dawn heleos ii mals detector  (Waters Corporation)

Journal: bioRxiv

Article Title: Resolving heterogeneity of targeted lipid nanoparticles through solution-based biophysical analyses

doi: 10.64898/2026.03.31.715590

Figure Lengend Snippet: ( a ) SAXS profiles of NT LNPs and tLNPs, showing the characteristic Bragg peak feature associated with internal lipid–RNA organization. ( b ) Fitting of the Bragg peak feature using a multiple-Lorentz model where red is the first-order Bragg peak fit, green represents higher-order particle disorder and blue is the cumulative fit of the two features. (c) In-line AF4-UV-Vis contour maps (200–300 nm) depicting wavelength-resolved absorbance of LNPs during separation. The 260 nm absorbance signal is characteristic of encapsulated RNA, enabling identification of RNA-containing LNP populations across the AF4 elution profile. Overlapping spectral features indicate the presence of multiple co-eluting populations with distinct compositional profiles, motivating subsequent chemometric deconvolution. ( d ) Absorbance at 260 nm (RNA-associated signal) and 280 nm (protein-associated signal) from ( c ) plotted against the corresponding 260:280 ratio for each LNP formulation. Deviations in the 260:280 ratio across elution time indicate heterogeneity in RNA and protein content, suggesting the presence of compositionally distinct subpopulations that cannot be resolved by bulk measurements alone. These data were further subjected to chemometric analysis (see Supplemental Figure 6). The determined ( e ) R h profiles derived from in-line DLS and ( f ) molar mass profiles derived from MALS analysis for NT LNPs (beige) and tLNPs (colors) overlaid with UV fractograms from in-line AF4 separation. ( g ) Peak 260:280 ratios from ( d ), shown for comparison across LNP groups. ( h ) In-line DLS R h and MALS-derived ( i ) mass, ( j ) radius of gyration, and ( k ) polydispersity plotted for comparison across LNP groups. Measurements are reported mean ± standard error for ( h–k).

Article Snippet: A DAWN TM HELEOS II MALS instrument with an integrated WyattQELS TM DLS detector (Wyatt Technology), an Optilab TM T-rEX differential refractometer (Wyatt Technology), and a G7165A UV-Vis multi-wavelength UV-Vis detector (Agilent Technologies) were used for online detection.

Techniques: Formulation, Derivative Assay, Comparison

Journal: bioRxiv

Article Title: Resolving heterogeneity of targeted lipid nanoparticles through solution-based biophysical analyses

doi: 10.64898/2026.03.31.715590

Figure Lengend Snippet: ( a ) Guinier analyses with corresponding residuals for SVD-resolved C1 and C2 components of NT LNPs and tLNPs, with the exception of F(ab’) 2 tLNPs, where only C3 is shown. White regions indicate components for which Guinier analysis failed due to large size (q min R g > 1.3). ( b ) P(r) analyses normalized by I(0) for the average profile and individual components (C1–C3) for NT LNPs and tLNPs. ( c ) Radius of gyration (R g ) and ( d ) maximum dimension (D max ) of the average profile and individual components derived from GNOM analysis for NT LNPs and tLNPs. Blank regions denote populations where GNOM analysis was invalid (q min D max > 4). ( e ) LNP shape factor calculated as D max / R g , where values of ∼2.58 and ∼3.0 correspond to spherical and prolate ellipsoid geometries, respectively. DENSS ab initio electron density reconstructions from the AF4-UV-DLS-MALS-SAXS profiles for ( f ) NT LNPs, ( g ) nanobody tLNPs, ( h ) DAPRin tLNPs, ( i ), F(ab’) 2 tLNPs, and ( j ) antibody tLNPs.

Article Snippet: A DAWN TM HELEOS II MALS instrument with an integrated WyattQELS TM DLS detector (Wyatt Technology), an Optilab TM T-rEX differential refractometer (Wyatt Technology), and a G7165A UV-Vis multi-wavelength UV-Vis detector (Agilent Technologies) were used for online detection.

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Resolving heterogeneity of targeted lipid nanoparticles through solution-based biophysical analyses

doi: 10.64898/2026.03.31.715590

Figure Lengend Snippet: Targeted mRNA delivery to the placenta is driven by tLNP structural subspecies. ( a-d ) DiR-labeled NT LNPs and tLNPs containing mCherry mRNA were incubated with placental BeWo b30 trophoblasts at a dose of 150 ng of mRNA per 150,000 cells. After ( a ) 1 h, ( b ) 4 h, and ( c ) 24 h, cellular accumulation was quantified. After ( d ) 24 h, mCherry expression was also quantified. Normalized DiR and mCherry MFI was calculated by normalizing to cells treated with NT LNPs. ( e–m ) NT LNPs and tLNPs containing FLuc mRNA were administered intravenously via retroorbital injection into pregnant and nonpregnant mice at a dose of 12 µg mRNA per mouse. After 6 h, mice were euthanized, and major organs were dissected. For pregnant mice, luminescence imaging of ( e ) livers and spleens and ( f ) placentas and fetuses were performed via an in vivo imaging system (IVIS). Luminescence from ( e-f ) was quantified via region of interest (ROI) analysis to obtain luminescence flux in the ( g ) liver, ( h ) spleen, ( i ) placentas, and ( j ) fetuses of pregnant mice. For nonpregnant mice, luminescence imaging of ( k ) livers and spleens was performed. Luminescence from ( k ) was quantified via region of interest (ROI) analysis to obtain luminescence flux in the ( m ) liver and ( m ) spleen of nonpregnant mice. Signal is reported mean ± SD from n = 3 biological replicates for ( a–d ) and n = 4 biological replicates for ( e–m). One-way ANOVA with post hoc Student’s t-tests using the Holm–Sídak correction for multiple comparisons was used to compare fluorescence in for ( a–d ) and luminescence in ( g–h, l–m ) across treatment groups. Nested one-way ANOVA with post hoc Student’s t-tests using the Holm–Sídak correction for multiple comparisons was used to compare luminescence in ( i–j ) across treatment groups. ( n–q ) Spearman correlations for ( n ) placental, ( o ) pregnant hepatic, and ( p ) nonpregnant hepatic luminescence values using the physicochemical parameters from traditional characterization methods, static SAXS analyses, and AF4-UV-DLS-MALS-SAXS analyses. ( q ) Heatmap representing the entire dataset. For Spearman correlation graphs, dotted lines represent r = –0.6 and 0.6.

Article Snippet: A DAWN TM HELEOS II MALS instrument with an integrated WyattQELS TM DLS detector (Wyatt Technology), an Optilab TM T-rEX differential refractometer (Wyatt Technology), and a G7165A UV-Vis multi-wavelength UV-Vis detector (Agilent Technologies) were used for online detection.

Techniques: Labeling, Incubation, Expressing, Injection, Imaging, In Vivo Imaging, Fluorescence

Journal: bioRxiv

Article Title: Resolving heterogeneity of targeted lipid nanoparticles through solution-based biophysical analyses

doi: 10.64898/2026.03.31.715590

Figure Lengend Snippet: ( a-b ) NT LNPs and tLNPs containing FLuc mRNA were administered intravenously via retroorbital injection into pregnant and nonpregnant mice at a dose of 12 µg mRNA per mouse. After 6 h, mice were euthanized, and serum was collected. Serum levels of C3a, TNF, IFN-γ, IL-6, ALT, and AST were quantified in ( a ) pregnant and ( b ) nonpregnant mice via ELISA. Measurements are reported mean ± SD from n = 3–4 biological replicates. One-way ANOVA with post hoc Student’s t-tests using the Holm–Sídak correction for multiple comparisons was used to compare cytokine levels across treatment groups. ( c–e ) Spearman correlations for ( c ) TNF, ( d ) IFN-γ, and ( e ) IL-6 serum levels in pregnant (top) and nonpregnant (bottom) mice using the physicochemical parameters from traditional characterization methods, static SAXS analyses, and AF4-UV-DLS-MALS-SAXS analyses. ( f ) Heatmap representing the entire dataset. For Spearman correlation graphs, dotted lines represent r = –0.6 and 0.6.

Article Snippet: A DAWN TM HELEOS II MALS instrument with an integrated WyattQELS TM DLS detector (Wyatt Technology), an Optilab TM T-rEX differential refractometer (Wyatt Technology), and a G7165A UV-Vis multi-wavelength UV-Vis detector (Agilent Technologies) were used for online detection.

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Representation and validation of the AF4‐MALS‐FLD method . (A) Overview of the workflow used for identification of EV surface proteins. PE‐conjugated antibodies were incubated with the sample (e.g. pre‐purified EVs, cell culture supernatant, urine, or plasma) and loaded into the AF4 channel. (B) The light scatter elution profile (in relative scale) (black, full line), UV elution profile (black, dotted line) and the size determination ( R rms in nm) (red) obtained by the multi‐angle light scattering (MALS) detector is plotted against time for labelling of SK‐BR‐3‐derived EVs with PE‐conjugated anti‐CD81 antibody. (C) The fluorescent light detector (FLD) signal (in relative scale) for SK‐BR‐3‐derived EVs labelled with PE‐conjugated anti‐CD9, anti‐CD63 and anti‐CD81 is plotted against time. (D) Transmission electron microscopy (TEM) images of different fractions of the AF4‐MALS‐FLD elution profile are shown (scale bar = 200 nm).

Article Snippet: Detection was performed by an ultraviolet (UV) detector at 280 nm (Shimadzu), a multi‐angle light scattering (MALS) detector DAWN HELEOS‐II using a laser at 658 nm (Wyatt Technology) and a fluorescence detector (FLD) with an excitation at 488 nm, emission at 578 nm and a gain of 16 (Agilent Technologies) (Geeurickx et al. ).

Techniques: Biomarker Discovery, Incubation, Purification, Cell Culture, Clinical Proteomics, Multi-Angle Light Scattering, Derivative Assay, Transmission Assay, Electron Microscopy

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: AF4‐MALS‐FLD analysis of EV surface proteins with biomarker potential in prostate and breast cancer . MCF‐7‐, MDA‐MB‐231‐ and SK‐BR‐3‐derived EVs were labelled with PE‐conjugated anti‐EpCAM antibodies and analysed by AF4‐MALS‐FLD. (A) The elution profile (in relative scale) of the multi‐angle light scatter (MALS) detector and the size ( R rms in nm) were plotted against time. The fluorescent light detector (FLD) signal for MCF‐7‐, MDA‐MB‐231‐ and SK‐BR‐3‐derived EVs labelled with (B) PE‐conjugated anti‐EpCAM and (C) PE‐conjugated anti‐HER2 antibodies were plotted. (D) From FLD elution profiles, the area under the curve for the EV peak (24–80 min) was determined. Unstained EV samples were used as a negative control. (E) Different concentrations (6 × 10 9 , 8 × 10 9 , 1 × 10 10 and 2 × 10 10 particles as measured by NTA) including a negative control of LNCaP‐derived EVs (high PSMA expression) were labelled with anti‐PSMA antibodies and analysed by the AF4‐MALS‐FLD protocol. (F) The area under the curve for the EV peak was determined for LNCaP‐derived EVs. Different concentrations (2 × 10 10 , 4 × 10 10 and 6 × 10 10 particles as measured by NTA) including a negative control of (G) MCF‐7‐derived EVs (high EpCAM expression) or (I) SK‐BR‐3‐derived EVs (high HER2 expression) were labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies respectively and analysed by the AF4‐MALS‐FLD protocol. The area under the curve for the EV peak (24–80 min) was determined for (H) MCF‐7‐ and (J) SK‐BR‐3‐derived EVs.

Article Snippet: Detection was performed by an ultraviolet (UV) detector at 280 nm (Shimadzu), a multi‐angle light scattering (MALS) detector DAWN HELEOS‐II using a laser at 658 nm (Wyatt Technology) and a fluorescence detector (FLD) with an excitation at 488 nm, emission at 578 nm and a gain of 16 (Agilent Technologies) (Geeurickx et al. ).

Techniques: Biomarker Discovery, Derivative Assay, Multi-Angle Light Scattering, Negative Control, Expressing

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Detection of EVs in complex matrices . (A) Different volumes of cell culture supernatant (0, 20, 40 and 60 µL) collected from the MCF‐7 cells were labelled with PE‐conjugated anti‐EpCAM antibodies and analysed by AF4‐MALS‐FLD. The area under the curve for the EV peak in complex matrices (40–80 min) was determined. (B) Different amounts of LNCaP‐derived EVs were spiked in 100 µL of concentrated urine, diluted 1:1 in PBS to reduce viscosity, labelled with PE‐conjugated anti‐PSMA antibodies, and analysed by AF4‐MALS‐FLD. The area under the curve for the EV peak was determined. Different amounts of (C) MCF‐7‐ or (D) SK‐BR‐3‐derived EVs were spiked in 100 µL of blood plasma, diluted 1:1 in PBS to reduce viscosity, and labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies, respectively. Labelled EVs were analysed by AF4‐MALS‐FLD and the area under the curve for the EV peak was determined. Different amounts of SK‐BR‐3 EVs were also spiked in blood plasma and labelled with isotype control antibodies. (E) Different concentrations of soluble EpCAM (1, 5 and 10 ng/mL) and soluble HER2 (50, 100 and 150 ng/mL) were spiked in blood plasma, labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies respectively, and analysed by AF4‐MALS‐FLD.

Article Snippet: Detection was performed by an ultraviolet (UV) detector at 280 nm (Shimadzu), a multi‐angle light scattering (MALS) detector DAWN HELEOS‐II using a laser at 658 nm (Wyatt Technology) and a fluorescence detector (FLD) with an excitation at 488 nm, emission at 578 nm and a gain of 16 (Agilent Technologies) (Geeurickx et al. ).

Techniques: Cell Culture, Derivative Assay, Viscosity, Clinical Proteomics, Control

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Validation of the AF4‐MALS‐FLD workflow on patient samples . Urine samples of five prostate cancer patients were labelled for PSMA and analysed by the AF4‐MALS‐FLD workflow. Fractions 40–80 min were collected, concentrated and processed for mass spectrometry‐based proteomic analysis. (A) EV markers Syntenin‐1, Flotillin‐1, CD63, CD9, CD81, Flotillin‐2, Alix and TSG101 were analysed (missing sample indicated in grey). Z ‐score transformation of intensities were plotted. (B) Targeted mass spectrometry analysed the presence of PSMA (FOLH1) in patient samples. The z ‐score transformation of intensities was plotted with the AF4‐MALS‐FLD peak area. (C) Blood plasma samples of healthy controls ( n = 7) and HER2 amplified breast cancer patients ( n = 10) were labelled with PE‐conjugated anti‐HER2 antibodies. (D) Blood plasma samples of healthy controls ( n = 6) and breast cancer patients ( n = 8) were labelled with PE‐conjugated anti‐EpCAM antibodies. The area under the curve values were normalised for the mean value in the healthy control group.

Article Snippet: Detection was performed by an ultraviolet (UV) detector at 280 nm (Shimadzu), a multi‐angle light scattering (MALS) detector DAWN HELEOS‐II using a laser at 658 nm (Wyatt Technology) and a fluorescence detector (FLD) with an excitation at 488 nm, emission at 578 nm and a gain of 16 (Agilent Technologies) (Geeurickx et al. ).

Techniques: Biomarker Discovery, Mass Spectrometry, Transformation Assay, Clinical Proteomics, Amplification, Control