Journal: ACS applied materials & interfaces

Article Title: Nanoparticle-Enabled Multiplexed Electrochemical Immunoassay for Detection of Surface Proteins on Extracellular Vesicles

doi: 10.1021/acsami.1c14506

Figure Lengend Snippet: Preparation and characterization of immunoprobes. (A) Steps required for functionalization of AuNPs. Attachment of Abs occurs via EDC–NHS bonding. Metal ions interact with Abs by coordination bonding. (B) NP-SIMS analysis of immunoprobes AuNPs/anti-CD63@Pb2+ (top) and AuNPs/anti-CD63@Cu2+ (bottom). Impacts were selected based on detection of Au2+ or Au3+, characteristic of the immunogold particles. The Y-axis represents the measured intensity divided by the number of measurements in each experiment. Selected regions of the mass spectra are shown highlighting secondary-ion characteristic to the Abs, silicon support, gold particles, and metal ions.

Article Snippet: Human recombinant CD63, CD81, and nephrin proteins were purchased from R&D Systems (Minneapolis, MN).

Techniques:

Journal: ACS applied materials & interfaces

Article Title: Nanoparticle-Enabled Multiplexed Electrochemical Immunoassay for Detection of Surface Proteins on Extracellular Vesicles

doi: 10.1021/acsami.1c14506

Figure Lengend Snippet: Electrochemical characterization of immunoprobes. (A) SWV response of different ratios of AuNPs/anti-CD63@Pb2+ to AuNPs/anti-CD81@Cu2+; (a) 1:0, (b) 0:1, (c) 1:1, and (d) 2:1. (B) Stability of AuNPs/Ab@M2+ immunoprobes when stored at 4 °C in HEPES buffer for 8 days. Peak current from SWV measurement on a given day (I) was divided by the value of the peak current immediately after electrode preparation (I0).

Article Snippet: Human recombinant CD63, CD81, and nephrin proteins were purchased from R&D Systems (Minneapolis, MN).

Techniques:

Journal: ACS applied materials & interfaces

Article Title: Nanoparticle-Enabled Multiplexed Electrochemical Immunoassay for Detection of Surface Proteins on Extracellular Vesicles

doi: 10.1021/acsami.1c14506

Figure Lengend Snippet: Construction of electrodes and detection of EVs. (A,B) Representative TEM images of EVs before (A) and after (B) incubation with immunoprobes. Scale bar, 100 nm. (C) Process flow for electrode modification and EV capture. (D) Characterization of individual steps in the electrode functionalization and EV capture using EIS: (a) Au electrode (insert), (b) Au/MUA, (c) Au/MUA/EDC–NHS, (d) Au/MUA/EDC–NHS/PLL, (e) Au/MUA/EDC–NHS/PLL/EVs, (f) Au/MUA/EDC–NHS/PLL/EVs/BSA, and (g) Au/MUA/EDC–NHS/PLL/EVs/BSA/AuNPs–anti-CD63@Pb2+. (E) Electrochemical (SWV) signals from EV-containing electrodes that were incubated with immunoprobes specific to CD63 (a), CD81 (b), a 1:1 mixture of both types of immunoprobes (c), and electrode without EVs after incubating with a 1:1 mixture of both types of immunoprobes (d).

Article Snippet: Human recombinant CD63, CD81, and nephrin proteins were purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Modification

Journal: ACS applied materials & interfaces

Article Title: Nanoparticle-Enabled Multiplexed Electrochemical Immunoassay for Detection of Surface Proteins on Extracellular Vesicles

doi: 10.1021/acsami.1c14506

Figure Lengend Snippet: Establishing detection limit and dynamic range for nanoparticle-enabled electrochemical immunoassay. (A) Electrochemical (SWV) analysis of electrodes containing different numbers of EVs after incubation with a mixture of AuNPs/anti-CD63@Pb2+ and AuNPs/anti-CD81@Cu2+ immunoprobes. (B) Total charge (Q) associated with each EV concentration. (C) Calibration curves of normalized charge Q¯ vs EV concentration for CD63 and CD81 constructed for an EV concentration range of 1.14 × 106–1.14 × 108 particles/mL. Here, Q¯=QEVs−Qisotype control Abs for detection of CD63 and CD81. The error bars represent the standard deviations from five different sensing electrodes (n=5).

Article Snippet: Human recombinant CD63, CD81, and nephrin proteins were purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Concentration Assay, Construct, Control

Journal: ACS applied materials & interfaces

Article Title: Nanoparticle-Enabled Multiplexed Electrochemical Immunoassay for Detection of Surface Proteins on Extracellular Vesicles

doi: 10.1021/acsami.1c14506

Figure Lengend Snippet: Quantifying CD63 and CD81 expression on EVs. (A) Calibration curves obtained after immobilizing different concentrations of human recombinant CD63 and CD81 proteins on the electrode surfaces. The linear plot of the normalized total charge (Q¯=Q−Q0) changes as a function of the logarithm of the concentration of recombinant CD63 or CD81 (0–500 ng/mL). The error bars represent the standard deviations from three different sensing electrodes (n=3). (B) Plots correlating concentration of CD63 or CD81 to the concentration of EVs allow to quantify surface marker expression. Values for normalized total charge Q¯ associated with immobilization of recombinant proteins were correlated with EV concentration using calibration curves from Figure 4C to construct plots presented here. The data points and error bars represent average and standard deviations of measurements from five different electrodes containing captured EVs (n=5).

Article Snippet: Human recombinant CD63, CD81, and nephrin proteins were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Recombinant, Concentration Assay, Marker, Construct

Journal: ACS applied materials & interfaces

Article Title: Nanoparticle-Enabled Multiplexed Electrochemical Immunoassay for Detection of Surface Proteins on Extracellular Vesicles

doi: 10.1021/acsami.1c14506

Figure Lengend Snippet: Using clinical samples to validate electrochemical immunoassay against flow cytometry. (A) Principle of assay operation. Electrodes were functionalized with anti-CD63 for capture of EVs. Immunoprobes targeting nephrin and podocin on urinary EVs were then used for labeling and electrochemical detection. (B) Representative electrochemical (SWV) analysis of EVs from a clinical sample captured on a working electrode. AuNPs/anti-nephrin@Pb2+ and AuNPs/anti-podocin@Cu2+ immunoprobes were used to label EVs and generate dual redox peaks. No redox activity was observed when electrodes containing EVs were labeled with isotype control immunoprobes (dashed line). (C) Representative flow cytometry analysis of nephrin and podocin expression in clinical EVs. The same sample was characterized by flow cytometry and electrochemical analysis. (D) plot of podocin/nephrin ratios obtained with electrochemical immunoassay (this method) and flow cytometry based on urine samples from six pregnant women (n=6). The results showed high correlation (R2=0.9001) between our method and flow cytometry.

Article Snippet: Human recombinant CD63, CD81, and nephrin proteins were purchased from R&D Systems (Minneapolis, MN).

Techniques: Flow Cytometry, Labeling, Activity Assay, Control, Expressing

Journal: eLife

Article Title: Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins

doi: 10.7554/eLife.86394

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , CD63 , Origene , Cat# TP301733 , .

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Purification, Chromatography

Journal: eLife

Article Title: Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins

doi: 10.7554/eLife.86394

Figure Lengend Snippet: ( A ) Levels of CD9, CD63, CD81, ApoB-100, and albumin were measured by Simoa after SEC of 1 ml plasma in each fraction using either Sepharose CL-2B, Sepharose CL-4B, or Sepharose CL-6B resin. ( B ) Extracellular vesicle (EV) yield is calculated in fractions 7–10 for Sepharose CL-2B, Sepharose CL-4B, or Sepharose CL-6B by averaging the ratios of CD9, CD63, and CD81. ( C ) Purity of EVs with respect to lipoproteins or free proteins is calculated by dividing relative EV yield (the average of the ratios of CD9, CD63, and CD81) by levels of ApoB-100 (top) or albumin (bottom). Error bars represent the standard deviation of four columns measured on different days with two technical replicates each. Figure 2—source data 1. Simoa data (protein concentrations) for fractions of SEC columns with different resins. Figure 2—source data 2. Simoa data (protein concentrations) for SEC column with different number of washes.

Article Snippet: Peptide, recombinant protein , CD63 , Origene , Cat# TP301733 , .

Techniques: Standard Deviation

Journal: eLife

Article Title: Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins

doi: 10.7554/eLife.86394

Figure Lengend Snippet: Levels of CD9, CD63, CD81, albumin, and ApoB-100 were measured by Simoa in individual 1 ml fractions (collected from the top) after density gradient centrifugation of plasma using an iodixanol gradient. Error bars represent the standard deviation of two replicates of each measurement. Figure 3—source data 1. Simoa data (protein concentrations) for different density gradient centrifugation fractions.

Article Snippet: Peptide, recombinant protein , CD63 , Origene , Cat# TP301733 , .

Techniques: Gradient Centrifugation, Standard Deviation

Journal: eLife

Article Title: Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins

doi: 10.7554/eLife.86394

Figure Lengend Snippet: Levels of CD9, CD63, CD81, ApoB-100 albumin were measured (in duplicate and then averaged) by Simoa to compare extracellular vesicle (EV) isolation from 1 ml plasma using density gradient (DG) centrifugation, SEC, or density gradient centrifugation followed by size exclusion chromatography (DG-SEC). For the DG and DG-SEC condition, fraction 10 was analyzed. Simoa measurements were used to quantify relative EV recovery ( A ), EV/ApoB-100 ratio ( B ), and EV/albumin ratio ( C ).

Article Snippet: Peptide, recombinant protein , CD63 , Origene , Cat# TP301733 , .

Techniques: Isolation, Centrifugation, Gradient Centrifugation, Size-exclusion Chromatography

Journal: eLife

Article Title: Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins

doi: 10.7554/eLife.86394

Figure Lengend Snippet: ( A ) Levels of CD9, CD63, CD81, and albumin were measured by Simoa after EV isolation from 1 ml plasma with size exclusion chromatography (SEC) using 0, 1, 2, or 3 in-column 10 ml PBS washes. Error bars represent the standard deviation from two technical replicates. ( B ) Percent recovery of EVs using average of ratios of CD9, CD63, and CD81 in SEC isolation relative to plasma.

Article Snippet: Peptide, recombinant protein , CD63 , Origene , Cat# TP301733 , .

Techniques: Isolation, Size-exclusion Chromatography, Standard Deviation

Journal: eLife

Article Title: Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins

doi: 10.7554/eLife.86394

Figure Lengend Snippet: ( A ) Schematic of the columns being compared: size exclusion chromatography (SEC) column comprised of 10 ml Sepharose CL-6B, dual-mode chromatography (DMC) columns comprised of 10 ml Sepharose CL-6B SEC resin atop 2 ml Fractogel cation exchange resin, Tri-Mode Chromatography (TMC) columns comprised of 10 ml Sepharose CL-6B SEC resin atop 2 ml 2:1 ratio of 2 ml Fractogel cation exchange resin to Capto Core 700 multimodal chromatography resin. ( B ) Electron microscopy of EVs isolated from plasma using SEC (left), DMC (middle), or TMC (right) columns. EVs indicated with red arrows (among background of lipoproteins). ( C ) EV recovery is calculated for EV isolation from plasma for SEC (fractions 7–10), DMC (fractions 9–12), or TMC (fractions 9–12). Simoa measurements in the designated fractions for CD9, CD63, and CD81 are taken as a ratio relative to measurements of these proteins from diluted plasma and these three ratios are then averaged to calculate recovery. ( D ) Purity of EVs with respect to free proteins is determined by dividing relative EV yield (the average of the ratios of CD9, CD63, and CD81) by relative levels of albumin in each condition. ( E ) Purity of EVs with respect to lipoproteins is determined by dividing relative EV yield (the average of the ratios of CD9, CD63, and CD81) by relative levels of ApoB-100 in each condition. Error bars represent the standard deviation of four column measured on different days with two technical replicates each. Figure 4—source data 1. Simoa data (protein concentrations) for TMC columns with different ratios of resins in the bottom layer. Figure 4—source data 2. Simoa data (protein concentrations) for different fractions of SEC and DMC columns. Figure 4—source data 3. Simoa data (protein concentrations) comparing SEC (fractions 7-10), DMC and TMC columns (fractions 9-12).

Article Snippet: Peptide, recombinant protein , CD63 , Origene , Cat# TP301733 , .

Techniques: Size-exclusion Chromatography, Chromatography, Electron Microscopy, Isolation, Standard Deviation

Journal: eLife

Article Title: Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins

doi: 10.7554/eLife.86394

Figure Lengend Snippet: Levels of CD9, CD63, CD81, ApoB-100, and albumin were measured (in duplicate and then averaged) by Simoa in extracellular vesicle (EV) samples isolated from 1 ml plasma to compare TMC columns with different volumes and ratios of Fractogel cation exchange resin to Capto Core 700 resin. All conditions describe the bottom layer under a 10 ml Sepharose CL-6B top layer. The 1, 2, or 4 ml volume of the bottom later indicates the volume of the solid resin mixture of Fractogel cation exchange resin and Capto Core 700 resin. The following fractions were collected for each: 8–11 for 1 ml bottom layer, 9–12 for 2 ml bottom layer, and 11–14 for 4 ml bottom layer.

Article Snippet: Peptide, recombinant protein , CD63 , Origene , Cat# TP301733 , .

Techniques: Isolation

Journal: eLife

Article Title: Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins

doi: 10.7554/eLife.86394

Figure Lengend Snippet: Levels of CD9, CD63, CD81, ApoB-100, and albumin were measured by Simoa in fractions 7–10 for SEC with 10 ml Sepharose CL-6B column and fractions 7–14 for DMC using a column with 2 ml Fractogel cation exchange bottom layer and 10 ml Sepharose CL-6B top layer. Error bars represent the standard deviation from two technical replicates.

Article Snippet: Peptide, recombinant protein , CD63 , Origene , Cat# TP301733 , .

Techniques: Standard Deviation

Journal: eLife

Article Title: Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins

doi: 10.7554/eLife.86394

Figure Lengend Snippet: Levels of CD9, CD63, CD81, ApoB-100, and albumin were measured by Simoa in extracellular vesicle (EV) samples isolated from 1 ml plasma using SEC (fractions 7–10), DMC (fractions 9–12), or TMC columns (fractions 9–12). Error bars represent the standard deviation of four column measured on different days with two technical replicates each.

Article Snippet: Peptide, recombinant protein , CD63 , Origene , Cat# TP301733 , .

Techniques: Isolation, Standard Deviation

Journal: eLife

Article Title: Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins

doi: 10.7554/eLife.86394

Figure Lengend Snippet: ( A ) CAD image of semi-automated SEC stand designed to hold eight columns at once with sliding collection tube holder that allows liquid to drip either into 2 ml collection tubes, or to waste. ( B ) Photograph of stand connected to a Tecan Cavro syringe pump controlled by a Raspberry Pi. ( C ) Simoa comparison of CD9, CD63, CD81, ApoB-100, and albumin when SEC was performed on 16 samples of 1 ml plasma using either manual SEC (8 samples) or SEC on the automated device (8 samples). Each point is the average of two Simoa measurements (technical replicates). Figure 5—source data 1. Simoa data (protein concentrations) comparing manual and automated SEC EV isolation (fractions 7-10).

Article Snippet: Peptide, recombinant protein , CD63 , Origene , Cat# TP301733 , .

Techniques: Comparison, Isolation

Journal: eLife

Article Title: Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins

doi: 10.7554/eLife.86394

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , CD63 , Origene , Cat# TP301733 , .

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Purification, Chromatography