Amplified Detection of Enzymes without Biological Antibodies

Sanku Mallik, North Dakota State University Fargo, DMR 0705767

Usually, small amounts of pathogenic enzymes are detected employing ELISA. Although this technique is very sensitive, it requires the use of biological antibodies. In addition to the expenses involved, since the antibodies are large biomolecules, proper handling and storage are required to maintain the activity. We are working on a new methodology for amplified detection of enzymes (similar to ELISA) without the use of biological antibodies. We have demonstrated the proof-of-concept by detecting low amounts (40 ng) of the cancer associated enzyme matrix metalloproteinase-9 (MMP-9) by using appropriately formulated liposomes.

The scheme for the amplified detection of MMP-9 without using biological antibodies. A rapid formation of brown color was observed in the presence of 40 ng of the enzyme; the assay solution remains clear in the absence of the enzyme.

Liposomes with synthesizedlipo-peptides encapsulatinghorse radish perioxidase (HRP)

MMP-9

Detect color by absorbance(410 nm)

OPDChromogenic HRP substrate

Excess OPD

Broader Impacts:

NDSU has started a summer internship program with Mississippi Valley State University (MVSU, an undergraduate institution in Itta Bena, MS). An Afro-American undergraduate student (Edna Lampkin) is working under Mallik’s supervision this summer. She will graduate from MVSU in 2010. She is also being mentored to enroll in the Graduate Program of the Pharmaceutical Sciences Department at NDSU following her graduation from MVSU.

A high school student (Sibinee Jokela) from rural North Dakota (Watford City High School, Watford City) is currently working in Mallik’s laboratory to get trained on liposome formation, UV-Vis and fluorescence spectroscopy. She is involved in conducting the optimization studies for the liposome-based enzyme detection methodology.

Amplified Detection of Enzymes without Biological Antibodies

Sanku Mallik, North Dakota State University Fargo, DMR 0705767

Amplified Detection of Enzymes without Biological Antibodies

Sanku Mallik, North Dakota State University Fargo, DMR 0705767

Composition of the liposomes: Liposomes were prepared in 25 mM HEPES buffer, pH = 8.0 with total lipid concentration of 1 mg/mL. The structures of the lipids used in the liposomes are shown below.

(1) Banerjee, J.; Hanson, A. J.; Gadam, B.; Elegbede, A. I.; Tobwala, S.; Ganguly, B.; Wagh, A.; Muhonen, W. W.; Law, B.; Shabb, J. B.; Srivastava, D. K.; Mallik, S. Bioconjugate Chem. in press.

(2) Elegbede, A. I.; Banerjee, J.; Hanson, A. A.; Tobwala, S.; Ganguli, B.; Wang, R.; Lu, X.; Srivastava, D. K.; Mallik, S. J. Am. Chem. Soc. 2008, 130, 10633-10642.

P

O-

O

O

N+O

H

O

O

O

O

POPC (70 mol%, commercially available)

NH

O

GPQG-IAGQR(GPO)4GG

(30 mol%, synthesized)cleavage sitefor MMP-9(target enzyme)