#7 An Ultra-Sensitive Diagnostic Kit for Prostate Cancer
NIH Title: Ultra-sensitive Diagnostic Detects fg/mL-pg/mL Pathogen/Disease Protein by Visual Color Change
NIH Reference Number: E-167-2014
Executive Summary:
General Description:
Early detection of cancer biomarkers is an unmet medical need because of the ultra low concentrations of the biomarkers in clinical samples. Current HRP-based ELISA immunoassays measure concentrations above 0.1 ng/mL in a sample. The more sensitive PCR assays require expensive and specialized equipment and reagents, skilled labor, and complex analysis techniques. Some AuNP-based immunoassays under development provided higher sensitivity. However, they were unable to quantitatively detect targets of interest; this limitation was likely associated with the narrow linear range of AuNP.
Researchers developed a novel assay that could overcome the above-mentioned shortcomings. In addition to detecting cancer biomarkers, this technology can potentially be used in early detection of other infectious disease agents such as norovirus or enterovirus.
Scientific Progress:
Researchers at NIH confirmed using UV-vis absorption spectra data that the extinction coefficient of 5 nm AuNPs is much lower than that of larger sized AuNPs. Using transmission electron microscopy they verified that the size of AuNPs grows with increasing concentration of H2O2. They further showed that at the same concentration the 5 nm AuNPs are colorless and the 15 nm AuNPs appears red on an ELISA plate. The experiment employed glucose oxidase (GOx) to generate H2O2. The amount of GOx is proportional to the concentration of detection targets sandwiched through an immunoreaction. When the researchers performed the immunoassay on the polystyrene (PS) substrate in a 96-well plate, the captured target pulled down the antibody–GOx–beads conjugates on the substrate, where the GOx catalyzes the oxidation of glucose to produce H2O2. The obtained H2O2 then induced growth of 5 nm sized AuNPs in the presence of AuCl4–. As a consequence, the colorless AuNP solution turns red. The NIH research team used the readout of absorption band at around 530 nm to determine the amount of the detection targets in solution. To verify the specificity and sensitivity of the assay, the researchers used both PSA-spiked solutions and the clinical samples as targets. They determined that the lowest detectable concentration of PSA for a visible readout was 10 fg/mL. This result was further validated using the readout on a conventional microplate reader. Lastly the researchers performed a comparative study of current invention with the FDA-approved HRP-based ELISA using 12 prostate cancer patient sera and 5 healthy controls. It clearly showed that the AuNP-based immunoassay provides 100% sensitivity and 100% specificity for PSA, while the commercial ELISA kit failed to detect two of the patient sera. Further dilution of patient sera by 10,000-fold confirmed that their detection kit prototype exceeded the commercial PSA ELISA kit by four orders of magnitude. These preliminary data suggest that the researchers have created a novel colorimetric assay that can detect cancer biomarkers from attomolar to picomolar levels. There is a potential that it could replace the current PSA ELISA kit for the early stage prostate cancer diagnostic.
Future Directions:
Strengths:
Weaknesses:
Patent Status:
US Application No. 61/994,622
US Application No. 62/052,866
Patent application in progress
Publications:
Liu D, et al. PMID: 24896231
Inventor Bio: Shawn Chen
Dr. Chen received his BS (1993) and MS (1996) in chemistry from Nanjing University. He then came to the United States, where he completed his PhD degree (1999) in 3 years at the University of Idaho, under the supervision of Prof. Chien M. Wai. He was involved in chelation chemistry of alpha-emitting radionuclides. He then moved to upstate New York and spent 16 months as a postdoc at Syracuse University working with Prof. Jon Zubieta, where he learned crystallography and coordination chemistry of technetium and rhenium. Although his second postdoc at the Washington University in St. Louis was short, he was profoundly influenced by his mentor, Prof. Michael J Welch, who is renowned for applying modern chemistry to the preparation of radiopharmaceuticals in medical imaging.
He joined the University of Southern California as an Assistant Professor in 2002. By working with Prof. Peter Conti and Prof. James Bading, he pioneered multimodality imaging of angiogenesis marker integrin αvβ3. In 2004, he moved to the Molecular Imaging Program at Stanford (MIPS) under the directorship of Prof. Sanjiv Sam Gambhir, and was promoted to Associate Professor in 2008. During his tenure at Stanford, he successfully translated 18F-labeled RGD peptide dimer into clinic for first-in-human imaging studies. In the summer of 2009, he joined the intramural research program of the National Institute of Biomedical Imaging and Bioengineering (NIBIB) as a Senior Investigator and Lab Chief.
He expanded the original PET Radiochemistry Group into the Laboratory of Molecular Imaging and Nanomedicine (LOMIN). LOMIN has three sections: the Chemistry and Radiochemistry Section (CRS); the Biological Molecular Imaging Section (BMIS); and the Theranostic Nanomedicine Section (TNS). CRS has research interests in the development of novel methods for incorporating radionuclides and fluorophores into molecules for the study of biologically important processes. BMIS focuses on identifying disease-specific biomarkers; developing new molecular imaging probes through cellular and molecular-biology-oriented methods; applying molecular probes in multimodality imaging; and characterizing novel imaging and therapeutic agents, both in vitro and in vivo. TNS creates and applies nanobiomaterials and devices that can provide personalized diagnosis, imaging, and therapy.
Dr. Chen has published over 500 peer-reviewed papers (H-index: 88; total citations: > 28,000) and numerous books and book chapters. He sits on the editorial board of over 10 peer-reviewed journals and is the founding editor of journal “Theranostics” (http://www.thno.org/).
NIH Reference Number: E-167-2014
Executive Summary:
- Invention Type: Diagnostic
- Patent Status: Patent pending
- Link: https://www.ott.nih.gov/technology/E-167-2014
- NIH Institute or Center: National Institute of Biomedical Imaging and Bioengineering (NIBIB)
- Disease Focus: Prostate Cancer
- Basis of Invention: A colorimetric assay based on an enzyme-catalyzed gold nanoparticle growth process
- How it works: This assay is dependent on the glucose oxidase (GOx)-catalyzed growth of small sized (5 nm in diameter) gold nanoparticle (AuNP) in the presence of H2O2 and AuCl4–. The target immunoreaction releases H2O2. The AuNPs solution is colorless at low concentration (<10nM), upon H2O2 induction the AuNPs will grow larger in size and the colorless solution can turn red which is a signal that can be quantitatively detected
- Lead Challenge Inventor: Xiaoyuan (Shawn) Chen (NIBIB)
- Inventors: Xiaoyuan (Shawn) Chen (NIBIB), Dingbin Liu (NIBIB)
- Development Stage: The immunoassay has been tested in the lab using both PSA spiked Fetal Bovine Sera and 12 clinical prostate cancer patient sera, which validated the specificity for PSA, comparative analysis with a commercial assay kit further showing superior sensitivity and specificity for PSA detection
-
Novelty:
- Quantitative AuNP-based immunoassay
- Highly sensitive compared to a commercial kit
- Easy readout with naked eyes
- Clinical Applications:
- Early detection of prostate cancer
- Early stage detection of norovirus or enterovirus infection
General Description:
Early detection of cancer biomarkers is an unmet medical need because of the ultra low concentrations of the biomarkers in clinical samples. Current HRP-based ELISA immunoassays measure concentrations above 0.1 ng/mL in a sample. The more sensitive PCR assays require expensive and specialized equipment and reagents, skilled labor, and complex analysis techniques. Some AuNP-based immunoassays under development provided higher sensitivity. However, they were unable to quantitatively detect targets of interest; this limitation was likely associated with the narrow linear range of AuNP.
Researchers developed a novel assay that could overcome the above-mentioned shortcomings. In addition to detecting cancer biomarkers, this technology can potentially be used in early detection of other infectious disease agents such as norovirus or enterovirus.
Scientific Progress:
Researchers at NIH confirmed using UV-vis absorption spectra data that the extinction coefficient of 5 nm AuNPs is much lower than that of larger sized AuNPs. Using transmission electron microscopy they verified that the size of AuNPs grows with increasing concentration of H2O2. They further showed that at the same concentration the 5 nm AuNPs are colorless and the 15 nm AuNPs appears red on an ELISA plate. The experiment employed glucose oxidase (GOx) to generate H2O2. The amount of GOx is proportional to the concentration of detection targets sandwiched through an immunoreaction. When the researchers performed the immunoassay on the polystyrene (PS) substrate in a 96-well plate, the captured target pulled down the antibody–GOx–beads conjugates on the substrate, where the GOx catalyzes the oxidation of glucose to produce H2O2. The obtained H2O2 then induced growth of 5 nm sized AuNPs in the presence of AuCl4–. As a consequence, the colorless AuNP solution turns red. The NIH research team used the readout of absorption band at around 530 nm to determine the amount of the detection targets in solution. To verify the specificity and sensitivity of the assay, the researchers used both PSA-spiked solutions and the clinical samples as targets. They determined that the lowest detectable concentration of PSA for a visible readout was 10 fg/mL. This result was further validated using the readout on a conventional microplate reader. Lastly the researchers performed a comparative study of current invention with the FDA-approved HRP-based ELISA using 12 prostate cancer patient sera and 5 healthy controls. It clearly showed that the AuNP-based immunoassay provides 100% sensitivity and 100% specificity for PSA, while the commercial ELISA kit failed to detect two of the patient sera. Further dilution of patient sera by 10,000-fold confirmed that their detection kit prototype exceeded the commercial PSA ELISA kit by four orders of magnitude. These preliminary data suggest that the researchers have created a novel colorimetric assay that can detect cancer biomarkers from attomolar to picomolar levels. There is a potential that it could replace the current PSA ELISA kit for the early stage prostate cancer diagnostic.
Future Directions:
- Second party validation of comparative analysis
- Optimize the assay to potentially increase the detection efficiency
- Explore the application of this technology in the field of norovirus or enterovirus detection
Strengths:
- Orders of magnitude are more sensitive than most ELISA (detects fg/mL to pg/mL)
- The limit of detection (LOD) for a PSA prototype exceeded the commercial ELISA by more than four orders of magnitude
- Plain sight color-based confirmation does not require complex equipment
- Designed for field use/point-of-care detection
- Besides H2O2, other reducing agents such as ascorbic acid and NADH can also induce AuNP growth, providing additional agents for assay design
Weaknesses:
- Current HRP-based PSA immunoassay has been approved and widely accepted, hence there will be a requirement to change the standard of care
Patent Status:
US Application No. 61/994,622
US Application No. 62/052,866
Patent application in progress
Publications:
Liu D, et al. PMID: 24896231
Inventor Bio: Shawn Chen
Dr. Chen received his BS (1993) and MS (1996) in chemistry from Nanjing University. He then came to the United States, where he completed his PhD degree (1999) in 3 years at the University of Idaho, under the supervision of Prof. Chien M. Wai. He was involved in chelation chemistry of alpha-emitting radionuclides. He then moved to upstate New York and spent 16 months as a postdoc at Syracuse University working with Prof. Jon Zubieta, where he learned crystallography and coordination chemistry of technetium and rhenium. Although his second postdoc at the Washington University in St. Louis was short, he was profoundly influenced by his mentor, Prof. Michael J Welch, who is renowned for applying modern chemistry to the preparation of radiopharmaceuticals in medical imaging.
He joined the University of Southern California as an Assistant Professor in 2002. By working with Prof. Peter Conti and Prof. James Bading, he pioneered multimodality imaging of angiogenesis marker integrin αvβ3. In 2004, he moved to the Molecular Imaging Program at Stanford (MIPS) under the directorship of Prof. Sanjiv Sam Gambhir, and was promoted to Associate Professor in 2008. During his tenure at Stanford, he successfully translated 18F-labeled RGD peptide dimer into clinic for first-in-human imaging studies. In the summer of 2009, he joined the intramural research program of the National Institute of Biomedical Imaging and Bioengineering (NIBIB) as a Senior Investigator and Lab Chief.
He expanded the original PET Radiochemistry Group into the Laboratory of Molecular Imaging and Nanomedicine (LOMIN). LOMIN has three sections: the Chemistry and Radiochemistry Section (CRS); the Biological Molecular Imaging Section (BMIS); and the Theranostic Nanomedicine Section (TNS). CRS has research interests in the development of novel methods for incorporating radionuclides and fluorophores into molecules for the study of biologically important processes. BMIS focuses on identifying disease-specific biomarkers; developing new molecular imaging probes through cellular and molecular-biology-oriented methods; applying molecular probes in multimodality imaging; and characterizing novel imaging and therapeutic agents, both in vitro and in vivo. TNS creates and applies nanobiomaterials and devices that can provide personalized diagnosis, imaging, and therapy.
Dr. Chen has published over 500 peer-reviewed papers (H-index: 88; total citations: > 28,000) and numerous books and book chapters. He sits on the editorial board of over 10 peer-reviewed journals and is the founding editor of journal “Theranostics” (http://www.thno.org/).
