阅读说明：本技术 单颗粒自动化拉曼捕集分析 (Single particle automated raman trapping analysis ) 是由 M·史蒂文斯 J·彭德斯 I·彭斯 于 2019-06-18 设计创作，主要内容包括：一种颗粒分析的自动化方法使用以下执行：电磁辐射源,所述电磁辐射源用于生成电磁辐射束；聚焦元件,所述聚焦元件用于将所述束引导到颗粒捕获区；检测器,所述检测器被配置成检测来自所述颗粒捕获区的信号响应；以及控制系统。将电磁辐射束聚焦到颗粒传送介质上,以限定用于将候选颗粒捕获在所述束内的所述颗粒捕获区。执行第一数据获取程序以对颗粒捕获进行测试。如果未检测到颗粒捕获,则重复所述第一数据获取程序。如果在所述束内检测到颗粒捕获,则执行第二数据获取程序以使用至少一种分析模态捕获颗粒数据,此后,将光束强度降低到子捕获水平,以从所述颗粒捕获区中释放所述颗粒。对所述颗粒传送介质中的连续颗粒重复步骤。(An automated method of particle analysis is performed using: an electromagnetic radiation source for generating a beam of electromagnetic radiation; a focusing element for directing the beam to a particle capture zone; a detector configured to detect a signal response from the particle capture zone; and a control system. Focusing a beam of electromagnetic radiation onto a particle transport medium to define the particle capture zone for capturing candidate particles within the beam. A first data acquisition procedure is performed to test for particle capture. If particle capture is not detected, the first data acquisition procedure is repeated. If particle capture is detected within the beam, a second data acquisition procedure is performed to capture particle data using at least one analysis modality, after which the beam intensity is reduced to a sub-capture level to release the particles from the particle capture zone. Repeating the steps for successive particles in the particle transport medium.)

1. An automated method of particle analysis, the method comprising:

(i) focusing a beam of electromagnetic radiation onto a particle transport medium to define a particle capture zone for capturing candidate particles within the beam;

(ii) performing a first data acquisition procedure to test for particle capture;

(iii) (iii) if no particle capture is detected, repeating step (ii);

(iv) if particle capture is detected, obtaining particle data from the captured particles;

(v) reducing the beam intensity to a sub-capture level to release the particles from the particle capture zone;

(vi) (vi) repeating steps (i) to (v) for successive particles in the particle transport medium.

2. The method of claim 1, wherein the first data acquisition procedure comprises performing a raman response data acquisition procedure sufficient to detect the presence of a predetermined spectral profile above a threshold indicative of particle capture.

3. The method of claim 1 or 2, wherein step (iv) further comprises:

a second data acquisition procedure is performed to capture the particle data using at least one analysis modality.

4.A method according to claim 3 when dependent on claim 2, wherein the second data acquisition procedure comprises performing a raman response data acquisition procedure that facilitates a signal-to-noise ratio that is greater than a signal-to-noise ratio of the first data acquisition procedure.

5. The method of claim 1, wherein the acquired particle data in step (iv) comprises data from the first data acquisition procedure.

6. The method of claim 1, wherein step (v) further comprises waiting a delay period after reducing the beam intensity to a sub-capture level, the delay period being sufficient to transport previously captured particles out of the particle capture zone and into the surrounding medium.

7. The method of claim 3, wherein the second data acquisition procedure comprises performing a data acquisition procedure comprising any one or more of: data capture for extended periods of higher signal-to-noise ratios; acquisition of spectrally extended data; collection of data using a different modality than the first data acquisition procedure; data capture for multiple time periods for averaging; data acquisition at varying laser power for increased signal-to-noise ratio.

8. The method of claim 7, wherein the different modalities include one or more of fluorescence spectroscopy and light absorption spectroscopy.

9. The method of claim 2, further comprising a calibration procedure for determining the threshold value used in step (ii), the calibration procedure comprising establishing a spectral signature that distinguishes between (i) a raman response of a target particle to the beam and (ii) a raman response of the particle transport medium to the beam in the absence of a target particle.

10. The method of claim 9, wherein the calibration procedure comprises determining one or more spectral features of the raman response that provide the discrimination, the spectral features comprising one or more of: a peak amplitude, a plurality of peak amplitudes, an area under one or more portions of the raman response spectrum, and a profile of at least a portion of the raman response spectrum.

11. The method of claim 2, further comprising a calibration procedure for determining the threshold value used in step (ii), the calibration procedure comprising: obtaining a reference spectrum from the particle transport medium without particles therein; obtaining a test spectrum from the particle transport medium having particles therein; determining a difference between the reference spectrum and the test spectrum; and determining that a particle has been captured if the difference is greater than a noise threshold of the reference spectrum.

12. The method of claim 11, further comprising establishing a spectral feature in the test spectrum that is greater than the noise threshold and thus distinguishes between (i) a raman response of a target particle to the beam and (ii) a raman response of the particle transport medium to the beam without a target particle.

13. The method of claim 3, further comprising determining a size of a captured particle using first data and/or second data acquired in the first and/or second data acquisition procedure.

14. The method of claim 13, wherein determining the size of the captured particles comprises determining a decrease in a spectral response signal when particle capture is detected, the spectral response signal being a characteristic of the particle transport medium without particles therein.

15. The method of claim 14, wherein the spectral signal characteristic of the particle transport medium comprises a spectral signal from a marker dispersed within the particle transport medium.

16. The method of claim 15, wherein the marker comprises perchlorate ions.

17. The method of claim 1, further comprising:

(iii) initiating a chemical change of the captured particles after step (iv) and repeating step (iv), or

(vi) initiating a chemical change of the particles in the particle transport medium after step (v) and repeating steps (i) to (v).

18. The method of claim 1, further comprising:

(iii) monitoring the captured particles for chemical changes by repeating step (iv), or

(vi) monitoring the chemical change of the particles in the particle transport medium by repeating steps (i) to (v).

19. The method of claim 1, wherein step (v) comprises disabling or obscuring the beam.

20. A particle capture and data acquisition apparatus, comprising:

an electromagnetic radiation source for generating a beam of electromagnetic radiation;

a focusing element for directing the beam to a particle capture zone;

a detector configured to detect a signal response from the particle capture zone;

a control system configured to implement the method of any one of claims 1 to 18.

21. A computer program distributable by electronic data transmission or by a computer readable medium, said computer program comprising computer program code means adapted to cause a particle trapping and data acquisition device to execute the program according to any one of claims 1 to 19 when said program is loaded on said device.

Other embodiments are intentionally within the scope of the accompanying claims.

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Other methods

SPARTA Raman micro spectrometer

Confocal raman spectrum acquisition was performed on a raman micro spectrometer (alpha300R +, WITec, ullm (Ulm, Germany)). The light source used was a 785nm laser (Toptica XTRA II) with a 63 x/1.0 NA water immersion microscope objective (W Plan-Apochromat, Zeiss, Germany, kouchhen, Germany). Scattered light was collected by a 100 μm optical fiber with a 600g/mm grating spectrograph (UHTS 300, WITec, Ulm, Germany) and acquired with a thermoelectrically cooled back-illuminated CCD camera (iDus DU401-DD, Andor, Belf asts, UK) with 3cm at the sample^-1Spectral resolution and spectrum of 85mW laser power. Laser control is performed remotely through a serial connection and custom Matlab (2016b) script.

Preparation of SPARTA Standard sample

For the SPARTA analysis, 200 μ l of the particle solution is typically required, about half of which is typically recovered depending on the measurement time. Determining the ideal particle concentration 1.10¹⁰-1·10¹²Between particles per milliliter or about 0.1-0.01% solids of the polystyrene particles. The sample solution was placed on a 22mm coverslip and fixed with a drop of Phosphate Buffered Saline (PBS) onto a standard microscopy slide. The sample was placed under water immersion for measurement.

SPARTA Standard data analysis

Will be as followsThe pre-processing procedure was applied to all spectra acquired using the SPARTA platform. The spectral center standard during acquisition was set to 1000cm^-1And truncating the original data to 350-^-1To omit the excitation signal. For measurements containing alkyne modifications, the spectral center was shifted to 1500cm^-1Thereby generating 606-2254 cm^-1The measurement range of (1). An automated script based on peak amplitude and derivative 2 was employed for cosmic spike removal followed by manual visual inspection. The spectral background was subtracted by curve fitting (Whittaker filter, λ ═ 100,000) and the noise was smoothed using a Savitzky-Golay filter (3 min, first order). The resulting spectra were normalized by the area under the curve, except for the SPARTA sizing analysis, as this analysis was incorporated by perchlorate ratio calculation. Subsequent statistical analyses (hierarchical clustering analysis, principal component analysis, multivariate curve regression, partial least squares discriminant analysis) were performed using PLS Toolbox (Eigenvector Research, Inc.).

Preparation of DPPC and d-DPPC liposomes

Liposomes were prepared according to the following standard procedure. Lipid (1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1, 2-dipalmitoyl-d 62-sn-glycero-3-phosphocholine (DPPC-d62, referred to herein as d-DPPC) (Avanti Polar Lipids Inc.), U.S.A. (AL, USA)) and cholesterol (Sigma-Aldrich, UK) stock solutions were prepared in chloroform and stored under argon at 20 degrees Celsius prior to use for DPPC liposomes a lipid film was prepared by adding 500. mu.l of DPPC stock solution and 43. mu.l of cholesterol stock solution in a 10ml round bottom flask, resulting in a molar ratio of 85:15 mol% DPPC: cholesterol for the d-DPPC liposomes 250. mu.l DPPC stock solution and 250. mu. l d-DPPC stock solution for the d-DPPC liposomes, thus, a 42.5:42.5:15 mol%// ratio of DPPC: d-DPPC: cholesterol was produced. Chloroform was evaporated under a stream of nitrogen to form a thin lipid film. Prior to rehydration, the lipid membrane was lyophilized overnight in a lyophilizer (Labconco, missouri (MO, USA)). The membrane was hydrated with 1ml PBS, shaken for 1 minute and sonicated for 1 minute. The solution was then extruded 31 times through a 200nm mesh size polycarbonate membrane (Avanti polar lipids, alabama, usa) at 60 ℃. The size distribution and particle concentration of the liposomes was determined by Nanoparticle Tracking Analysis (NTA).

Polymer vesicle preparation

From poly (2-methyloxazoline-b-dimethylsiloxane-b-2-methyloxazoline) (M)_n·10³0.5-b-4.8-b-0.5) triblock copolymer (P18140D-MOXZDMSMOXZ, Polymer Source Inc, Quebec, Canada) PMOXA-b-PDMS-b-PMOXA polymersome (further denoted ABA). 1ml of a 6mg/ml stock solution of triblock copolymer in ethanol was added to a 5ml round bottom flask. ABA-heparin polymer vesicles were prepared by mixing in a synthetic 25 wt% PDMS-b-heparin (Mw ═ 5kDa-b-11kDa) block copolymer as previously described by Najer et al. (Najer, A. et al, "Nanometric modeling of host cell membranes blocked invasion and exposed invasive Plasmodium" ACS Nano8, 12560-12571 (2014)). In a 5ml round bottom flask, 1ml of a 6mg/ml ABA stock solution was combined with 0.5ml of a 4mg/ml stock solution of PDMS-b-heparin block copolymer in ethanol. The polymer solution was dried at 50 ℃ on a rotary evaporator at 20mbar for approximately 15 minutes. Subsequently, the polymer membrane was rehydrated in 1.2ml PBS for 72 hours under vigorous agitation. The polymer solution was filtered through a 0.45 μm syringe filter (Millex-HV 13mm PVDF, Merck group (Merck KGaA), germany) and extruded 5 times through a polycarbonate membrane with a mesh size of 200nm (Avanti polar lipids, usa) and subsequently 31 times through a polycarbonate membrane with a mesh size of 100 nm. The polymersomes were further purified by Size Exclusion Chromatography (SEC) (10X 300mm column filled with Sepharose 2B (Sigma-Aldrich, UK)) in PBS and 1ml fractions were collected. The polymersome size distribution was analyzed by DLS and NTA.

Nanoparticle Tracking Analysis (NTA)

Nanoparticle tracking analysis (NS300, 532nm laser, Malvern (Malvern), uk) was performed by taking 330 seconds of video of a 1ml sample in PBS. Maintaining the camera level at 13 and 14, where the screen gain is 1, and the detection threshold is set to 5. The samples were diluted to 1X 10 per ml⁸1 to 10⁹The optimal measurement range of each particle for measurement. The measurements were analyzed using Nanosight NTA3.0 software (marvens, uk, 2014).

Dynamic Light Scattering (DLS)

Dynamic light scattering (ZEN3600 Zetasizer, malvern, uk) was performed in a disposable semi-micro cuvette (Brand GMBH, germany) using 400 μ l of solution by taking 3 measurements at 173 ° scattering angle by NIBS and averaging the measurements (10-15 acquisitions each). Measurements were obtained using Zetasizer software v.7.02 (marvens, uk 2013). Number distributions were used to verify and compare particle size distributions.

Preparation of cysteine-tyrosine (CYY) tripeptide

CYY tripeptide was synthesized by standard solid phase peptide synthesis using Fmoc protecting group chemistry on Rink-amide MBHA resin and protected cysteine and tyrosine amino acids (AGTC Bioproducts Ltd.). Briefly, Fmoc deprotection was performed using 20 v% piperidine in DMF for 10 min, followed by two washes with DMF and DCM. Amino acid coupling was performed with Fmoc protected amino acid (4 eq), HBTU (3.75 eq) and DIEA (6 eq) in DMF for 2 h, and then the procedure was repeated sequentially. The peptide was cleaved from the resin and deprotected with 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane and 2.5% water for 4 hours. TFA was removed using rotary evaporation and the peptide was precipitated and washed with cold diethyl 200mL and 2 x 50 mL. For purification, the peptide was dissolved in a solution of 4.9% ACN in ultrapure water and 0.1% TFA and used with a pore size of 5 μm and a particle size ofAnd C18 Gemini 150X 21.2mm column (Phenomenex, Calif.) by reversed phase preparative high performance liquid chromatography (HPLC; Shimadzu corporation, Japan (Japan)). The mobile phase was ultrapure water containing 0.1% TFA @15 ml/min, andthe modified concentrations of ACN with 0.1% TFA in the mobile phase were 0% 0-3 min, 0-100% 3-12 min, 100% 12-13 min, and 0% 13-15 min during the 15 min run. HPLC fractions were checked for correct mass using liquid chromatography-mass spectrometry (LCMS, Agilent (Agilent), CA, USA) (observed MW 447.2, predicted [ CYY ═ 447.2)]H⁺447.16) and the pure peptide fractions were combined, rotary evaporated to remove ACN and lyophilized by freeze drying (Labconco, MO, USA).

Series functionalization of polystyrene particles

Amine-functionalized 0.2 μ M polystyrene particles (Polybead amino 0.20 μ M, Polysciences Inc.). further functionalized with 2-iminothiolane reaction buffer of 2mmol EDTA in PBS was prepared and adjusted to pH 8 with 2M NaOH, thereby preparing a 6mg/ml 2-iminothiolane solution for functionalization 780 μ l of reaction buffer was combined with 200 μ l of 0.20 μ M polystyrene particles (2.6% solids (w/v)) and 20 μ l of 2-iminothiolane solution and kept reacting overnight at room temperature, which resulted in a 0.5% solids (w/v) solution of thiol-functionalized particles, which was further diluted 10 times in PBS and purified, unless otherwise stated purification was performed by centrifugation at 14,000rcf for 10 minutes, then redispersed in a pellet for 30 seconds and sonicated for 1 minute, thereby obtaining a clear solution. After each purification step, Dynamic Light Scattering (DLS) measurements were performed before SPARTA to verify that aggregation was not present. A minimum of 100 successful trapping spectra were acquired using the SPARTA experimental parameters set to 1 second iterative acquisition time, 10 second high SNR acquisition time, and 1 second laser disable time. The particles were further treated with 10mg of 5, 5-dithio-bis- (2-nitrobenzoic acid) (DTNB) to form a disulfide bond between the thiol-functionalization and TNB anion. The particles were purified and SPARTA was performed to verify disulfide bond formation. By using 2mg of CYY (M)_w446.16 g/mole, 4.5mM) treatment of the TNB functionalized particles resulted in tripeptide functionalization. After purification, SPARTA was performed to observe tripeptide functionalization. To demonstrate the reversibility of the functionalization, 100. mu.l of tris (2-carboxyethyl) phosphine (TCEP, 0.5M)Neutral pH Bond-Breaker^TMThe solution turned bright yellow when the seemer heschel Scientific company (thermo fischer Scientific)) added to TNB functionalized particles, indicating disulfide cleavage. Similarly, disulfide bonds between the particles and tripeptides are cleaved. After purification, the recovery of thiol functionalization was verified by SPARTA.

Dynamic click reaction of polystyrene particles

Carboxyl functionalized 0.2 μm polystyrene particles (Polybead carboxylate 0.20 μm, euphorbia, Inc (Polysciences, Inc.)) were functionalized with propargylamine using EDC-NHS coupling. Solutions were made from PBS containing 20mg/ml 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 20mg/ml n-hydroxysuccinimide, and 40. mu.l of each solution was added to 200. mu.l polystyrene particles (2.6% solids) and 800. mu.l PBS. The solution was shaken on a hot mixer at room temperature and 20. mu.l of neat propargylamine was added after 30 minutes. The reaction was allowed to proceed overnight with continuous shaking. The synthesis solution was diluted 10 times and purified. Purification was performed by centrifugation at 14,000rcf for 10 minutes, after which the pellets were redispersed in PBS. Redispersion was aided by vortexing for 30 seconds and sonicating for 1 minute. After purification, DLS traces were obtained before SPARTA to verify that aggregation was not present. Solutions were prepared from PBS containing 100mM copper sulfate, 100mM sodium ascorbate, and 0.5M potassium bicarbonate. The azidoacetate was prepared by adding 1M sodium hydroxide to a solution of molar equivalents of 2-azidoacetic acid (Sigma-Aldrich, UK). The population click reaction was performed by forming triazoles by adding 2.88 μ l 100mM sodium ascorbate, 1.80 μ l 100mM copper sulfate and 0.5 μ l pure azidoacetic acid to 200 μ l alkyne-functionalized polystyrene particles, diluted 100-fold in PBS. Use of 0.5M KHCO₃The solution was adjusted to pH 7. Single particle conservation click reactions were performed with the addition of 7.46. mu.l of azidoacetate (equivalent to 0.5. mu.l of pure azidoacetic acid) without further pH adjustment.

Click reaction monitoring by UV-Vis analysis

3-azido-7-hydroxycoumarin (Jena Bioscience GmbH, Germany) was used to verify whether the CuAAC reaction would occur on alkyne-functionalized polystyrene nanoparticles. For this purpose, the fluorescence of the produced triazole product was monitored by UV-Vis spectroscopy (Abs/Em ═ 404/477 nm). In a 96-well plate, 200. mu.l of a 1000 Xdilution of purified alkyne-functionalized polystyrene particles in PBS was combined with 1. mu.M solution containing 2.88. mu.l of 100mM sodium ascorbate, 1.80. mu.l of 100mM copper sulfate and 5. mu.l of 3-azido-7-hydroxycoumarin in water. As a control, measurements were performed while excluding 3-azido-7-hydroxycoumarin or copper sulfate. Fluorescence was monitored over 30 minutes and measurements were taken at 15 second intervals.

Analysis of hybrid vesicles by SPARTA

Hybrid vesicles, Janus dendrimers and sphingomyelin-cholesterol liposomes were prepared for purification as required by a thin film hydration method in DPBS by sequential vortexing and subsequent size exclusion. Stability measurements at room temperature indicate stability over a period of one month. The sample concentration was measured using nanoparticle tracking analysis and the samples were stored at 4 ℃ prior to measurement.

The SPARTA protocol utilizes 150 μ L of sample in PBS at about 1013 particles/mL. The trapping evaluation time was set to 1.5 seconds, after which trapping above the threshold was recorded with an integration time of 10 seconds to obtain a high SNR spectrum. The capture sequence typically begins with 200 captures and proceeds automatically until completion. The acquired spectra were processed using a custom MATLAB (2016a/b) script for cosmic spike removal, background subtraction, smoothing, and normalization. NNLS fitting was performed based on pure spectra obtained from individual components by natural and custom Matlab scripts.

Cubic analysis of SPARTA

Compositions comprising monoolein, cholesterol, and a combination of phosphatidylcholine and phosphatidic acid are formed using sonication and homogenization. Briefly, the formation of cubes by sonication involves co-dissolving lipids of the desired composition in a solvent, evaporating the solvent under nitrogen, and removing excess water using a lyophilizer to form a lipid film. Subsequently hydrated in the desired buffer, pre-dissolved stabilizing polymer is added and tip sonication/homogenization is performed to form a dispersion. Due to volume limitations, a small volume ultrasonic tip is utilized. The cubes were kept at room temperature prior to performing the SPARTA analysis. Phospholipase D (derived from Streptomyces chromofuscus) was stored frozen at-80 ℃ prior to use. For a separate experimental run, the total lipid concentration was 0.1Mg/mL per 250 μ L sample, mixed with the appropriate amount of Mg/Ca buffered DPBS and PLD at a known concentration to monitor enzyme activity-cube composition and lipid conversion.

SPARTA measurements were performed on 200. mu.l samples of enzyme-doped cubic samples with a high SNR integration time of 10 seconds, an iteration time of 1 second, and a laser disabling time of 1 second. Typically, 200 traps were initialized, but this number will vary depending on the desired activity and the concentration of enzyme added to the sample. Spectra were obtained in a grab and release (population sampling) and catch and hold (single particle time course) method, enabling monitoring of enzyme kinetics. The acquired spectra were processed using a custom MATLAB (2016a/b) script for cosmic spike removal, background subtraction, smoothing, and normalization.

Extracellular vesicle analysis by SPARTA

The MDA-MB-231 and MCF10A cell lines were obtained from ATCC (Manassas, VA, USA, Va., USA). JIMT-1 was obtained by DSMZ (German Breenrelix (Braunschweig, Germany)). MDA-MB-231 and JIMT-1 cells (Gibco, Saimer Feishel, UK) were cultured in DMEM supplemented with 10% (v/v) FBS, 20mM HEPES and 1 XPicillin/streptomycin. MCF10A cells were maintained in DMEM/F12(Gibco) supplemented with 5% (v/v) horse serum (Gibco), 20ng/mL EGF (Peprotech), 0.5. mu.g/mL hydrocortisone (Sigma Aldrich), 100ng/mL cholera toxin (Sigma Aldrich), 10. mu.g/mL insulin (Sigma Aldrich) and 1 XPS/streptomycin (Gibco) cells were cultured at 37 ℃ and replaced with 5% CO2 with medium every 2 days. Prior to EV separation, cells were cultured in serum-free medium for 2 days to confluence, and then the conditioned medium was collected, centrifuged at 1500x g for 5 minutes, and filtered using a 0.45 μm bottle top filter. The medium was concentrated to approximately 500-fold using rotary filtration (Amicon Ultra-15, 100kDa) and 500. mu.L was purified by size exclusion chromatography on a 1cm diameter, 30cm long Sepharose CL2B column (Sigma Aldrich UK). The 1mL column fractions were collected and the EV containing fractions were determined using nanoparticle tracking analysis (Nanosight NS300) and correlated with BCA protein quantification (semer feishel, uk). The EV was kept frozen at-80 ℃ prior to performing the SPARTA assay.

SPARTA measurements were performed on 200 μ L samples of purified EV with a high signal-to-noise integration time of 20 seconds, an iteration time of 1 second, and a laser disable time of 1 second. 200 traps were initialized and PBS background was collected for 10 measurements on each measurement day. The acquired spectra were processed using a custom MATLAB (2016a/b) script for cosmic spike removal, background subtraction, smoothing, and normalization. PLSDA modeling was performed using PLS Toolbox (Eigenvector research Inc.).