume, rV = V, can be used to determine this. 样品 / V 腔 Multiply by the average chord length in the cavity c̅ = 4V 腔 / A 腔 (where A) 腔 (The surface area of ​​the cavity) multiplied by the enhancement factor M To approximate. Approximate path length. L =rV c̅M Control the lower limit of detectable optical density; for example, for samples with low absorbance, it is desirable to make L Maximize: i) M For cavity surface reflectivity ρ →1 and cavity port fractions f →0 becomes the maximum, ii) rV increases with relative sample volume and approaches -1 when the sample completely fills the sphere, iii) c̅ For spherical cavities, this is the largest. A spherical cavity that is completely filled with the sample, with the highest surface reflectivity and the smallest port opening, can be the optimal setting for detecting ultra-low concentrations.

[0440] • Selecting a combination of parameters (cavity and sample geometry, port locations, numerical aperture, etc.) that suits the requirements of effectiveness, reproducibility, and user convenience is not straightforward. Design choices can be nontrivial compromises. For example, the device 1 described above is suitable for standard liquid baths, including baths with short optical path lengths for strongly absorbing liquids.

[0441] Related to the measurement of combined extinction absorbance: • Extinction measurements are performed within the integrating cavity; this is geometrically constrained because the sample wall must be perpendicular to the incident beam, requiring a square or flat-walled liquid bath. Liquid baths with curved surfaces (e.g., cylindrical) are also possible, but will require specialized optics to resist refractive effects.

[0442] • It is necessary to constrain the available numerical aperture in both transport and detection to avoid diffuse illumination of the sample and minimize the detection of multiple scattered light.

[0443] • Combined transmission and detection optics are required that can switch between absorption and extinction paths. The arrangement of these paths must ensure that they do not interfere with each other.

[0444] Device calibration / measurement / control example details Details of exemplary calibration methods that can be used to calibrate the spectrometer apparatus described above are presented in the appendix.

[0445] The spectrometer can be configured to measure the spectrum of a liquid sample selected from any one or more of the following: a.water; b. Wine; c. Beverages; d. Edible liquid or partially liquid products; e. Coatings; f. Water, such as seawater; g. Nanoparticles; h. Emulsion; i. Blood In one example, the spectrometer device could therefore be a wine testing device.

[0446] The above list is non-restrictive.

[0447] Unless the context clearly requires otherwise, throughout the specification, the words “contains”, “comprising”, etc., shall be interpreted in the sense of inclusion, as opposed to exclusivity or delimitation (that is, in the sense of “including but not limited to”).

[0448] Although the invention has been described by way of example and with reference to possible embodiments thereof, it should be understood that modifications or improvements may be made thereto without departing from the scope of the invention. The invention can also be broadly defined as any or all combinations of two or more of the portions, elements, and features referenced or indicated in the specification of this application, individually or collectively. Furthermore, where specific components or integers of the invention having known equivalents have been referenced, such equivalents are incorporated herein as if described separately.

[0449] Any discussion of prior art throughout the specification should never be considered as an admission that such prior art is widely known or forms part of common knowledge in the art. Appendix – Example Calibration Method Calibration procedure for combined extinction and absorption spectra Background Technology

[0450] In standard UV-VIS transmission measurements, the extinction of the sample being measured is... Using Beer-Lambert's law and extinction coefficient, the sample... and the path length through the sample The relevant information is given in the following formula: in, It is the ratio of transmitted light to incident light at a given wavelength. If the extinction image is caused by a specific analyte in the sample (e.g., dye molecules), then the extinction coefficient is related to the concentration of the analyte. and the molar extinction coefficient of the analyte Proportional. Therefore, when the path length is known, the measured extinction is directly proportional to the sample extinction coefficient, which can be used to calculate the analyte concentration using the following formula: This shows the quantitative energy change measured by UV-VIS, because for a standard UV-VIS setup, the path length is limited by the length of the liquid bath used, typically 1 cm. In reality, extinction is the sum of scattering and absorption by the sample, thus... It can be divided into two parts: one from absorption and one from scattering. These are then referred to as the molar absorptivity, respectively. and scattering coefficient This makes the Bill-Lambert expression: This allows for three typical cases, where the sample can: 1. Absorb only, that is and 2. Only scattering, i.e. and 3. Absorption and scattering, i.e. and For most samples, case 1 applies, where Furthermore, Equation 2 can be used to determine the concentration or molar absorptivity of the analyte. However, when Case 3 applies, the standard UV-VIS configuration cannot be used because it is difficult to separate the contribution from scattering and absorption individually. Therefore, if the analyte of interest has... But embedded in nonzero In liquids containing analytes, the concentration of the analyte cannot be determined by extinction measurement due to the contribution of scattering. The solution to this problem lies in embedding the sample within an integrating cavity as described in the original invention to eliminate the effect of scattering on the measured signal.

[0451] Integrating cavity path length In non-standard transmission configurations, such as those shown below, where the sample is embedded within an integrating cavity such that light interacting with the sample is diffusely reflected within the cavity walls, the path length within the sample is no longer simply defined as the thickness of the liquid bath, because the light can pass through the sample many times at different angles before leaving the cavity and entering the spectrometer. Thus, using Equation 2 to determine the molar absorptivity of the sample is no longer valid (for the sake of argumentation, we will limit ourselves to case 1 here). And will Written as In this case, the absorption measured in the cavity is instead given by the following formula: in, It is the effective cavity path length, and It is the “true absorption” signal measured in a standard transmission measurement with a path length of 1 cm. It is clearly the wavelength (due to the non-flat reflection of the cavity material) and The function. Rearranging equation 4 gives: This indicates that by measuring a range of absorption samples in both transmission and absorption modes (as described in the original invention) and taking... and Compared to directly tracing the cavity path It is determined to be a function of wavelength and sample (real) absorption.

[0452] A method is needed to determine the cavity path length so that the absorption measured in this configuration can be... This can be converted into equivalent absorption to be measured in a transmission setting with a 1 cm path length. As a result, once the path length is known, compared to the transmission setting... Corrections can be made for increases (or decreases) in path length, and Equation 2 can be applied to determine the absorption coefficient (or concentration) of the analyte.

[0453] Integrating cavity calibration The method outlined for determining the effective integration cavity path length is implemented below using the setup described in the original invention and the dye Eosin B as the analyte. A series of dye concentrations is measured to determine the path length within a certain range of sample absorption. In all cases, It is the "true" absorption measured in transmission mode, and This is the absorption in the cavity and the wavelength-dependent path length. Measurement details are described in the Methods section.

[0454] Wavelength [nm] Figure 1 Absorption spectra of Eosin concentration series in absorption mode Wavelength [nm] Figure 2 Absorption spectra of Eosin concentration series in transmission mode The invention obtained using the series of Eosin B concentrations described herein and Spectrum in Figure 1 and Figure 2The figure shows the dye concentration in each measurement. When the spectra appear similar in both configurations, there is a significant difference in the magnitude of the spectra measured at each concentration. As expected by the Beer-Lambert law, in the extinction configuration (not shown), in the absence of scattering, It scales linearly with the absorption coefficient. However, in the cavity configuration, due to the nonlinear response of the cavity path length to increased absorption, the magnitude deviates rapidly from a linear relationship with the concentration.

[0455] Figure 3 The absorption was measured at 517 nm. The function Figure 4 At 517 nm, it is considered as the true absorption. The function use Figure 1 and Figure 2 The data in the equation is obtained by taking the values ​​given in equation 4. and The ratio is calculated at any wavelength. The path length factor obtained from Eosin data at 517 nm is... Figure 3 The measured absorption and targeting Figure 4 True absorption draw. The intermediate value was then found through linear interpolation between the measured data points shown. It ranged from 0.001 to 1.39 at a single wavelength. The cavity path length was successfully determined within the specified range. For the specific cavity geometry used, at 517 nm, At the lowest absorption ( It reaches a maximum value of 7 cm at (=0.007), and at the highest absorption ( =1.51) decreases to a minimum of 1.1 cm. Used to obtain The method is intended to be extended to all wavelengths, where the dye is fully absorbed.

[0456] Cavity path length in the presence of scattering Scattering pairs when the sample has a non-zero scattering coefficient (and thus) The effect has been targeted Figure 5 and 6Two cases are illustrated. It is evident in the two cases with significantly different dye concentrations that scattering in solution has a negligible effect on the measured absorption spectrum, demonstrating that the cavity path length is insensitive to sample scattering. Therefore, the calibration method outlined above for zero scattering can be applied to samples with arbitrary scattering coefficients. For the corresponding extinction of the 488nm Eosin series... Spectrum in Figure 7 As shown, scattering due to silicon particles is evident in the broad Rayliegh-type spectrum presented (note that the Eosin absorption curves with subscripts are partially visible in some spectra where scattering and absorption are comparable).

[0457] Wavelength [nm] Figure 5 488 nM Eosin in solutions with increased scattering coefficient The spectrum, in which silicon particles are diluted from the raw material, is shown in the legend.

[0458] Wavelength [nm] Figure 6 7.8 μM Eosin in a solution with increased scattering coefficient The spectrum, in which silicon particles are diluted from the raw material, is shown in the legend.

[0459] Wavelength [nm] Figure 7 For the corresponding extinction spectra of the 488 nM+ silicon concentration series The dilution of silicon particles from the raw material is shown in the legend.

[0460] I. Applying cavity calibration to any solution: Calibrated cavity spectrometer (by Characterization can now be used to quantitatively determine the absorbance of any sample (which has already been measured). The extinction and (true) absorption spectra within the range. This is in Figure 8 As shown in Figure 9, measurements were performed on two food dyes (Blue1 and Red3, respectively) with different absorption spectra in a cavity spectrometer. The dyes absorb at significantly different wavelengths, thus ensuring the accuracy of the measurement calibration method. The measurements were calculated using Eosin-based calibration data. A suitable mathematical model can be used to recalibrate the measured absorption. To generate a calibrated absorbance, the calibrated absorbance shown in the figure is relative to the true absorbance. The absorption spectra are always identical (obtained via transmission mode). These two cases demonstrate the effectiveness of the proposed method for returning equivalent absorption spectra from measured absorption in absorption mode to transmission mode. Therefore, the method remains effective for samples with arbitrary scattering coefficients and will correctly produce the true absorption spectra of the samples. And simultaneously produce its extinction spectrum .

[0461] Figure 8 1.25 μM Blue1 in H2O Spectrum and original absorption spectrum (Scaled 4 times for comparison) and The calibrated version.

[0462] Figure 9. 1.25 μM Red3 in H2O Spectrum and original absorption spectrum (Scaled 4 times for comparison) and The calibrated version.

[0463] Appendix A – Measurement Procedure The following steps are used to perform a complete measurement of a sample using this invention: 1. The user sets the spectral integration time and the total number N of spectral acquisitions required for the measurement. For each step (reference, sample, and dark), the average spectrum is calculated by averaging the N spectra.

[0464] 2. The software is used to set the instrument to absorption mode.

[0465] 3. The intensity of transmitted light in absorption mode was measured by placing 2 mL of reference solution (in this case, water) in a 1 cm × 1 cm liquid tank inside the cavity and measuring the intensity (recorded by software) as... To obtain the reference spectrum.

[0466] 4. The software is used to switch the instrument to extinction mode.

[0467] 5. Measurements were performed using the same reference solution in extinction mode, where the intensity of transmitted light was recorded by the software. .

[0468] 6. Remove the liquid tank, remove the reference solution, and replace it with 2 mL of sample solution (i.e., the lowest concentration of dye used). The liquid tank is replaced with the cavity in the same position as the reference solution.

[0469] 7. In extinction mode, measure the intensity of light transmitted through the sample, and record it by the software. .

[0470] 8. The software is used to switch the instrument to absorption mode and measure the intensity of light transmitted through the sample, which is then recorded by the software. .

[0471] 9. The software was set to dark mode (i.e., the light source was turned off) and the dark spectrum was measured and recorded by the software. .

[0472] 10. For the entire set of samples in the dilution series, repeat steps 5-7 from the lowest to the highest concentration of dye, taking a new reference sample if there is a problem with light drift.

[0473] The extinction and (measured) absorption spectra of the nth sample are calculated as follows: as well as Where n is the number of samples in the dilution series, indicating that n=1 is the lowest concentration and n=n is the highest concentration.

[0474] For the extinction and absorption spectra shown (zero scattering case), each spectrum is then post-processed by first subtracting a constant background. In the case of Eosin B, this is done by averaging each spectrum in the range of 700 to 750 nm. Each spectrum is then smoothed to remove noise using a Savizky-Golay filter with a bandwidth of 61 and a second-order polynomial. The measured absorption spectra are then converted to true absorption spectra, as listed in Equation 5, thus producing the 2-spectrum, i.e., the extinction spectrum. and true absorption spectrum .

[0475] Appendix B – Experimental Details The Eosin B sample from SigmaAlrich was prepared by diluting the raw material in water to 500 μM. The raw material solution was prepared from the received powder. The scattering solution of 300 nm silicon particles was prepared by diluting the powder in 50 mg mL of water. -1The scattering solution is prepared by diluting the raw material solution with an aqueous solution (e.g., received from the supply). The scattering solution is then mixed with an equal amount of Eosin B to achieve the desired final dye + scattering concentration. This method implies the assumption that the dye molecules and scattering particles will not interact through electrostatic absorption of the molecules onto the particle surface, nor through chemical interactions. For this reason, it is desirable to use dyes with the same charge as the particle surfaces; in this case, Eosin B is negatively charged and the silica particles have COOH surface groups that will be negatively charged in solution, therefore there should be no interaction between the two species, as indicated by the similarity between the absorption spectra of the dye dissolved in H₂O and the absorption spectra of the dye dissolved in the silica solution. All samples are prepared immediately before measurement in the instrument. After each sample is measured in a dilution series, the tank is thoroughly rinsed with water, then ethanol, and then water (if necessary), and a fresh aqueous reference solution is taken if required.

[0476] References Claims 1. A spectrometer apparatus for measuring the spectrum of a liquid sample, the apparatus comprising: An integrating cavity, the integrating cavity including one or more reflective inner walls, and the integrating cavity being configured to house a liquid tank containing a liquid sample within the integrating cavity. The integrating cavity includes at least one optical inlet port and at least one optical outlet port. The optical inlet port or each optical inlet port is configured to receive light from at least one light source, and the optical outlet port or each optical outlet port is configured to transmit light to a spectrometer. The device further includes an optical path adjuster configured to selectively adjust the optical path through the integrating cavity, such that at least two different optical paths are provided; wherein When the optical path modulator is in the first configuration, the device is in transmission mode, in which light from the light source follows a first optical path from the light inlet port or one of the light inlet ports to the liquid sample, such that the light from the light source directly illuminates the liquid sample before the light transmitted by the sample is transmitted through the light outlet port or one of the light outlet ports and received by the spectrometer for wavelength analysis to provide the extinction spectrum of the liquid sample; and When the optical path adjuster is in the second configuration, the device is in diffuse reflection mode, in which light from the light source follows a second optical path into the integrating cavity from the inlet port or one of the optical inlet ports. The light from the light source is incident on one or more reflective inner walls of the integrating cavity and undergoes diffuse reflection within the integrating cavity, such that the light from the light source illuminates the liquid sample before being transmitted through the optical outlet port or one of the optical outlet ports and received by the spectrometer for wavelength analysis to provide the absorption spectrum of the liquid sample contained in the liquid sample.

[0477] 2. The apparatus of claim 1, wherein the apparatus is arranged such that, when in the second configuration, light from the second optical path is transmitted: a. Directly from the inlet port to one or more walls of the integrating cavity; and / or b. Directly from the inlet port to the sample and subsequently to one or more walls of the integrating cavity via the sample.

[0478] 3. The apparatus of claim 1 or 2, wherein the inlet port used in the first configuration and the outlet port used in the first configuration are directly opposite each other, such that when in the first configuration, the first optical path extends directly across the integrating cavity.

[0479] 4. The apparatus according to any one of claims 1 to 3, further comprising the light source.

[0480] 5. The apparatus according to any one of the preceding claims further includes a controller configured to control the optical path modulator to selectively adjust the path of light passing through the apparatus.

[0481] 6. The apparatus of claim 5, wherein the controller is an integral part of the apparatus and communicates directly with the apparatus.

[0482] 7. The apparatus of claim 5, wherein the controller is located remotely from the apparatus and configured to communicate wirelessly with the transceiver of the apparatus.

[0483] 8. The apparatus according to any one of claims 5 to 7, wherein the controller is configured to control the spectrometer, and more specifically, to process the light received by the spectrometer for wavelength analysis of the light to provide an extinction spectrum and / or absorption spectrum of the liquid sample contained in the tank.

[0484] 9. The apparatus of claim 5, further comprising the spectrometer.

[0485] 10. The apparatus according to any one of claims 5 to 9, wherein the controller is configured to control one or more of the following: a. Switch between the first configuration and the second configuration; b. Obtain the spectrum from the integrating cavity; c. Select operating conditions; d. Display the spectrum on the display of the device or the display of the controller, or on a display that communicates with the device or the controller; e. Store the data in the memory of the device or the memory of the controller, or in a memory that communicates with the device or the controller; f. The user interface of the device or the user interface of the controller, or the user interface communicating with the device or the controller, which interacts with the device and allows the user to control the position of the optical path adjustment mechanism.

[0486] 11. The apparatus according to any one of the preceding claims, wherein the optical path adjuster includes at least one movable optical element configured to manipulate light incident on the optical element from the light source, and the optical path adjuster is configured to adjust the movable optical element to selectively provide the first optical path and the second optical path.

[0487] 12. The apparatus of claim 11, wherein the optical element is adjustable by moving the optical element relative to the integrating cavity from a first position where the light travels along the first optical path and a second position where the light travels along the second optical path.

[0488] 13. The apparatus of claim 12, wherein the integrating cavity comprises orthogonal longitudinal, vertical, and transverse axes, and any one or more of the following positional characteristics of the optical element can be adjusted with respect to any one or more of these axes: a. Vertical position; b. Vertical position; c. Lateral position; d. Orientation; e. Inclination angle.

[0489] 14. The apparatus according to any one of claims 11 to 13, wherein a plurality of movable optical elements are provided.

[0490] 15. The apparatus of claim 11, wherein the movable optical element is selected from: a. Prism; b. Lens; c. Reflector; d. Diffraction grating; e. Fiber optic cable; f. The light source.

[0491] 16. The apparatus according to any one of claims 11 to 15, wherein the optical path adjuster comprises at least one fixed optical element that is not adjustable relative to the integrating cavity.

[0492] 17. The apparatus of claim 16, wherein the fixed optical element is configured to manipulate light from the light source prior to the light inlet port.

[0493] 18. The apparatus of claim 16 or 17, wherein the fixed optical element is configured to manipulate light from the light exit port.

[0494] 19. The apparatus according to any one of claims 16 to 18, wherein the fixed optical element is selected from: a. Prism; b. Lens; c. Reflector; d. Diffraction grating; e. Fiber optic cable; f. The light source.

[0495] 20. The apparatus according to any one of the preceding claims, wherein the optical path modulator includes at least one electronic controller operable to selectively operate one or more light sources to selectively provide the first optical path and the second optical path.

[0496] 21. The apparatus of claim 20, comprising at least a first light source and a second light source, wherein the controller is configured to independently control each light source.

[0497] 22. The apparatus according to any one of the preceding claims, wherein the optical path adjuster is positioned between the light source and the optical entry port.

[0498] 23. The apparatus according to any one of the preceding claims, wherein the optical path modulator is positioned between the spectrometer and the optical output port.

[0499] 24. The apparatus according to any one of the preceding claims, wherein a plurality of optical path adjustment mechanisms are provided.

[0500] 25. The apparatus according to any one of the preceding claims, wherein a plurality of optical entry ports are provided, and the optical path modulator is configured to provide a first optical path by guiding light from the light source through a first optical entry port, and to provide a second optical path by guiding light from the light source through a second optical entry port.

[0501] 26. The apparatus according to any one of the preceding claims, wherein a plurality of optical exit ports are provided, the first optical path directing light from the integrating cavity through the first optical exit port, and the second optical path directing light from the integrating cavity through the second optical exit port.

[0502] 27. The apparatus according to any one of the preceding claims, wherein the integrating cavity comprises any one of the following: a. Diffuse reflection spherical integrating cavity; b. Cylindrical cavity; c. A cubic or square cavity.

[0503] 28. The apparatus according to any one of the preceding claims, wherein the integrating cavity includes an internal coating configured to provide any one or more of the following: a. Specular reflection; b. Diffuse reflection; c. Reflection in the UV light spectrum; d. Reflection in the visible light spectrum; e. Reflection in the infrared spectrum.

[0504] 29. The apparatus according to any one of the preceding claims, wherein the light source comprises any one or more of the following: a. Quartz halogen source; b. LED; c. Laser; d. Any multi-color light source.

[0505] 30. The apparatus according to any one of the preceding claims, wherein the shape of the liquid tank is: a. square; b. Plate-shaped; c. Cylindrical; d. Sphere-shaped.

[0506] 31. The apparatus according to any one of the preceding claims, wherein the apparatus is a UV-VIS spectrometer apparatus.

[0507] 32. The apparatus according to any one of the preceding claims further includes a sample holder configured to hold a liquid tank containing a liquid sample within the integrating chamber.

[0508] 33. The apparatus according to any one of the preceding claims, wherein the light source comprises a first LED light source and a second LED light source, and the light path modulator comprises a controller configured to control the first LED light source and the second LED light source such that, in the first configuration, the first LED light source is controlled to provide light in the first light path, and in the second configuration, the second LED light source is controlled to provide light in the second light path.

[0509] 34. The apparatus of claim 33, wherein light from each LED light source is transmitted to the integrating cavity via a corresponding optical fiber cable.

[0510] 35. The apparatus of claim 33 or 34, wherein each LED light source transmits light to a corresponding light inlet port.

[0511] 36. The apparatus according to any one of claims 33 to 35, wherein each optical path transmits light through a corresponding optical exit port.

[0512] 37. The apparatus according to any one of claims 33 to 36, wherein the first LED light source is associated with a collimating lens positioned between the first LED light source and a light inlet port associated with the LED light source.

[0513] 38. The apparatus of any one of claims 33 to 37, further comprising a first exit port and a second exit port, and a beam splitter configured to selectively allow light from the first exit port and the second exit port to be transmitted into the spectrometer.

[0514] 39. A spectrometer apparatus for measuring the spectrum of a liquid sample, specifically wherein the obtained spectrum is the absorption spectrum and extinction spectrum of the sample, the apparatus comprising: An integrating cavity, the integrating cavity including one or more reflective inner walls, and the integrating cavity being configured to house a liquid tank containing a liquid sample within the integrating cavity. The integrating cavity includes at least one optical inlet port and at least one optical outlet port. The optical inlet port is configured to receive light from a light source, and the optical outlet port is configured to transmit light to a spectrometer. The device further includes an optical path adjuster configured to selectively adjust the optical path through the integrating cavity, such that at least two different optical paths are provided; wherein: When the optical path modulator is in the first configuration, the device is in transmission mode, in which light from the light source follows a first optical path from the light inlet port to the liquid sample, such that the light from the light source directly illuminates the liquid sample before the light transmitted by the sample is collected via the light outlet port and received by the spectrometer for wavelength analysis to provide the extinction spectrum of the liquid sample; and When the optical path adjuster is in the second configuration, the device is in diffuse reflection mode, in which light from the light source follows a second optical path from the inlet port into the integrating cavity, and the light from the light source is incident on one or more reflective inner walls of the integrating cavity or directly onto the liquid sample; The light transmitted and / or scattered by the sample is transmitted through the outlet port, and the device is configured such that light directly transmitted by the sample is reflected by one or more inner walls of the cavity before it is transmitted through the outlet port and received by the spectrometer for wavelength analysis to provide the absorption spectrum of the liquid sample contained in the liquid sample.

[0515] 40. A spectrometer apparatus for measuring the spectrum of a liquid sample, the apparatus comprising: An integrating cavity, the integrating cavity including one or more reflective inner walls, and the integrating cavity being configured to house a liquid tank containing a liquid sample within the integrating cavity. The integrating cavity includes a first optical inlet port, a second optical inlet path, and at least one optical outlet port. The first optical inlet port is configured to receive light from a first LED light source, and the second optical inlet port is configured to receive light from a second LED light source. At least one optical outlet port is provided and configured to transmit light to a spectrometer. The device further includes an optical path adjuster configured to selectively adjust the optical path through the integrating cavity, such that at least two different optical paths are provided; wherein When the optical path modulator is in the first configuration, the device is in transmission mode, in which light from the first LED light source follows a first optical path from the first light inlet port to the liquid sample, such that the light from the first LED light source directly illuminates the liquid sample before the light transmitted by the sample is transmitted through the light outlet port and received by the spectrometer for wavelength analysis to provide the extinction spectrum of the liquid sample; and When the optical path adjuster is in the second configuration, the device is in diffuse reflection mode, in which light from the second LED light source follows a second optical path from the second inlet port into the integrating cavity. The light from the second LED light source is incident on one or more reflective inner walls of the integrating cavity and undergoes diffuse reflection within the integrating cavity, such that the light from the light source illuminates the liquid sample before being transmitted through the light outlet port or another light outlet port and received by the spectrometer for wavelength analysis to provide the absorption spectrum of the liquid sample contained in the liquid sample.

[0516] 41. The spectrometer apparatus according to any one of the preceding claims, wherein the apparatus is configured to measure the spectrum of a liquid sample selected from any one or more of the following: a.water; b. Wine; c. Beverages; d. Edible liquid or partially liquid products.

[0517] 42. A spectrometer apparatus, which is substantially as described herein and shown in the accompanying drawings.

[0518] 43. A method for measuring the spectrum of a liquid sample, the method using the apparatus according to any one of claims 1 to 41, the method comprising the steps of: a. Activate the light source; b. Controlling the optical path adjuster to be in either transmission mode or diffuse reflection mode; and c. Wavelength analysis of the light transmitted through the light exit port via the spectrometer is used to provide the absorption spectrum of the liquid sample contained in the liquid tank.

Claims

1. A method for determining at least one property of a therapeutic payload and / or a nanoparticle delivery carrier, said therapeutic payload and nanoparticle delivery carrier forming a diffusely scattering liquid suspension sample, said method comprising: Position the sample within the integrating cavity; The probe light is guided to the first port of the integrating cavity; A portion of the probe light from the second port of the integrating cavity is received after being scattered from the sample. Measure one or more spectral characteristics of the probe light received at the second port; as well as Based on one or more spectral characteristics measured from the probe light received at the second port, at least one characteristic of the therapeutic payload and / or nanoparticle delivery carrier in the liquid suspension is determined.

2. The method according to claim 1, wherein, Determining at least one characteristic of the therapeutic payload and / or nanoparticle delivery carrier in the liquid suspension includes: determining one or more absorption spectral characteristics of the corresponding therapeutic payload and / or nanoparticle delivery carrier based on one or more spectral characteristics measured by the probe light.

3. The method according to claim 2, wherein, Determining one or more absorption spectral characteristics of the therapeutic payload and / or nanoparticle delivery carrier includes applying a calibration transformation to one or more measured spectral characteristics of the probe light to determine the absorption spectral characteristics of the corresponding therapeutic payload and / or nanoparticle delivery carrier.

4. The method according to claim 3, wherein, The calibration transformation is based on the calibration of the integrating sphere and / or the absorption coefficient of the corresponding therapeutic payload and / or nanoparticle delivery carrier at the probe wavelength.

5. The method according to any one of the preceding claims, wherein, The at least one characteristic of the therapeutic payload and / or nanoparticle delivery carrier includes a measure related to the amount or concentration of the payload and / or nanoparticle delivery carrier in the sample.

6. The method according to any one of the preceding claims, wherein, The at least one characteristic of the therapeutic payload includes the absorption spectrum of the payload and / or the nanoparticle delivery carrier.

7. The method according to claim 6, further comprising: Based on the absorption spectrum of the payload and / or nanoparticle delivery carrier, determine one or more characteristics associated with the corresponding payload and / or nanoparticle delivery carrier, and identify the payload and / or nanoparticle delivery carrier based on the characteristics.

8. The method according to any one of the preceding claims, wherein, The probe light enters the integrating cavity through the first port along a first axial direction and is received at the second port along a second axial direction, wherein the second axial direction deviates from the first axial direction, such that the probe light received at the second port is scattered from the integrating cavity and / or the sample.

9. The method according to any one of the preceding claims, further comprising: The probe light is directed between the third and fourth ports of the integrating cavity, the fourth port being arranged opposite the third port such that the fourth port receives the probe light transmitted directly from the third port, the transmitted light is measured, and the extinction value of the probe light received at the fourth port is determined.

10. The method of claim 9, further comprising: The scattering amount of the suspended sample is quantified based on the extinction value and the determined absorption spectral characteristics.

11. The method according to claim 8 or claim 9, wherein, The third port is the first port, so that the fourth port receives probe light transmitted directly from the first port.

12. The method according to claim 8 or claim 9, wherein, The fourth port is the second port, which enables the second port to receive probe light transmitted directly from the third port.

13. The method according to any one of the preceding claims, wherein, The detection light includes one or more wavelengths in the ultraviolet-visible-near-infrared spectrum, such as light covering the range of 240 nm to 750 nm, 240 nm to 650 nm, at about 260 nm, at discrete wavelengths between 240 nm and 320 nm, or scanning the wavelength range of 240 nm to 750 nm, 240 nm to 650 nm, or 240 nm and 320 nm.

14. The method according to any one of the preceding claims, wherein, The nanoparticle delivery carrier carries the therapeutic payload and includes one or more of the following: lipid-based nanoparticles carrying RNA, antibody-drug conjugates, viral vectors (e.g., lentiviruses and AAVs), liposomes, exosomes, polymer nanoparticles, liposome-based systems (e.g., lipid complexes), metal nanoparticles, SPIONs, polymer micelles, dendritic polymers, nanoemulsions, silica particles, and nanogels.

15. The method according to any one of the preceding claims, wherein, The nanoparticle delivery carrier is a lipid-based nanoparticle, and the payload includes RNA, such as mRNA, siRNA, or ssRNA.

16. The method according to any one of the preceding claims, wherein, The payload includes one or more of the following: oligonucleotides, DNA, drugs, vaccines, tumor therapies and gene editing therapies, and / or small molecule drugs.

17. A method for analyzing or measuring / characterizing nanoparticle carriers, comprising: Acquire / receive at least one or more scatter-corrected absorption measurements / data or scatter-corrected absorption spectroscopy measurements / data for liquid samples or solutions containing multiple nanoparticle carriers. The method includes the method described in any of the preceding claims, and The liquid sample or solution includes the diffuse scattering liquid suspension sample, and the step of acquiring / receiving at least one or more scattering-corrected absorption measurement results / data or scattering-corrected absorption spectral measurement results / data includes measuring one or more spectral characteristics of the probe light.

18. The method according to claim 17, wherein, Determining at least one characteristic of the therapeutic payload in the liquid suspension includes: using the at least one scatter-corrected absorption measurement / data or scatter-corrected absorption spectroscopy measurement / data, and utilizing the extinction coefficient of the nanoparticle carrier's payload at the target absorption wavelength to determine the payload capacity of the nanoparticle carrier in the liquid sample or solution.

19. The method of claim 17, wherein, Determining at least one characteristic of the therapeutic payload in the liquid suspension includes: using the at least one scatter-corrected absorption measurement / data or scatter-corrected absorption spectroscopy measurement / data, and utilizing the extinction coefficient of the payload of the nanoparticle carrier and the extinction coefficient of the carrier component of the nanoparticle carrier at the target absorption wavelength, to determine the ratio of the payload of the nanoparticle carrier to the carrier component / part of the liquid sample or solution.

20. The method of claim 17, wherein, Determining at least one characteristic of the therapeutic payload in the liquid suspension includes: using the at least one scattering-corrected absorption measurement / data or scattering-corrected absorption spectroscopy measurement / data, and utilizing the extinction coefficients of the constituent carrier elements of the carrier component / part of the nanoparticle carrier, to determine the molar ratio of the constituent carrier elements of the carrier component / part of the nanoparticle carrier.

21. The method according to claim 17, wherein, Determining at least one characteristic of the therapeutic payload in the liquid suspension includes: using the at least one scatter-corrected absorption measurement / data or scatter-corrected absorption spectroscopy measurement / data and the measured or determined nanoparticle carrier size to determine the nanoparticle carrier concentration in the liquid sample or solution.

22. The method according to claim 17, wherein, Determining at least one characteristic of the therapeutic payload in the liquid suspension includes: determining the nanoparticle carrier concentration in the liquid sample or solution using at least one scattering spectrum or scattering spectrum data and the measured or determined nanoparticle carrier size.

23. An apparatus for determining at least one property of a therapeutic payload and / or a nanoparticle delivery carrier, said therapeutic payload and nanoparticle delivery carrier forming a diffusely scattering liquid suspension, said apparatus comprising: An integrating chamber is configured to receive the sample of the liquid suspension entering the sample space; A light source is arranged to guide probe light through a first port into the integrating cavity; A detector for measuring probe light received at a second port of the integrating cavity, the second port being arranged such that the light is received after being scattered from the sample in the sample space; as well as The analyzer is configured to determine at least one characteristic of the therapeutic payload and / or nanoparticle delivery carrier in the liquid suspension based on one or more spectral characteristics of the probe light received at the second port.

24. The apparatus according to claim 23, wherein, The analyzer is further configured to determine one or more absorption spectral characteristics of the therapeutic payload and / or nanoparticle delivery carrier based on one or more measured spectral characteristics of the probe light, and the at least one characteristic of the corresponding therapeutic payload and / or nanoparticle delivery carrier is determined based on the one or more absorption spectral characteristics of the therapeutic payload and / or nanoparticle delivery carrier.

25. The apparatus according to claim 23, wherein, The analyzer is configured to determine the absorption spectral characteristics of the therapeutic payload and / or nanoparticle delivery carrier by applying a calibration transformation to one or more spectral characteristics measured on the probe light.

26. The apparatus according to claim 25, wherein, The calibration transformation is based on the calibration of the integrating sphere and / or the absorption coefficient of the corresponding therapeutic payload and / or nanoparticle delivery carrier at the probe wavelength.

27. The apparatus according to any one of claims 23-26, wherein, The light source is configured to guide probe light along a first axial direction through the first port into the integrating cavity, and the second port is arranged to receive probe light scattered from the integrating sphere and / or the sample along a second axial direction, wherein the second axial direction deviates from the first axial direction.

28. The apparatus according to claim 27, wherein, The integrating sphere also includes a third port and a fourth port, the fourth port being arranged opposite the third port to receive probe light transmitted from the third port, and the analyzer being further configured to determine the extinction value of the probe light received at the fourth port.

29. The apparatus according to claim 28, wherein, The analyzer is further configured to quantify the scattering amount of the suspended sample based on the extinction value and the determined absorption spectral characteristics.

30. The apparatus according to claim 28 or claim 29, wherein, The third port is the first port, such that the fourth port is configured to receive probe light transmitted from the first port.

31. The apparatus according to claim 28 or claim 29, wherein, The fourth port is the second port, such that the second port is configured to receive probe light transmitted from the third port.

32. The apparatus according to any one of claims 23 to 31, wherein, The detection light includes one or more wavelengths in the ultraviolet-visible-near-infrared spectrum, such as light covering the range of 240 nm to 750 nm, 240 nm to 650 nm, at about 260 nm, at discrete wavelengths between 240 nm and 320 nm, or scanning the wavelength range of 240 nm to 750 nm, 240 nm to 650 nm, or 240 nm and 320 nm.

33. The apparatus according to any one of claims 23 to 32, wherein, The at least one characteristic of the therapeutic payload is a measure of the amount or concentration of the payload present in the sample.

34. The apparatus according to any one of claims 23 to 33, wherein, The analyzer is configured to determine at least one quantitative characteristic of a nanoparticle delivery carrier carrying a therapeutic payload, wherein the nanoparticle delivery carrier carrying the therapeutic payload includes one or more of the following: lipid-based nanoparticles carrying RNA, antibody-drug conjugates, viral vectors (e.g., lentiviruses and AAVs), liposomes, exosomes, polymer nanoparticles, liposome-based systems (e.g., lipid complexes), metal nanoparticles, SPIONs, polymer micelles, dendritic polymers, nanoemulsions, silica particles, and nanogels.

35. The apparatus according to any one of claims 23 to 34, wherein, The analyzer is configured to determine at least one quantitative characteristic of a nanoparticle delivery carrier carrying a therapeutic payload, wherein the nanoparticle delivery carrier is a lipid-based nanoparticle and the payload includes RNA, such as mRNA, siRNA, or ssRNA.

36. The apparatus according to any one of claims 23 to 35, wherein, The analyzer is configured to determine at least one quantitative characteristic of a nanoparticle delivery carrier carrying a therapeutic payload, wherein the payload includes one or more of the following: oligonucleotides, DNA, drugs, vaccines, tumor therapies and gene editing therapies, and / or small molecule drugs.

37. A nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device, comprising the apparatus according to any one of claims 23 to 36. in, The integrating cavity includes one or more reflective inner walls, and the integrating cavity is configured to receive a cuvette, the cuvette being configured to contain a liquid sample or solution comprising at least one or more nanoparticle carriers, the liquid sample or solution including the diffuse scattering liquid sample; The integrating cavity includes at least one optical inlet port and at least one optical outlet port, the one or more optical inlet ports being configured to receive light from the light source, and the one or more optical outlet ports being configured to deliver light to a spectrometer, wherein the spectrometer includes the detector; The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device may optionally be a lipid-based drug delivery carrier analyzer or a lipid-based drug delivery carrier measurement / characterization device, and is configured to operate in diffuse reflection mode. In this mode, light from the light source enters the integrating cavity from one or more inlet ports along an optical path, is incident on the reflective inner wall of the integrating cavity, and is diffusely reflected within the integrating cavity. This allows the light from the light source to irradiate the liquid sample before being transmitted through one or more optical outlet ports and received by the spectrometer for wavelength analysis to provide the absorption spectrum of the at least one or more nanoparticle carriers contained in the liquid sample or solution.

38. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to claim 36, wherein, The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device is used or configured to be used for: Determine the effective loading amount or effective loading concentration of the nanoparticle carrier, and / or Determine the effective load of the nanoparticle carrier to the carrier content / concentration ratio, and / or Determine the ratio or fractional content of the constituent carrier components of the nanoparticle carrier, and / or Determine the concentration of nanoparticle carriers in the solution or formulation.

39. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to claim 37 or claim 38, wherein, The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device is a lipid-based nanoparticle analyzer or lipid-based nanoparticle measurement / characterization device, used or configured to determine the concentration of lipid-based nanoparticles in a solution or formulation, or used or configured to determine the concentration of lipid-based drug delivery carriers in a solution or formulation.

40. The nanoparticle carrier analyzer or nanoparticle carrier measurement / characterization device according to any one of claims 37 to 39, wherein, The lipid-based nanoparticle carrier comprises (solid) lipid nanoparticles and / or liposomes or is composed of (solid) lipid nanoparticles and / or liposomes.

Images