Method of determining lipoprotein concentration in solution using light scattering

A technology of light scattering and lipoprotein particles, which is applied to the measurement of scattering characteristics, analysis of suspensions and porous materials, and material analysis through optical means, and can solve problems such as expensive, incomplete information on lipoprotein particles, and limited applications

Pending Publication Date: 2020-09-18
OXFORD UNIV INNOVATION LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods are time-consuming, expensive, provide incomplete information about lipoprotein particles, and / or have limited application in clinical settings

Method used

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  • Method of determining lipoprotein concentration in solution using light scattering
  • Method of determining lipoprotein concentration in solution using light scattering
  • Method of determining lipoprotein concentration in solution using light scattering

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0233] Example 1 - Detection of purified lipoprotein particles by iSCAT

[0234] Materials / Methods

[0235] Substrate preparation and measurements

[0236] Borosilicate glass coverslips (No 1.5, 24 x 50 mm, VWR) were sequentially rinsed with MilliQ water, then with ethanol and again with MilliQ water. They were then dried under a stream of dry nitrogen. CultureWell silicone spacers (Grace Bio-Labs) were cut and placed on freshly cleaned coverslips to provide four separate 30-50 μl sample chambers on the same substrate.

[0237] Purified HDL and LDL lipoprotein samples were obtained from Lee Biosystems (LDL cat#360-10; HDL cat#361-10), see for example https: / / www.leebio.com / product / 984 / low-density-lipoprotein - described in ldl-human-serum-360-10. HDL and LDL were separated by ultracentrifugation. The samples were diluted 500,000-fold and 50,000-fold, respectively.

[0238] Data acquisition and analysis were then performed by iSCAT, while non-specific binding of lipopro...

Embodiment 2

[0241] Example 2 - Detection of Purified Lipoprotein Particles in Blood and Serum by iSCAT

[0242] The iSCAT method as described in Example 1 was performed on serum and blood samples from human individuals. To obtain serum, blood was allowed to clot in an upright position for at least 30 minutes and then centrifuged (30 minutes, 1500 xg). Transfer serum to plastic screw cap vials. Blood and serum samples were prepared as follows: 1 μL finger prick blood or serum samples were diluted 2000-fold in HEPES / KCl buffer containing 5 mM EDTA (to prevent clotting) (see below). 10 μl of the diluted sample was added to 40 μl of 25 mM HEPES buffer (pH 7.4, containing 100 mM KCl) to obtain a final 10,000-fold diluted blood.

[0243] Figure 5 Detection of HDL and LDL in plasma samples is shown. Imaged plasma samples simultaneously exhibited two signals corresponding to HDL and LDL. As expected, HDL particles appeared more frequently than LDL (see histogram below).

[0244] Figure...

Embodiment 3

[0245] Example 3 - Calibration to calculate HDL and LDL concentrations

[0246] To calculate the mass / concentration of HDL and LDL in the blood samples of Example 2, we constructed a calibration curve in which proteins of known mass / concentration were measured, giving a linear relationship between molecular weight and iSCAT contrast.

[0247] For concentration calibration, we used known concentrations of MSP1D1 DMPC lipid nanodiscs. Nanodiscs are synthetic model membrane systems composed of a lipid bilayer of phospholipids with a hydrophobic edge screened out by two amphipathic proteins. These proteins are called membrane scaffold proteins (MSPs) and are arranged in double bands. Nanodisc samples were diluted to the nM range and added to the buffer. The non-specific binding of nanodiscs to glass was recorded using iSCAT, as shown in Figure 7 shown. As the number of particles in solution decreases and the number of accessible binding sites decreases, the frequency of bin...

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Abstract

The invention relates the use of single particle light scattering, preferably interferometric scattering microscopy (also referred to herein as iSCAT), to measure the concentration of a particle in asolution. The invention furthermore relates to the use of light scattering to detect lipoprotein particles in a sample, and to related diagnostic and treatment methods.

Description

technical field [0001] The present invention involves the use of single particle light scattering, preferably Interferometric Scattering Microscopy (also referred to herein as iSCAT), to measure the concentration of particles in solution. Particles can be present in simple or complex solutions, such as biological or environmental samples. The present invention can be used to determine the absolute concentration of particles in solution, and this capability provides a means to study the stoichiometric relationship between different particles in solution. As the inventors have demonstrated, iSCAT also provides the means for reliable and precise detection, mass quantification, imaging and characterization of particles as small as a single molecule, and thus the ability to use the same technique for concentration measurements is highly beneficial . [0002] Furthermore, the present invention relates to the detection of lipoprotein particles in samples using light scattering, pre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/92G01N33/68
CPCG01N33/6887G01N33/92G01N2800/32G01N2015/1454G01N2015/1043G01N15/1463G01N15/06G01N15/0205G01N33/487G01N2800/50G01N21/49G01N2021/513
Inventor 乔安娜·安德烈卡菲利普·库库拉加文·杨贾斯汀·贝尼希
Owner OXFORD UNIV INNOVATION LTD
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