Apparatus and method for measuring dynamic light scattering of a sample

Abstract

The present disclosure describes a method and apparatus for measuring dynamic light scattering of a sample. In one embodiment, the apparatus includes a platen, a light source located below the platen and configured to emit luminescence through the platen and into the sample, collection optics located below the platen and configured to capture scattered light, and a light absorber configured to contact the sample, absorb transmitted light, and redirect reflected light away from the collection optics. In one embodiment, the method includes placing the sample on the platen, emitting luminescence from the light source below the platen through the platen into the sample, capturing the scattered light with the collection optics below the platen, contacting the sample with the light absorber, absorbing the transmitted light with the absorber, and redirecting the reflected light away from the collection optics with the absorber.

Description

[Technical Field]

[0001] Priority This application claims priority to U.S. Patent Application No. 17 / 017,671, filed September 10, 2020.

[0002] background The present disclosure relates to dynamic light scattering, and more particularly to measuring dynamic light scattering of a sample. Summary of the Invention [Means for solving the problem]

[0003] overview The present disclosure describes a method and apparatus for measuring dynamic light scattering of a sample. In an exemplary embodiment, the apparatus includes: (1) a platen including a top surface and a bottom surface, the top surface configured to hold a sample; (2) a light source located below the platen and configured to emit light through the platen and into the sample; (3) collection optics located below the platen and configured to capture scattered light scattered by the sample; and (4) a light absorber located above the platen, the light absorber configured to contact the sample, absorb transmitted light transmitted through the sample, and redirect reflected light reflected from the interface between the sample and the bottom surface of the absorber away from the collection optics. In an exemplary embodiment, the method includes the steps of: (1) placing a sample on a platen having a top surface and a bottom surface, the top surface configured to hold the sample; (2) emitting light from a light source below the platen through the platen and into the sample; (3) capturing scattered light scattered by the sample with collection optics below the platen; (4) contacting the sample with a light absorber above the platen; (5) absorbing transmitted light transmitted through the sample with the absorber; and (6) redirecting reflected light reflected from the interface between the sample and the bottom surface of the absorber away from the collection optics with the absorber. [Brief explanation of the drawings]

[0004] [Figure 1A] 1 illustrates an apparatus in accordance with an exemplary embodiment; [Figure 1B] 1 illustrates an apparatus according to one embodiment. [Figure 1C] 1 illustrates an apparatus according to one embodiment. [Figure 2] 1 illustrates a method according to an exemplary embodiment. [Figure 3A] 1 illustrates a graph according to one embodiment. [Figure 3B] 1 illustrates a graph according to one embodiment. [Figure 3C] 1 illustrates a graph according to one embodiment. [Figure 4] 1 illustrates a graph according to one embodiment. [Figure 5] 1 shows data according to one embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0005] Detailed Description The present disclosure describes a method and apparatus for measuring dynamic light scattering of a sample. In an exemplary embodiment, the apparatus includes: (1) a platen including a top surface and a bottom surface, the top surface configured to hold a sample; (2) a light source located below the platen and configured to emit light through the platen and into the sample; (3) collection optics located below the platen and configured to capture scattered light scattered by the sample; and (4) a light absorber located above the platen, the light absorber configured to contact the sample, absorb transmitted light transmitted through the sample, and redirect reflected light reflected from the interface between the sample and the bottom surface of the absorber away from the collection optics. In an exemplary embodiment, the method includes the steps of: (1) placing a sample on a platen having a top surface and a bottom surface, the top surface configured to hold the sample; (2) emitting light from a light source below the platen through the platen and into the sample; (3) capturing scattered light scattered by the sample with collection optics below the platen; (4) contacting the sample with a light absorber above the platen; (5) absorbing transmitted light transmitted through the sample with the absorber; and (6) redirecting reflected light reflected from the interface between the sample and the bottom surface of the absorber away from the collection optics with the absorber.

[0006] In one embodiment, the device and method perform measurements entirely in a backscattering configuration and do not adjust the path length. In one example, when the absorber contacts the top surface of the platen, a sandwich is formed consisting of the lower window / platen, the sample, and the upper absorber. For example, the device has free interfaces only on the sides, which are essentially the overflow area, so that the laser and scattered light pass only through well-polished optical surfaces and the sample, while the transmitted beam is collected by the absorber and the laser does not pass through the uncontrolled meniscus. Also, for example, the top and bottom optical surfaces do not need to be parallel, as they may be positioned at a known angle that destroys etaloning and directs light reflected from the top surface away from the measurement sensor.

[0007] definition particle Particles can be a component of a liquid sample. Such particles can be molecules, nanoparticles, virus-like particles, liposomes, emulsions, bacteria, and colloids of various types and sizes. The size of these particles can range from nanometers to several microns.

[0008] Analysis of macromolecular or particle species in solution Analysis of macromolecular or particulate species in solution can be accomplished by preparing a sample in an appropriate solvent and then injecting an aliquot into a separation system, such as a liquid chromatography (LC) column or field-flow fractionation (FFF) channel, which separates the various particulate species contained in the sample into their various components. Once separated, typically based on size, mass, or column affinity, the sample can be subjected to analysis by light scattering, refractive index, ultraviolet absorbance, electrophoretic mobility, and viscosity response.

[0009] Current Technology Current techniques describe measurements performed in transmission. For example, current sample holders can dynamically adjust the gap between the top and bottom windows to find the optimal spacing for absorption measurements to balance measurement linearity with sensitivity, with short path lengths providing linear and low sensitivity and long path lengths providing good sensitivity and nonlinear results. Therefore, path length adjustment is important for such current absorption techniques.

[0010] Other current techniques, such as blade cells, allow light to pass through the gas-liquid interface, allowing the light to bounce around the droplet in an uncontrolled manner. Such current techniques are susceptible to acoustic pickup from vibrations in the laboratory. Path length adjustment is irrelevant for backscattered dynamic light scattering / static light scattering (DLS / SLS) measurements.

[0011] Therefore, there is a need to measure the dynamic light scattering of a sample in a reproducible manner. 1A, 1B, and 1C, in an exemplary embodiment, the apparatus includes a platen 110 including a top surface 112 and a bottom surface 114, with the top surface 112 configured to hold a sample; a light source 120 located below the platen 110 and configured to emit light through the platen 110 to the sample; collection optics 130 located below the platen 110 and configured to capture scattered light scattered by the sample; and a light absorber 140 located above the platen 110, the light absorber 140 configured to contact the sample and absorb transmitted light transmitted through the sample and to direct reflected light reflected from an interface 150 between the sample and a bottom surface 142 of the absorber 140 away from the collection optics 130.

[0012] Referring to FIG. 2 , in an exemplary embodiment, the method includes step 210 of placing a sample on a platen including a top surface and a bottom surface, the top surface configured to hold the sample; step 220 of emitting light from a light source below the platen through the platen to the sample; step 230 of capturing scattered light scattered by the sample with collection optics below the platen; step 240 of contacting the sample with a light absorber above the platen; step 250 of absorbing transmitted light transmitted through the sample with the absorber; and step 260 of redirecting reflected light reflected from the interface between the sample and the bottom surface of the absorber away from the collection optics.

[0013] Platen In one embodiment, the platen 110 includes a window. In certain embodiments, the platen 110 is a window. In one embodiment, the top surface 112 of the platen 110 and the bottom surface 114 of the platen 110 include polished optical surfaces. In one embodiment, the top surface 112 of the platen 110 and the bottom surface 114 of the platen 110 are polished optical surfaces. In one embodiment, the platen 110 is transparent. In one embodiment, the top surface 112 of the platen 110 and the bottom surface 114 of the platen 110 include flat surfaces. In one embodiment, the top surface 112 of the platen 110 and the bottom surface 114 of the platen 110 are flat surfaces.

[0014] For example, the platen 110 may be fused silica or sapphire to ensure durability of the optical surfaces.

[0015] light source In one embodiment, the light source 120 is configured to focus the emitted light onto the sample. In certain embodiments, the light source 120 is configured to focus the emitted light into the interior of the sample. In one embodiment, the light source 120 includes a laser. In certain embodiments, the light source 120 is a laser. In one embodiment, the laser is coupled to a single-mode polarization-maintaining optical fiber. In one embodiment, the light source 120 is configured to focus the emitted light to have a beam waist of less than 0.5 mm within the sample. In certain embodiments, the light source 120 is configured to focus the emitted light to have a beam waist of less than 0.5 mm within the sample.

[0016] Light-collecting optical system In one embodiment, the collection optics 130 includes (a) an optical fiber and (b) at least one lens coupled to the fiber, where one of the fiber and the at least one lens is aligned with a focal point of the light source 120 within the sample. In certain embodiments, the collection optics 130 includes (a) an optical fiber and (b) at least one lens coupled to the fiber, where one of the fiber and the lens is aligned with a focal point of the light source 120 inside the sample. In one embodiment, the fiber includes one of a single-mode optical fiber and a multimode optical fiber. In certain embodiments, the fiber is one of a single-mode optical fiber and a multimode optical fiber.

[0017] Light absorber In one embodiment, the light absorber 140 is selected to be capable of absorbing the energy of the transmitted light within the light absorber 140, and the light absorber 140 is selected to be capable of directing the energy away from the sample. In one embodiment, the light absorber 140 is selected to be capable of absorbing the energy to an optical depth of 5 mm or less within the light absorber 140. In one embodiment, the light absorber 140 comprises neutral density glass. In certain embodiments, the light absorber 140 is neutral density glass.

[0018] Light absorber carrier 1B and 1C, in a further embodiment, the apparatus further includes a light absorber carrier 160, where the carrier 160 includes the light absorber 140. In one embodiment, the carrier 160 includes one of at least one swing arm 162, a vertical sliding block, and a horizontal sliding block. In a specific embodiment, the carrier 160 is one of at least one swing arm 162, a vertical sliding block, and a horizontal sliding block.

[0019] wiper In a further embodiment, the apparatus further includes a wiper configured to wipe the upper surface 112 of the platen 110. In one embodiment, the apparatus further includes a cleaning fluid injector configured to eject a cleaning fluid onto the upper surface 112 of the platen 110. In one embodiment, the wiper is configured to clean the upper surface 112 of the platen 110 by wiping the cleaning fluid against the upper surface 112.

[0020] temperature adjustment In a further embodiment, the device further includes at least one temperature regulator configured to control the temperature of at least one of the absorber 140 and the platen 110, thereby adjusting the sample temperature of the sample. In a further embodiment, the method further includes adjusting the sample temperature of the sample by controlling the temperature of at least one of the absorber and the platen by the temperature regulator to a temperature value, the temperature value being one of a set value and a variable value that varies over time. For example, the device and method may be well suited for rapid thermal ramping to study protein aggregation or unfolding because the sample is believed to be in good thermal contact with the top and bottom plates, which are themselves good thermal conductors. The device and method also allow the gap between the bottom and top windows to be adjusted to 1 mm or less, enabling measurements of <1 μL.

[0021] One problem with thermal ramping measurements is that dissolved gases tend to come out of solution as the temperature increases. Devices with small sample volumes minimize the size and number of bubbles that may form. Bubble formation is a relatively slow process. A sufficiently fast ramping device and method may allow ramping to be completed before bubbles appear or before evaporation can change the sample concentration. [Example]

[0022] Example For example, FIG. 3A shows dynamic light scattering measurements performed by the present apparatus and method on BSA, demonstrating the reproducibility of data collected by the present apparatus and method. In a further example, FIG. 3B shows dynamic light scattering measurements performed by the present apparatus and method on a large particle sample. After measurements were performed on BSA and the top surface 112 of the platen 110 and the bottom surface of the absorber 140 were cleaned, 100 nm nanospheres (i.e., polystyrene latex beads) demonstrate the continued reproducibility of data collected by the present apparatus and method. In a further example, FIG. 3C shows dynamic light scattering measurements performed by the present apparatus and method on BSA after measurements were performed on a large particle sample, i.e., 100 nm nanospheres (i.e., polystyrene latex beads), and the top surface 112 of the platen 110 and the bottom surface of the absorber 140 were cleaned, demonstrating the continued reproducibility of data collected by the present apparatus and method. [Example]

[0023] In another example, Figure 4 shows an overlay of 94 50-second data collections (1.3 hours) of Pierce BSA measured by the present device and method, demonstrating the reproducibility of data collected by the present device and method. Additionally, Figure 5 demonstrates that the present device and method can accurately measure the radius of particles in a variety of samples.

[0024] The description of various embodiments of the present disclosure is presented for illustrative purposes and is not intended to be exhaustive or to be limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terms used herein are selected to explain the principles of the embodiments, practical applications, or technical improvements over technologies found in the market, or to enable those skilled in the art to understand the embodiments disclosed herein.

Claims

1. a platen having a top surface and a bottom surface, the top surface configured to hold a sample on the top surface; a light source positioned below the platen and configured to emit light through the platen onto the sample; a collection optical system located below the platen and configured to capture scattered light scattered by the sample; a light absorber positioned above the platen; Equipped with the light absorber is configured to contact the sample while the sample is held on the top surface of the platen, to absorb transmitted light that has passed through the sample, and to redirect light that has reflected from an interface between the sample and a bottom surface of the light absorber away from the collection optics; The sample partially has a free interface on a side surface of the sample, the platen includes a window; the top surface of the platen and the bottom surface of the platen comprise polished optical surfaces.

2. The apparatus of claim 1 , wherein the top surface of the platen and the bottom surface of the platen comprise flat surfaces.

3. The apparatus of claim 1 , wherein the light source is configured to focus the emitted light onto the sample.

4. The apparatus of claim 3 , wherein the light source comprises a laser.

5. 5. The apparatus of claim 4, wherein the laser is coupled to a single-mode polarization-maintaining optical fiber.

6. 4. The apparatus of claim 3, wherein the light source is configured to focus the emitted light to have a beam waist of less than 0.5 mm within the sample.

7. The focusing optical system includes: An optical fiber; at least one lens coupled to said optical fiber; Equipped with The apparatus of claim 1 , wherein one of the optical fiber and the at least one lens is aligned to a focal point of the light source within the sample.

8. The apparatus of claim 7 , wherein the optical fiber comprises one of a single-mode optical fiber and a multimode optical fiber.

9. the light absorber is selected so as to be able to absorb the energy of the transmitted light within the light absorber; The apparatus of claim 1 , wherein the optical absorber is selected to be capable of directing the energy away from the sample.

10. 10. The apparatus of claim 9, wherein the optical absorber is selected to absorb the energy to an optical depth within the optical absorber of 5 mm or less.

11. 10. The apparatus of claim 9, wherein the light absorber comprises a neutral density glass.

12. further comprising a light absorber carrier; The device of claim 1 , wherein the light absorber carrier comprises the light absorber.

13. The apparatus of claim 12 , wherein the light absorber carrier comprises at least one of a swing arm, a vertical sliding block, and a horizontal sliding block.

14. The apparatus of claim 1 , further comprising a wiper configured to wipe the top surface of the platen.

15. The apparatus of claim 14 , further comprising a cleaning fluid injector configured to emit a cleaning fluid onto the upper surface of the platen.

16. 10. The apparatus of claim 1, further comprising at least one temperature regulator configured to control a temperature of at least one of the optical absorber and the platen, thereby regulating a sample temperature of the sample.

17. placing the sample on a platen having a top surface and a bottom surface, the top surface configured to hold the sample on the top surface; The sample partially has a free interface on a side surface of the sample, the platen includes a window; the top surface of the platen and the bottom surface of the platen comprise polished optical surfaces; and after the step of placing the sample, contacting the sample with a light absorber above the platen; and emitting luminescence from a light source below the platen through the platen onto the sample; capturing scattered light scattered by the sample with collection optics below the platen; absorbing the transmitted light transmitted through the sample by the light absorber; and redirecting, by the light absorber, reflected light reflected from the interface between the sample and a bottom surface of the light absorber away from the collection optics.

18. further comprising the step of adjusting a sample temperature of the sample by controlling a temperature of at least one of the light absorber and the platen to a certain temperature value by a temperature regulator; 18. The method of claim 17, wherein the temperature value is one of a set value and a time-varying variable value.

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