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Fluorescence Assays for Serine Proteases

Serine, cysteine, and aspartic proteases are markers for the ability of many cancers to grow and to form metastases. Several serine, cysteine and aspartic proteases are over-expressed by numerous cancer cell lines. Elevated expression levels of urokinase and several other components of the plasminogen activation system are found to be correlated with tumor malignancy.

This diagnostic assay is comprised of protease-sensitive cleavage sequences for up to 30 proteases, which are used as linkers between two fluorophores (nanoparticles and/or organic or inorganic dyes). Depending on the nanoparticles and dyes used, optical (fluorescence), magnetic (MRI), and x-ray imaging of the tumor location and extension, together with quantitative determination of the proteases’ activities, can be performed. This assay method is useful for the quantitative determination of any enzyme in blood and tissue that can cleave a specific linker between two fluorophores.

-Assays can be performed in vivo & in vitro
-Eliminates the need for invasive biopsies
-More rapid diagnostic
-Much less time demanding
-Easier to use
-Less costly
-Cancer can be detected in earlier stages than using conventional technologies (e.g. classic MRI or mammograms, PSA test etc.)

-Quantitative determination of the protease-activities of all cancers that over-express one or several proteases (protease signature).
-Coupling of optical (fluorescence), magnetic (MRI), and x-ray imaging of the tumor location and extension, together with a quantitative determination of the urokinase activity.
-Application within the brain tissue (intercranial infiltration).
-Method is suitable to be applied within the brain tissue (intercranial infiltration)

-U.S. patent #8,969,027 issued on March 3, 2015.
-International patents issued in Australia, France, Germany and United Kingdom.
-International Patent Protection filed in Canada in September 2010.


Initially, we tested 32 separate participants in various stages of breast or non-small cell lung cancer. Data was collected from 20 people with breast cancer -- ranging in age from 36 to 81 years old -- and 12 people with lung cancer -- ranging in age from 27 to 63 years old. Twelve people without cancer were also tested as a control group. This group ranged in age from 26 to 62 years old.

Blood samples from each participant were tested three times. Analysis of the data showed a 95 percent success rate in detecting cancer in participants, including those with breast cancer in stages 2 and 3 and those with lung cancer in stages 1 and 2.

These studies were followed by trials with larger patient cohorts (> 250 cancer patients and 200 healthy human subjects). Analysis of the data indicated that breast and lung cancer could be detected in stage 1 with 98 percent success rate. Thyroid cancer was detectable with 95 percent success in stage 1 as well. The recent trials, performed at Kunming Medical University, China, clearly indicate that this detection method is ideal for early cancer recognition. It can also be used to follow the progress of cancer treatment, and to detect the recurrence of cancer earlier than with other methods.

The test is adapted to plate reader technology and requires 25 microliters of blood serum per enzyme. A result can be obtained within 30 min. The detection of other solid tumors (prostate, ovarian, pancreatic) is currently being tested.

Additional Details


Kansas State University

Intellectual Property Protection

Pending Patent

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