WO2014058371A1 - Spectroscopie par corrélation d'interférence de diffusion (sics) - Google Patents

Spectroscopie par corrélation d'interférence de diffusion (sics) Download PDF

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WO2014058371A1
WO2014058371A1 PCT/SE2013/000155 SE2013000155W WO2014058371A1 WO 2014058371 A1 WO2014058371 A1 WO 2014058371A1 SE 2013000155 W SE2013000155 W SE 2013000155W WO 2014058371 A1 WO2014058371 A1 WO 2014058371A1
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analytes
sample
fluctuations
scattering
detection volume
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PCT/SE2013/000155
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Stefan Wennmalm
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Stefan Wennmalm
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/02—Investigating particle size or size distribution
    • G01N15/0205—Investigating particle size or size distribution by optical means
    • G01N15/0211—Investigating a scatter or diffraction pattern
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N15/075—Investigating concentration of particle suspensions by optical means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N2015/0038—Investigating nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N2015/0042—Investigating dispersion of solids
    • G01N2015/0053—Investigating dispersion of solids in liquids, e.g. trouble
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/02—Investigating particle size or size distribution
    • G01N15/0205—Investigating particle size or size distribution by optical means
    • G01N15/0211—Investigating a scatter or diffraction pattern
    • G01N2015/0222—Investigating a scatter or diffraction pattern from dynamic light scattering, e.g. photon correlation spectroscopy

Definitions

  • the invention relates to analysis of diffusing particles and biomolecules in solution or in cells.
  • biophysics, biochemistry, and cell biology methods are needed for analyzing the interaction of biomolecules.
  • a particular requirement on such methods is the possibility to measure interactions even at low concentrations, down to nano-molar and lower concentrations.
  • the invention also relates to the field of analyzing particles that may not be biological, for example particles in solutions and emulsions. For example there is a need to determine the concentration and size of particles in engine-fuels, for environmental and health purposes. Another example is analysis of aggregation of particles in for example cosmetic products such as skin lotions.
  • DLS dynamic light scattering
  • QELS quasi- elastic light scattering
  • laser diffraction for example utilized in instruments from the company Malvern, which utilizes the fact that the angle relative to the incoming laser in which light is scattered from particles in solution is dependent on the particle size.
  • a third technique is laser Doppler velicometry, which can be used together with phase analysis light scattering (PALS), and these are used to estimate the velocity of particles in a flow. None of these techniques can determine the particle concentration, and for nanoparticles (smaller than 200 nm diameter), no technique can estimate particle concentration independently from particle size; existing techniques require pre-knowledge about particle size in order to estimate concentration, and vice versa.
  • PALS phase analysis light scattering
  • FCS fluorescence correlation spectroscopy
  • FCS detects transient fluorescence signals from fluorescently labelled particles (most commonly biomolecules) in solution as they diffuse through an open detection volume that usually has a size in the range 0.2 femto liter to a few femtoliter.
  • the duration of the generated fluorescence bursts is indicative of the size of the diffusing species, since the diffusion coefficient is inversely proportional to the particle radius.
  • the detection volume is restricted by the size of the laser focus and also by the confocal detection with a pinhole in the image plane.
  • FCS resembles the invention, because the invention also detects a signal from particles as they diffuse through a detection volume, and the detection volume is, or can at least be, restricted as in FCS by the dimensions of the laser focus and by the pinhole (the detection may however be different, because the forward scattered light may be analyzed, which requires a second lens or objective, on the other side of the sample. However, the detection may also be similar to that of FCS in that back scattered light may be detected by the same objective as used for the incoming laser).
  • the invention also resembles FCS in that the signal may (but does not have to) be analyzed by autocorrelation or cross-correlation.
  • FCS instruments are in 2012 built and sold at least by Zeiss, Leica, Picoquant, ISS and
  • FCS has become an important tool in biophysics and cell biology, in academia as well as in industry.
  • a laser is focused by a microscope objective, which generates a focus inside the liquid sample.
  • the axial radius of the laser focus cannot be smaller than about 0.2 ⁇ due to the diffraction limit.
  • the focus can however be enlarged, to have a radius of several micrometers.
  • Fluorescent molecules for example organic fluorophores, labelled biomolecules, or fusions of a protein with a fluorescent protein like GFP, generate fluorescence bursts as they transit through the excitation focus. A part of the emitted fluorescence is collected by the same objective, focused through a pinhole in the image plane, and thereafter focused again onto detectors.
  • the collected fluorescence can be spectrally discriminated from scattered laser light by using dichroic mirrors and emission filters between the objective and the pinhole.
  • the final detection volume is restricted both by the dimensions of the laser focus and by the size of the pinhole in the image plane, which in the diffraction limited case results in a detection volume of -0.3 fl (fig 1).
  • the detected fluorescence bursts can give information about the mobility and
  • Fig 1 Description of a common experimental setup for FCS.
  • Laser light blue
  • the emitted fluorescence light green
  • An emission filter selects fluorescence emission and blocks scattered laser light.
  • the emission is then focused through a pinhole, which discriminates out-of-focus photons, and focused onto a photo detector.
  • the autocorrelation function ACF is calculated. Fitting of the ACF to an appropriate model gives information about concentrations and mobilities of the diffusing particles.
  • FCS is for example used to analyze interactions between biomolecules: When a small, fluorescently labeled molecule interacts with, or binds to, a larger, unlabeled molecule or particle, the mobility of the smaller molecule will decrease since large molecules have lower mobility than small molecules. In this way the process of binding over time between the smaller, labeled, molecule and the larger, unlabelled molecule can be detected and analyzed.
  • FCS Fluorescence Cross-Correlation Spectroscopy, where two excitation foci of different wavelength (often 488 nm and 633 nm) are superimposed in the sample. Interacting partner-molecules are labeled with a 488-excitable dye and a 633-excitable dye respectively, and their respective emissions are spectrally filtered and detected by two separate detectors. This allows analysis of interacting molecules independent of their respective sizes (see any of several review papers by Petra Schwille). Summary of the invention (Abstract):
  • a drawback of present scattering based methods is that they cannot estimate the size in two ways simultaneously, for example from the diffusion coefficient and from the scattering intensity simultaneously. This results, for example, in that they cannot determine the particle concentration independently from the particle size, because these two both affect the scattering from the sample.
  • the invention does estimate the particle size both from the diffusion coefficient and from a signal based on scattering, and can thus estimate not only the particle size but also simultaneously estimate the particle concentration (see the manuscript below for details).
  • a laser is focused to form a detection volume inside a liquid sample, and while most of the laser light is transmitted through the sample, some light is scattered in the forward direction from particles or biomolecules inside the detection volume. This scattered light interferes with the transmitted laser light, forming the detected fluctuations which give information about the particles or biomolecules that gave rise to the scattering.
  • any sample parameter can be investigated that affects the two measures of particle size differently.
  • the invention can conveniently be combined with FCS, and allows thereby unlabelled and labelled particles or biomolecules to be analysed simultaneously, which enables the fraction of labelled particles or biomolecules to be estimated. Furthermore, the affinity between unlabelled particles or biomolecules and fluorescently labelled ligands can be estimated from a single measurement, as described in detail in the manuscript.
  • a method for analysing particles or biomolecules in a liquid sample comprising:
  • the signal is laser light transmitted through the detection volume and the fluctuations are reductions or increases in the signal due to the presence of particles or biomolecules in the detection volume; and analyzing the detected fluctuations to obtain information about the analytes in the sample.
  • the fluctuations caused by the presence of particles or biomolecules in the detection volume may arise due to scattering of the incoming laser light from the particles or biomolecules which then interferes with the transmitted laser light.
  • a Scattering Interference Correlation Spectroscopy system comprising a laser, a coverslip sandwich in between which a sample resides, focusing means for focusing the laser inside the sample inside the coverslip sandwich, means for collecting the scattering and interference signals and any fluorescence signals from analytes within the coverslip sandwich, detectors for detecting the scattering and interference signals and any fluorescence signals, and means for autocorrelating the detected signals.
  • the system described in the third aspect of the invention may be combined with Fluorescence Correlation Spectroscopy (FCS) in a single instrument, such that while the SICS-part of the instrument analyses the size and concentration of the analytes present in the sample (labeled as well as unlabeled), the FCS-part of the instrument analyses simultaneously the fluorescently labeled fraction.
  • FCS Fluorescence Correlation Spectroscopy
  • the fourth aspect of the invention ie the combination of SICS and FCS, allows determining the fraction of labeled analytes in the sample. Furthermore, if unlabeled particles or biomolecules are analyzed in the presence of labeled, small ligands, where the ligands bind to the unlabeled particles or biomolecules, the affinity (K d ) can be estimated from one single measurement, as described in the manuscript below.
  • a device for analyzing the detected signal comprising:
  • a data storage device which in addition may be a data analysis device, for storage and possibly also analysis of the detected signal
  • the analysis may be in the form of autocorrelation analysis, where the amplitude and the decay time of the autocorrelation function give two separate estimations of the size of the analytes in the sample, and the amplitude of the autocorrelation function also can give information about the concentration of analytes in the sample, or the analysis may be in the form of intensity distribution analysis or analysis of photon counting histograms, which can give information about the size of the analytes in the sample, and the shape of the analytes in the sample, or the analysis may be in the form of cross-correlation with another signal.
  • the detector should consist of a photo diode which can detect count rates as high a 10 15 photons per second or higher, or a photo multiplier tube running in dc mode, capable of detecting 10 12 photons per second or more.
  • An important feature of the invention is that the signal from fluctuations caused by interference of the scattered light with the transmitted or reflected laser light is detected from a limited detection volume. This allows analysis resembling that in FCS, to determine the average transit time of particles through that detection volume, and also to determine the average number of particles in this detection volume.
  • the detection volume may be restricted by the dimensions of a focused laser beam, for example a diffraction limited laser focus, and also by a pinhole or similar positioned between the sample and the detector, for example at the image plane of the detection focus, and likely positioned in the forward direction due to forward scattering, but back scattering may also be analyzed, in which case the scattered light is collected by the same focusing means as was used for creating the detection focus, and the scattered light is then focused through a pinhole in the backward direction.
  • a focused laser beam for example a diffraction limited laser focus
  • a pinhole or similar positioned between the sample and the detector for example at the image plane of the detection focus, and likely positioned in the forward direction due to forward scattering, but back scattering may also be analyzed, in which case the scattered light is collected by the same focusing means as was used for creating the detection focus, and the scattered light is then focused through a pinhole in the backward direction.
  • a fibre-coupled detector may also be used, in which case the fibre opening may be used instead of the pinhole or similar, and the fibre opening may for example have a diameter of 5, 10, 20 or 50 ⁇ .
  • a fibre coupled detector was used, even though the fibre cannot be seen in the drawing of fig 1A.
  • Another important feature of the invention is that the diffusion coefficient and the signal based on scattering give two separate and independent measures of particle size. Due to this it is possible to determine parameters that are affected differently by these two size measures, for example the shape of particles.
  • particle size and particle concentration can be determined using a single technique. If the instrument is calibrated, then size and concentration of an unknown sample (with known refractive index and fairly spherical particles) can in principle be determined in a single measurement. This is done by taking the size as estimated from the diffusion coefficient and inserting this size estimate into the expression for the amplitude of the autocorrelation function, which allows the concentration to be derived.
  • Another important feature of the invention is that the technique is very similar to FCS but does not require fluorescence labelling. This will allow the invention to be combined with FCS, and allow simultaneous analysis of labelled and unlabeled particles. This will for example make possible to determine the percentage of particles or biomolecules that carry a fluorescently labelled ligand, or the percentage of particles of biomolecules that are fluorescently labelled.
  • FCS Combining the invention and FCS may be accomplished using an instrument for the invention (called SICS in the manuscript attached below) as is described in fig la in the manuscript, but where not only the forward scattered light (label-free detection) is utilized, but also the fluorescence signal from fluorescently labelled particles in the sample is detected.
  • the mirror that reflects the laser light from the laser down into the objective may be a dichroic mirror, as in the FCS instrument in fig 1 above.
  • a fluorescence signal generated in the sample may be collected by the objective, passed through the dichroic mirror, focused through the pinhole and finally focused again onto the detector, as shown in fig 1 above (green line for the collected fluorescence emission).
  • FCS instrument in fig 1 above is taken as starting point for combining FCS and the invention
  • combination of FCS and the invention may be accomplished by collecting the transmitted and scattered laser light below the sample in fig 1 above, as described in the manuscript (plus using a "coverslip sandwich" as described in the manuscript).
  • the instrument in fig 1 above is already an FCS instrument, so adding the collection and analysis of the transmitted and scattered light would yield a combination of the invention and FCS.
  • the pinholes or similar and detectors may be optimized such that both detectors (the detector that detects the interference signal from scattering and the transmitted beam, and the detector that detects fluorescence) detect a signal from the same, or almost the same open volume.
  • FCS signals and the signals detected by the invention may either be analyzed separately and compared, which in a single measurement would yield the total concentration of particles and the concentration of label-carrying particles, which allows the percentage of label-carrying particles to be determined.
  • the two signals can be cross-correlated, which may yield additional information.
  • this could as in inverse-FCS (see references in the manuscript below) allow the volume of the analyzed labelled particles to be determined, since the SICS-signal is proportional to the particle volume (and the amplitude of the autocorrelation function is proportional to the square of the particle volume).
  • Such cross-correlation analysis may also be a good way to determine that binding between a small, fluorescently labelled ligand and a larger non-labeled particle has occurred.
  • a larger if a larger,
  • fluorescently labelled particle binds to a smaller, non-labeled ligand, this may also be advantageously analyzed by cross-correlaiton analysis, since the volume change upon binding will have a strong effect on the fluctuations detected in SICS+FCS which are proportional to particle volume, stronger than the effect that the same binding event would have on the diffusion time in an FCS measurement, since the diffusion time only scales with the cubic root of the particle mass.
  • volume of particles in SICS may also be derived by fitting of single-species or multiple-species models to intensity distribution histograms.
  • SICS could also be applied to analysis of particles on a surface, which may be a solid surface or a fluid surface. This could either be realized by sweeping the laser over surface, or moving the surface relative to the laser.
  • photo detectors capable of detecting higher count rates than what APDs are capable of should allow higher laser powers to be used. This will reduce the relative influence of noise (called shot noise or photon noise) relative to the signal and thus allow SICS to analyze even smaller particles than what is presently possible using APDs.
  • Such photodetectors may be PMTs in DC-mode (ie, not single photon counting PMTs), or photo diodes, both which may be used together with analogue to digital converters.
  • Lock-in detection is a common approach which circumvents the problem that small fluctuations of interest may drown in other, larger fluctuations, for example caused by the laser.
  • measurement with the SICS technique is to split the laser light before the sample, such that one fraction of the beam (probably the majority of the original intensity) is focused inside the sample as usual, but the other fraction is not, and the intensity of the latter can then be recorded with one detector simultaneously as the SICS-measurement is performed, which allows slow fluctuations from the laser to be subtracted from the SICS- signal, or otherwise compensated for.
  • Figure 1 describes a standard FCS setup.
  • Figure 1 A describes a combined SICS- and FCS-instrument, where SICS is the part of the instrument below the sample and FCS is the part of the instrument above the sample.
  • Keywords nanoparticles, label-free, interferometry, particle sizing, light scattering, fluorescence correlation spectroscopy
  • DLS dynamic light scattering 1
  • laser diffraction measures the particles' projected cross-section. None of the techniques estimate however the concentration of particles, and they cannot easily be combined with fluorescence techniques.
  • interferometric techniques for analysis of single metal and polymer nanoparticles (NPs) and viruses have gained much interest. They offer high sensitivity detection of unlabeled nano-sized objects 3 ' 4 , but also allow metal NPs to be used as an alternative label, free from fluorescence bleaching, blinking and saturation 5"8 .
  • PCS photothermal correlation spectroscopy 9
  • PhACS photothermal absorption correlation spectroscopy 10
  • scattering interference correlation spectroscopy is introduced as a label- free technique, where fluctuations are likely caused by interference between the phase shifted forward scattering from NPs and the transmitted laser light (reference beam) as in PCS and PhACS (fig 1 a).
  • Autocorrelation of the forward scattered and transmitted light yields information not only about the NPs' hydrodynamic radius, but also about their effective cross-section and concentration.
  • FCS fluorescence correlation spectroscopy
  • FIG. 1 ACF curves from four measurements on 62 nm (left) and 26 nm diameter (middle) unlabeled NPs. Insets: G(0)-1 vs N for the respective NP sizes. A q for all four NP sizes was obtained from the slope which equals A q 2 . Right: A q plotted versus the NP diameter for the four NP sizes. A q scales with the NP volume, evidencing that the fluctuations are caused by interference.
  • N 0.18 NPs resided in the detection volume
  • a histogram of the detected intensity for 93 nm NPs shows a broader distribution with a tail towards lower counts per bin, indicating that NPs transiting the detection volume give rise to negative fluctuations 11 (fig lc). Positive fluctuations may also be present, but this cannot be concluded given the limited signal to noise ratio in these measurements.
  • ACF autocorrelation function
  • the decay-time dependence of the ACF curves on particle size was investigated by measurements on the 210, 93, 62 and 26 nm diameter NPs.
  • the respective diffusion times were 26, 8.6, 6.4, and 2.8 ms (fig 3, upper).
  • the diffusion coefficient gives a separate estimate of the particle size, which can be used together with eq. 2 to derive the particle concentration from the ACF amplitude.
  • the instrument can even be calibrated to yield the diffusion time, normalized effective cross- section A q and concentration of an unknown sample, by utilizing that the diffusion time for point like particles is linear with the particle diameter d, and that A q scales as d 3 .
  • SICS + FCS was also used to measure the affinity of 62 nm negatively charged nonfluorescent NPs to ligands in the form of the positively charged fluorophore FIL488 at pH 7.3 (fig 4, right).
  • Nonfluorescent NPs at 55 nM were mixed with ligands at concentrations varying from 8 ⁇ down to 1 nM, and each sample was analyzed by SICS + FCS.
  • the FCS-curves give the concentration of free ligand [L] and of the ligand- NP complexes [L*NP] respectively, while the SICS-curves give the total concentration of NPs.
  • K d [NP][L]/[L*NP] can be measured from each single measurement, which is not possible using single color FCS.
  • the eight measurements yielded K ⁇ r 3.1 ⁇ 3.6 nM (fig 4, right).
  • the fluctuations detected in SICS scale with the NP volume (fig 2, right), which indicates that they are caused by interference of the forward scattered laser light with the transmitted laser light 3 ' 5 ' 7. Similar interference signals have been utilized for single NP detection 16 , for NP correlation analysis 1 , and in phase analysis light scattering for measurement of particle velocity 18 . Also in PCS 9 and PhACS 10 the generated signal is attributed to interference between the scattered and the transmitted light, however, recently an alternative theory interprets the photothermal fluctuations as originating from a nano-lensing effect 19 .
  • the S/N should increase as the square root of the laser power and accordingly, use of photo diodes which can sustain count rates higher than 10 16 Hz should substantially enhance the S/N ratio and sensitivity in SICS.
  • the normalized effective cross-section A q by performing multi-component analysis of intensity distribution histograms (fig 1C ). Such analysis will be important for very non-spherical particles, whose size cannot be estimated from the diffusion coefficient. For such particles, comparison of A q with the diffusion coefficient will then yield information about the particles' shape 2 .
  • the theoretically estimated A q corresponds to that of a sphere of 70 nm diameter. Such a sphere would have had a diffusion time of 7-8 ms in the instrument used here, however the measured diffusion times of the phages were almost ten times longer, indicating an extremely elongated shape .
  • a related technique i •s inverse-FCS 12 ' 21 ' 22 which also combines analysis of labelled and unlabeled NPs.
  • Inverse-FCS allows the absolute volume of particles and even protein molecules in solution to be measured using zero-mode waveguides 11 .
  • SICS as presented here has however an advantage in that NPs and possibly biomolecules can be analyzed in a simpler, diffraction limited detection volume.
  • SICS allows analysis of both size and concentration of unlabeled nanoparticles in solution. Furthermore, simultaneous analysis of labeled and unlabeled nanoparticles was shown by combining SICS and FCS. Measurements were performed on Ml 3 phage viruses and on unlabeled and labeled polystyrene NPs down to 24 nm diameter. The contrast in SICS likely arises from interference between the scattered light from particles and the transmitted laser light, as indicated by the fact that the fluctuations scale with the particle volume.
  • SICS and FCS allows the percentage of label-carrying particles or viruses to be determined, and single-measurement estimation of K D though only one species is labeled.
  • the discussed possibilities for improvement should allow analysis of even smaller NPs and biomolecules, which for example will allow the success of post- translational labelling of protein molecules to be measured.
  • FCS fluorescence correlation spectroscopy

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Abstract

L'invention concerne une nouvelle technique de spectroscopie de fluctuation basée sur l'interférométrie. La technique, dénommée spectroscopie par corrélation d'interférence de diffusion (SICS), assure l'autocorrélation du signal de la lumière à diffusion frontale et de la lumière laser transmise émanant de nanoparticules (NP) en solution. La SICS a deux caractéristiques importantes : Tout d'abord, pour des nanoparticules non marquées ayant un indice de réfraction connu, elle permet l'analyse non seulement du coefficient de diffusion mais aussi de la section transversale effective et de la concentration, en une seule mesure. Ensuite, elle peut être combinée à une spectroscopie par corrélation de fluorescence (FCS) pour une analyse simultanée de nanoparticules marquées et non marquées. La SICS est ici démontrée sur des phages M13 non marqués et sur des nanoparticules non marquées d'un diamètre de 210 nm à 26 nm. Il est également démontré comment la combinaison de la SICS et de la FCS permet de déterminer la fraction de nanoparticules fluorescentes dans un mélange, et d'évaluer Kd à partir d'une seule mesure de liaison.
PCT/SE2013/000155 2012-10-13 2013-10-14 Spectroscopie par corrélation d'interférence de diffusion (sics) WO2014058371A1 (fr)

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Publication number Priority date Publication date Assignee Title
CN107305177A (zh) * 2016-04-21 2017-10-31 易幼文 一种微粒物可视化装置和便携式微粒物检测系统

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