Tangential and Flow-through Capture of Protein G on NPN

ByGreg Madejski

We have long maintained that we can capture more analyte from a sample using a flow-through configuration compared to a tangential configuration. We can confirm this view quantitatively by measuring the amount of material captured on the membrane with equal source flow rates. Additionally, one of the advantages of a silicon-based platform is that we can use silicon-based functionalization chemistries to adhere other molecules to the sensor surface.

This experiment is intended to show the ability of a flow-cell design to improve capture for a basic antibody-based assay. We use a vapor of epichlorohydrin to bind a immunoglobulin G molecule to the surface of the NPN membrane, then flow in Protein G, which is captured by the IGG. To determine the amount of protein captured, we develop a fluorescence measurement from the membrane surface, using an alkaline-phosphatase based reaction with a 4-MUP substrate.

Experimental Details

Chips were obtained from Wafer 1299, 4-slot, 10.9% porosity, 44 nm average pores, 100 nm NPN)

  • Piranha clean Chips (3:1 H2SO4:H2O2) (30 minutes)
  • Rinse in DI water, N2 gas dry
  • 1 ml of epichlorohydrin, vacuum evaporation (2 hrs)
    • Vial in warm water bath during evaporation
  • Mouse IGG 5 mg/ml in PBS-ET (1x PBS, .05% Tween 20, 5 mM EDTA) (30 min)
  • Block with 5% FBS in PBS-ET (30 min)
  • Flow Target solution
    • Chips were inserted into flow cell
    • 1 ug/ml Protein G in PBS-ET
      • Tangential (40 ul/min) (30 min)
      • Flow-Through (40 ul/min + 20 uL/min)
    • 1 ug/ml protein G + Alkaline Phosphatase
      • Tangential (40 ul/min) (30 min)
      • Flow-Through (40 uL/min + 20 uL/min)
    • Wash with PBS-ET (10 min)
  • Develop chips in 4-MUP solution (10 mM in 1x Tris HCl, a pH 7.8 buffer solution)
  • Measure Fluorescence of developed solution using plate reader (360 nm illumination, 440 nm emission)
Experimental Setup. The orange bar represents the membrane sandwiched between two channels of a flow-cell system.

Results

 

4-MUP first test 12-18-2018. Flow indicates ‘flow-through’, the other is ‘tangential’. Developer solution 4-MUP without any exposure to the membranes is also shown.

Our first test showed an increase in capture of Protein G across the membrane. The background is high, but we can see an increase in fluorescence for the flow-through treatments over the tangential treatments. The amount of fluorescence in the non-alkaline-phosphatase-containing samples is high; the PBS buffer exposure might be contributing to the measurement. We see an increase in signal for the alkaline phosphatase containing sample compared to the Protein G samples alone, controlling for the type of flow.

I reran this experiment last week to try and get a sense of the error.

Error Bars are standard error of the mean (n=2).

 

Normalized to 4-Mup Signal. Error Bars are standard error of the mean (n=2).

This experiment showed a much stronger 4-MUP signal than I found in January, but the conclusion remained the same. Moving forward, we should try to optimize this protocol by running a number of different concentrations of Protein G and flow rates/ratios.

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