Amplified Detection of Enzyme Activity without Biological Antibodies

Sanku V. Mallik, North Dakota State University Fargo, DMR 0705767

Matrix metalloproteinases (MMPs) are involved in the progression and metastasis of a large number of cancers. The total concentrations of MMP-2 and -9 in cancerous tissues and in serum are usually detected by ELISA. The use of antibodies makes these methods costly for the analysis of MMPs. It is difficult to produce the antibodies on a large scale (> 500 mg). Activity-based detection of these enzymes employing the commercially available kits is time consuming (18–20 h). We are developing a new, rapid, in vitro detection strategy for active forms of MMP-2 and -9 which does not require the use of an antibody and incorporate “ELISA-like” signal amplification protocol employing lipid nanoparticles (liposomes). These nanoparticles are easy to produce, even on a large scale (> 500 mg).

The scheme for the amplified detection of cancer cell secreted active MMP-9 without using antibodies. A rapid formation of brown color was observed in the presence conditioned cell culture media from the MCF7 cells. HT-29 cells do not secrete large amount of the enzyme and the color formation was not rapid or intense. The assay solution remains clear in the presence of the media before cell culture.

Liposomes with synthesizedlipo-peptides encapsulatinghorse radish perioxidase (HRP)

MMP-9

Detect color by absorbance(410 nm)

OPDChromogenic HRP substrate

Excess OPD

0 5 10 15 20 25 300.0

0.3

0.6

0.9

1.2

Medium only

HT-29

Abs

orba

nce

Cha

nge

(410

nm

)

Time (min)

MCF-7

Broader Impacts:

NDSU has started a summer internship program for under-represented minority students. An undergraduate student (Jane Leung, NDSU) worked under Mallik’s supervision this summer. She is majoring in Biochemistry.

A local high school student (Brett Johnson, Shanley High School, Fargo, ND) worked in Mallik’s laboratory during the summer months to get trained on peptide synthesis, purification, liposome formation, UV-Vis and fluorescence spectroscopy.

Amplified Detection of Enzyme Activity without Biological Antibodies

Sanku V. Mallik, North Dakota State University Fargo, DMR 0705767

Amplified Detection of Enzyme Activity without Biological Antibodies

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) Amplified detection of cell secreted matrix metalloproteinase-9 without using antibodies. Banerjee, J.; Hanson, A. A.; Nyren-Erickson, E.; Ganguly, B.; Wagh, A.; Muhonen, W. W.; Shabb, J. B.; Srivastava, D. K.; Mallik, S. J. Chem. Soc. Chem. Commun. 2010, 46, 3209-3211.

(2) Microwave assisted synthesis of triple helical collagen-mimetic peptides. Banerjee, J.; Hanson, A. J.; Mallik, S. Nature Protocols 2010, 5, 39-50.

(3) Release of liposomal contents by cell-secreted matrix metalloproteinase-9. 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. 2009, 20, 1332-1339.

P

O-O

O

N+OH

O

O

O

OPOPC (70 mol%, commercially available)

NH

OGPQG-IAGQR(GPO)4GG

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

Sanku V. Mallik, North Dakota State University Fargo, DMR 0705767