Reduction chemistry-assisted nanopore determination method for immunoglobulin isotypes

Area of Science:

• Biochemistry
• Nanotechnology
• Immunology

Background:

• Immunoglobulins (IgG and IgM) are vital for immune defense.
• Deficiencies or altered concentrations of IgG and IgM indicate immune disorders.
• Accurate and sensitive detection methods are crucial for diagnosing immune deficiencies.

Purpose of the Study:

• To develop a reduction chemistry-assisted nanopore method for quantifying IgG and IgM.
• To differentiate between IgG and IgM in blood serum using nanopore sensing.
• To establish a label-free, single-molecule approach for diagnosing immunoglobulin-related diseases.

Main Methods:

• Utilized TCEP (tris(2-carboxyethyl)phosphine) for disulfide bond reduction, cleaving Ig proteins into fragments.
• Employed an αHL nanopore to detect distinguishable current signals from separated polypeptide fragments.
• Integrated molecular dynamics (MD) simulations to understand fragment capture mechanisms via electrostatic potentials and H-bonds.

Main Results:

• Achieved distinguishable current signals corresponding to cleaved IgG and IgM fragments within an αHL nanopore.
• Successfully differentiated between IgG and IgM in blood serum samples without nanopore adsorption or clogging.
• Demonstrated label-free, single-molecule sensing with high sensitivity and selectivity.

Conclusions:

• The reduction chemistry-assisted nanopore method provides a robust platform for IgG and IgM determination.
• This approach offers a promising diagnostic tool for immune deficiency disorders characterized by immunoglobulin variations.
• The method's label-free and single-molecule nature enhances its utility in clinical diagnostics.