Method for the detection of enzymatic activity with magnetically functionalized substrates

A method for detecting the integrity of a substrate for an enzyme, consisting of the steps in order:

• selecting at least one macromolecule, wherein at least one of the at least one macromolecule is selected from the group consisting of a protein, a polypeptide, a modified protein, a modified polypeptide, a glycoprotein, a lipoprotein, collagen, one or more collagen fragments, a mixture of collagen with one or more collagen fragments, and gelatin;
• creating the substrate, the substrate is a plurality of magnetic particles covalently bound, by means of a process that involves silanization, to the at least one macromolecule;
• incorporating the substrate in a sample;
• measuring through a magnetic based detection method a magnetic property of the sample to obtain a first measurement;
• measuring, through the magnetic based detection method and after obtaining the first measurement, a magnetic property of the sample to obtain a second measurement;
• comparing the first measurement to the second measurement;
• and
• determining there is degradation of the substrate by an enzyme in the sample if there is a change between the first measurement and the second measurement;
• wherein said change is indicative of at least one of (a) aggregation of said plurality of magnetic particles, (b) dispersion of said plurality of magnetic particles, (c) a change in the size of said plurality of magnetic particles, and (d) a change in the size of said substrate.

A method for detecting the activity of at least one enzyme in a sample, consisting of the steps in order:

• selecting at least one macromolecule, wherein at least one of the at least one macromolecule is selected from the group consisting of a protein, a polypeptide, a modified protein, a modified polypeptide, a glycoprotein, a lipoprotein, collagen, one or more collagen fragments, a mixture of collagen with one or more collagen fragments, and gelatin;
• forming a substrate, the substrate is a plurality of magnetic particles covalently bound, by means of a process that involves silanization, to the at least one macromolecule;
• incorporating the substrate in the sample;
• measuring through a nuclear magnetic resonance-based detection method a T1 or T2 relaxation time of the sample to obtain a first measurement;
• measuring, through the nuclear magnetic resonance-based detection method and after obtaining the first measurement, the T1 or T2 relaxation time of the sample to obtain a second measurement;
• comparing the first measurement to the second measurement;
• and
• determining there is an aggregation of the magnetic particles as a consequence of enzymatic digestion of the substrate thus indicating the presence of enzymatic activity in the sample.