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