Immobilized Enzymes — Multifunctional Reagents: Which statement is NOT a method excluded by the phrase “using multifunctional reagents” for producing immobilized enzymes?

Introduction:
“Multifunctional reagents” (often bifunctional crosslinkers like glutaraldehyde or carbodiimides with activators) provide chemical handles to tether enzymes to supports or to each other. The question asks which listed method is not applicable; in fact, the first three options all leverage multifunctional chemistry in common immobilization workflows.

Given Data / Assumptions:

• Crosslinkers can connect enzyme–enzyme (aggregated enzymes) or enzyme–support.
• Supports may be pre-activated (functionalized) to react with enzyme residues.
• Physical entrapment is considered separately from chemical crosslinking.

Concept / Approach:

Typical strategies include (i) adsorption followed by crosslinking to lock enzymes onto a surface, (ii) support activation (e.g., epoxy, aldehyde, NHS esters) that covalently bind enzyme groups, and (iii) direct enzyme–enzyme crosslinking to form insoluble aggregates (CLEAs). All of these use multifunctional chemistry. Therefore, none of the listed methods (A–C) are excluded by the definition; the correct choice is “None of the above.”

Step-by-Step Solution:

Verification / Alternative check:

Process schemes for carriers like agarose–aldehyde, EDC/NHS-activated matrices, or glutaraldehyde-CLEAs confirm the use of multifunctional chemistry.

Why Other Options Are Wrong:

A–C are indeed methods using multifunctional reagents. E is a different immobilization class (physical entrapment) and not what the question is targeting for exclusion among A–D.

Common Pitfalls:

Confusing “entrapment” (no covalent crosslinker needed) with “chemical immobilization.” Read wording carefully.

Final Answer:

None of the above (all listed approaches are valid ways involving multifunctional reagents)