#4 An apparatus containing microarray binding sensors for gene expression and nucleic acid binding assays
NIH Title: Apparatus for Microarray DNA Binding Sensors Using Carbon Nanotube Transistors
NIH Reference Number: E-056-2007
Executive Summary:
General Description:
Traditional DNA microarray used in clinical applications is laborious, time-consuming, and requires complex protocols involving large amounts of reagents. In addition, results have limited sensitivity and suffer from optical degradation. There is a need for a method of detecting DNA that overcomes these obstacles. In this invention, the researchers provide for a method of electronically detecting biological probe-target binding. They also describe a method of growing carbon nanotubes using iron nanoparticle catalysts, a lithographic process for fabricating electronic chips, hardware and software for data retrieval and characterization procedures.
Scientific Progress:
The researchers describe a method of fabricating transistor DNA binding sensors by growing carbon nanotubes mats on iron-coated silicon oxide substrates. They have demonstrated that top-gated transfer characteristic through Ag/AgCL counter, (i.e. a shift in threshold voltage which signified DNA hybridization) showed high repeatability upon device washing and change of buffer solution. In order to verify that the threshold shift in electrical signal was due to target binding, not non-specific binding, they first used a fluorescent labeled target DNA as a positive control at a high salt concentration (required for hybridization) and then replaced the buffer to a low salt concentration and were successful in measuring a threshold shift in the electrical measurement. The researchers also used a simple model to estimate the amount of DNA that was bound to the sensor surface. To determine the sensitivity and selectivity of the device, they measured the response at different target concentrations, and concluded that the sensitivity of the assay is between 10 to 100 nM. Improvement of the sensitivity is feasible through improved probe immobilization, optimized hybridization protocols, and usage of single CNT transistors. These data using the device prototype indicate that the sensor is a useful tool for DNA-DNA detection. It is also less prone to false positives, is highly specific, and is sensitive to concentrations as low as 30 nM of 61-mer DNA. The researchers also propose that this device could be used for other biomolecules such as nucleosides, proteins, drugs, and cells using aptamer probes.
Future Directions:
Strengths:
Weaknesses:
Patent Status:
U.S. Pat: 8,017,938 issued 19 March 2011
PCT Application No. PCT/US2007/06809 filed on 19 Mar 2007
European Patents also issued in France, Germany, Ireland, and Great Britain
Publications:
Pandana H, Aschenbach KH, Lenski DR, Fuhrer MS, Khan J, Gomez RD. A versatile biomolecular charge-based sensor using oxide-gated carbon nanotube transistor arrays. IEEE Sens J. 2008 June;8(6):655-660. [DOI: 10.1109/JSEN.2008.922724]
Aschenbach KH, Pandana H, Lee J, Khan J, Fuhrer M, Lenski D, Gomez RD. Detection of nucleic acid hybridization via oxide-gated carbon nanotube field-effect transistors. Proceedings of SPIE MEMS and Nanotechnologies, Volume 6959 (2008). [DOI: 10/1117/12.778531]
Subramanian, K. Aschenbach, J. Evangelista, M. Najjar, W. Song and R.D. Gomez, “Rapid, sensitive and label free detection of Shiga-toxin producing E. coli O157 using carbon nanotube biosensors, “Biosensors and Bioelectronics”, 32 (2012), 69-75.
Inventor Bio:
Dr. Khan obtained his bachelor's degree in 1984 and his master's degrees in 1989 in immunology and parasitology at England's University of Cambridge. He subsequently obtained his MB BChir (M.D. equivalent) there and the postgraduate degree of MRCP (Membership of the Royal College of Physicians), equivalent to board certification in the United States. After clinical training in internal medicine and pediatrics as well as other specialties, he received a Leukemia Research Fellowship. In May 2001, Dr. Khan joined the Pediatric Branch, NCI, as a tenure track investigator. Dr. Khan and colleagues have published a model for diagnosis of cancer using artificial neural networks (ANN), a form of artificial intelligence, and microarray technology. Dr. Khan is currently the Deputy Chief of the Genetics Branch and Senior Investigator and Head of the Oncogenomics Section in the Genetics Branch at the National Cancer Institute, NIH, and has worked at the NIH for the last 20 years. He has one of the largest groups in the NIH campus in applying Next Generation Sequencing (NGS) strategies to investigate cancers. He is the principal investigator of a clinical protocol to identify germ line and somatic mutations in cancers using NGS. He is developing a clinical protocol utilizing, exome, panel and transcriptome sequencing to enable precision therapy. This will be done in a CLIA laboratory of which he is the co-director. He is a Co-PI of the TARGET neuroblastoma efforts as well as a joint Children’s Oncology Group-NCI NGS project for rhabdomyosarcoma. He is a board certified in pediatrics and pediatric hematology and oncology, and attends on clinical service twice a year at the pediatric oncology branch.
Inventor Bio:
Romel D. Gomez is a Professor of Electrical and Computer Engineering at the University of Maryland. He teaches undergraduate and graduate level courses. As an associate chair, strengthened the ECE curriculum in communications, embedded systems, cyber security, and power. He is also a researcher in the areas of micromagnetism and biosensing and co-authored over 80 peer-reviewed publications, several book and book chapters, and 3 U.S. Patents. He is the co-inventor of several other devices including a low-cost Dengue detection apparatus based on LAMP, and instrumentation system for K-12 scientific experiments. He received the ECE Corcoran Award for education, the NSF CAREER award, the Clark School Kent Faculty Award, the Clark School Keystone Professorship, the Clark School Faculty Service Award, and the Distinguished Alumni in Science and Technology of the University of the Philippines. He earned his PhD from the University of Maryland in 1990, MS from Wayne State in University in 1984 and BS from the University of the Philippines in 1980, all in Physics.
NIH Reference Number: E-056-2007
Executive Summary:
- Invention Type: Diagnostic
- Patent Status: Patent granted
- Link: https://www.ott.nih.gov/technology/e-056-2007
- NIH Institute or Center: National Cancer Institute (NCI)
- Disease Focus: Cancer, infectious diseases
- Basis of Invention: Microarray binding sensors contain biological probe materials and carbon nanotube transistors (CNTs)
- How it works: Each transistor is associated with a distinct probe and interaction is measured by the transconductance between the source and drain electrodes before and after the hybridization event which facilitates binding
- Lead Challenge Inventor: Javed Khan (NCI)
- Inventors: Javed Khan (NCI); Romel Gomez (UMD)
- Development Stage: Prototype available
-
Novelty:
- Complete isolation of the CNTs from chemical reactions concomitant with probe immobilization and target capture
- It uses a gate oxide overlayer on top of the carbon nanotubes, CNT functions only as charge sensors
- Eliminates the need for chemical labeling and enzymatic manipulation
-
Clinical Applications:
- High-throughput monitoring of genome-wide DNA and mRNA copy number changes for research, diagnostic and prognostic uses
- Sequencing entire genes, replacing the current gel-based sequencing techniques
- Monitoring miRNA levels in cancer
- Detecting DNA copy number changes and deletions in chromosomal regions
- Detecting Single Nucleotide Polymorphisms
- Identifying targets of transcription factors
- Detecting pathogens in the air, blood and body secretions
General Description:
Traditional DNA microarray used in clinical applications is laborious, time-consuming, and requires complex protocols involving large amounts of reagents. In addition, results have limited sensitivity and suffer from optical degradation. There is a need for a method of detecting DNA that overcomes these obstacles. In this invention, the researchers provide for a method of electronically detecting biological probe-target binding. They also describe a method of growing carbon nanotubes using iron nanoparticle catalysts, a lithographic process for fabricating electronic chips, hardware and software for data retrieval and characterization procedures.
Scientific Progress:
The researchers describe a method of fabricating transistor DNA binding sensors by growing carbon nanotubes mats on iron-coated silicon oxide substrates. They have demonstrated that top-gated transfer characteristic through Ag/AgCL counter, (i.e. a shift in threshold voltage which signified DNA hybridization) showed high repeatability upon device washing and change of buffer solution. In order to verify that the threshold shift in electrical signal was due to target binding, not non-specific binding, they first used a fluorescent labeled target DNA as a positive control at a high salt concentration (required for hybridization) and then replaced the buffer to a low salt concentration and were successful in measuring a threshold shift in the electrical measurement. The researchers also used a simple model to estimate the amount of DNA that was bound to the sensor surface. To determine the sensitivity and selectivity of the device, they measured the response at different target concentrations, and concluded that the sensitivity of the assay is between 10 to 100 nM. Improvement of the sensitivity is feasible through improved probe immobilization, optimized hybridization protocols, and usage of single CNT transistors. These data using the device prototype indicate that the sensor is a useful tool for DNA-DNA detection. It is also less prone to false positives, is highly specific, and is sensitive to concentrations as low as 30 nM of 61-mer DNA. The researchers also propose that this device could be used for other biomolecules such as nucleosides, proteins, drugs, and cells using aptamer probes.
Future Directions:
- Microfluidics
- Developing hand held device for working the field
- Signal processing Pattern recognition algorithm
- Detection of protein levels using antibodies or Aptamers
- Mutational detection
Strengths:
- Increased sensitivity and ease of use
- Elimination of chemical labeling and enzymatic manipulation
- Ability to streamline performance of numerous laboratory procedures, including sequencing of DNA and identification of miRNA levels in cancer
- Less expensive and more efficient
- Rapid results
Weaknesses:
- Further miniaturization of the device may be needed
- Loading and hybridization conditions need to be optimized for sensitivity improvement
Patent Status:
U.S. Pat: 8,017,938 issued 19 March 2011
PCT Application No. PCT/US2007/06809 filed on 19 Mar 2007
European Patents also issued in France, Germany, Ireland, and Great Britain
Publications:
Pandana H, Aschenbach KH, Lenski DR, Fuhrer MS, Khan J, Gomez RD. A versatile biomolecular charge-based sensor using oxide-gated carbon nanotube transistor arrays. IEEE Sens J. 2008 June;8(6):655-660. [DOI: 10.1109/JSEN.2008.922724]
Aschenbach KH, Pandana H, Lee J, Khan J, Fuhrer M, Lenski D, Gomez RD. Detection of nucleic acid hybridization via oxide-gated carbon nanotube field-effect transistors. Proceedings of SPIE MEMS and Nanotechnologies, Volume 6959 (2008). [DOI: 10/1117/12.778531]
Subramanian, K. Aschenbach, J. Evangelista, M. Najjar, W. Song and R.D. Gomez, “Rapid, sensitive and label free detection of Shiga-toxin producing E. coli O157 using carbon nanotube biosensors, “Biosensors and Bioelectronics”, 32 (2012), 69-75.
Inventor Bio:
Dr. Khan obtained his bachelor's degree in 1984 and his master's degrees in 1989 in immunology and parasitology at England's University of Cambridge. He subsequently obtained his MB BChir (M.D. equivalent) there and the postgraduate degree of MRCP (Membership of the Royal College of Physicians), equivalent to board certification in the United States. After clinical training in internal medicine and pediatrics as well as other specialties, he received a Leukemia Research Fellowship. In May 2001, Dr. Khan joined the Pediatric Branch, NCI, as a tenure track investigator. Dr. Khan and colleagues have published a model for diagnosis of cancer using artificial neural networks (ANN), a form of artificial intelligence, and microarray technology. Dr. Khan is currently the Deputy Chief of the Genetics Branch and Senior Investigator and Head of the Oncogenomics Section in the Genetics Branch at the National Cancer Institute, NIH, and has worked at the NIH for the last 20 years. He has one of the largest groups in the NIH campus in applying Next Generation Sequencing (NGS) strategies to investigate cancers. He is the principal investigator of a clinical protocol to identify germ line and somatic mutations in cancers using NGS. He is developing a clinical protocol utilizing, exome, panel and transcriptome sequencing to enable precision therapy. This will be done in a CLIA laboratory of which he is the co-director. He is a Co-PI of the TARGET neuroblastoma efforts as well as a joint Children’s Oncology Group-NCI NGS project for rhabdomyosarcoma. He is a board certified in pediatrics and pediatric hematology and oncology, and attends on clinical service twice a year at the pediatric oncology branch.
Inventor Bio:
Romel D. Gomez is a Professor of Electrical and Computer Engineering at the University of Maryland. He teaches undergraduate and graduate level courses. As an associate chair, strengthened the ECE curriculum in communications, embedded systems, cyber security, and power. He is also a researcher in the areas of micromagnetism and biosensing and co-authored over 80 peer-reviewed publications, several book and book chapters, and 3 U.S. Patents. He is the co-inventor of several other devices including a low-cost Dengue detection apparatus based on LAMP, and instrumentation system for K-12 scientific experiments. He received the ECE Corcoran Award for education, the NSF CAREER award, the Clark School Kent Faculty Award, the Clark School Keystone Professorship, the Clark School Faculty Service Award, and the Distinguished Alumni in Science and Technology of the University of the Philippines. He earned his PhD from the University of Maryland in 1990, MS from Wayne State in University in 1984 and BS from the University of the Philippines in 1980, all in Physics.
