PROTEIN CHARGE

A protein's charge depends on pH

pH > pI → net negative

Each protein has an isoelectric point, pI, the pH at which its net charge is zero. Above the pI it carries net negative charge, and below it net positive. A protein with pI 5 is negative in pH 7.4 buffer, which raises an NMOS threshold when it binds.

The sign of the signal depends on the target's pI and the buffer pH.

PICK THE CONDITIONS

Match the buffer to the target

pI below the pH Net negative: an NMOS threshold rises on binding

pI above the pH Net positive: an NMOS threshold falls on binding

pI near the pH Almost no net charge, so almost no signal

Buffer pH It also has to keep the receptor working, so choose a compromise

Both the sign and the size of the signal come from pI versus pH.

A MODEL SYSTEM

Biotin and streptavidin

biotin + streptavidin

Streptavidin binding to a biotin-coated gate is a classic test of a new BioFET. The bond is strong and highly specific, so it isolates the sensor's behavior from the chemistry's. Streptavidin has a pI near 5 to 6, so in neutral buffer it adds negative charge.

A well-understood pair separates sensor problems from chemistry problems.

DOSE RESPONSE

Signal rises with concentration

ΔVth = ΔV_max · c / (c + K_D)

Combine the Langmuir curve with the charge signal: ΔVth = ΔV_max · c/(c + K_D), where ΔV_max is the shift with every receptor occupied. Plotting ΔVth against concentration gives a calibration curve you can invert to read an unknown sample.

A calibration curve turns millivolts into concentration.

REPORTED PERFORMANCE

What published devices claim

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Wide range

Many reports span several decades of concentration

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Low limits

Femtomolar to picomolar detection is often reported, usually in diluted buffer

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Read carefully

Results in low-salt buffer may not carry over to blood or serum

Always ask what sample the claimed limit was measured in.

UNIT 21 STUDY COMPLETE

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Ready for the Fab Challenge?

You've covered how antibody-based BioFETs read a protein, and why buffer pH matters.