Integrated Circuit Chromatography Chip (I3C)
by
Marlon Williams, C.E.O.
Williams Integrated Technologies, LLC, Delaware C-Corp
Table of Contents
I. Introduction
II. Significance of Problem
III. Rationale for Research
IV. Scope
V. Growth Potential
VI. Implementation (Work Plan)
VII. Proposed Budget
VIII. Research Facilities
IX. Key Personnel
X. Conclusion
Abstract
Cancer continues to be one of the biggest killers worldwide, and the number of new cases each year will grow for the foreseeable future, with nearly 24 million new cases annually expected by 2030. These cases not only can lead to pain and death among individuals but also are prohibitively expensive to diagnose, treat and monitor. The diagnostics tests alone for monitoring a cancer patient’s disease state can cost $240,000 annually. Next-generation technologies are needed in cancer treatment to increase patient wellbeing, lower costs and reduce the overall burden on the patient and healthcare system.
Williams Integrated Technologies, LLC (“WIT” and “the Company”) is developing a new technology to monitor and track cancer in the body through an innovative, first-of-its-kind non-invasive device. The Integrated Circuit Chromatology Device (I3C) represents a breakthrough in the treatment of cancer, as a small, handheld and portable device that can track the response of patients to cancer therapy in real-time as well as monitor a patient’s remission status. The I3C uses a special porous biosilicon to test for the presence of specific cancer antigens in the blood, relaying results via a proprietary near infrared (NIR) transceiver to the device. The device then uploads the data to a server for analysis and monitoring real-time response to treatment as well as tracking remission and recurrence.
The I3C would replace the current complex, expensive, and time-consuming process for tracking cancer treatment. Patients currently must make regular, expensive visits to a doctor or lab and undergo blood tests, with results sometimes taking weeks to get. Other cancer-tracking methods include X-rays, CT scans, PET scans, mammography, and ultrasound technology, which are even more expensive and have limited availability. As a result, these procedures are administered infrequently, which impedes timely tracking of vital recovery and remission markers. Importantly, the I3C is non-invasive, and once fully developed, will be administered by holding the device against the surface of the skin. The novel NIR transceiver is also designed to bypass the limitations of skin penetration to ensure that signal is not lost during its application, reducing the possibility of error.
The market for oncology diagnosis and treatment was valued at $286 billion globally in 2021 and is projected to more than double to over $581 billion by 2030, according to a report from Precedence Market Research. North America accounts for nearly half of the global market. The I3C device will not only substantially speed up testing results but can quickly and inexpensively treat large numbers of patients, increasing access to testing and substantially lowering the ongoing costs of testing and monitoring progress, thereby reducing the overall burden on individuals, the healthcare industry, insurance companies and the economy in the U.S. and globally.
The data obtained by the I3C device can also be analyzed to track and monitor macro-level drug-response trends to develop better future outcomes. On a broad impact level, the technology is also designed to be adaptable for the detection of any proteins in the blood, so its application can be expanded to the detection of other diseases for which non-invasive testing methods are not available, such as Alzheimer’s disease and others. The technology, when fully developed, will create market opportunities in multiple multibillion-dollar global industries.
WIT is currently in Phase I of development for the I3C technology and device. Phase I will thoroughly investigate the stability of the porous biosilicon and the ability of the novel NIR transceiver to accurately measure the amount of cancer antigens or markers in the body. The biosilicon has already been shown through prototype testing to display generic signals from in Simulated Human Plasma. Investigating if the prototype will show specific results for cancer antigens will be achieved within an estimated 12 months of receiving Phase I study funding.
WIT is based in Florida and is led by a high-performing team of engineers and scientists who have the necessary experience to execute this type of research and bring the product to market. WIT was founded by Marlon Williams, a biomedical engineer with over 20 years of experience in systems and network analysis. He is an expert in laser systems for the CT, MRI, PET, Linac, and Cyberknife imaging systems and routinely interacts with oncologists and hospital directors. The team also includes Dr. Sunil Krishnan of the Mayo Clinic Jacksonville, who is an expert in radiation therapy and patient-centered research via clinical trials. In addition, Will Blake, the senior engineer, has over 20 years of experience in embedded systems and firmware, including expertise in diagnostic devices. The advisory team also includes a microbiologist, professors and researchers, and consultants with access to state-of-the-art laboratories and equipment.
Funding of $4 million is currently being sought to facilitate Phase I of development, which is expected to result in an I3C prototype within 18 months, followed by clinical testing and the FDA application process in years 4-5. Within 5 years, the Company expects the I3C to be a global standard in cancer management and a growing corporate entity that positively impacts Florida’s economy and research institutions.
The aim of this research is to:
Commence full-scale R&D, clinical trials and device development with simulated human plasma
Commence full-scale R&D, clinical trials and device development with simulated human plasma Create a next-generation prototype for clinical trials
Create a next-generation prototype for clinical trialsFile patent protections and attain other IP protections
File patent protections and attain other IP protectionsLaunch FDA application and trial process
Launch FDA application and trial processMarket the I3C device to doctors, hospitals, insurance companies and others
Market the I3C device to doctors, hospitals, insurance companies and othersDevelop addition applications and identify ancillary markets with unmet testing needs
Develop addition applications and identify ancillary markets with unmet testing needsBecome recognized as a leading global screening device manufacturer
Become recognized as a leading global screening device manufacturerI. Introduction
Cancer patients spend on average up to $240,000 annually on the costs of diagnostic tests to monitor their disease state1. Currently, the routine method of testing how patients respond to cancer treatment relies on in vitro laboratory tests that detect specific cancer antigens present in the blood of patients. Accurate quantification of these cancer markers is imperative in determining how well the patient is responding to their treatment regimen. Currently, cancer patients undergo repeated blood tests to monitor their response to treatment. As a patient goes from one drug to another in the quest to find optimal response to a specific therapy, the patient becomes exhausted both physically and financially. These additional costs also become a burden to insurance companies that provide coverage of repeated tests and trials of various cancer drugs until a drug that the patient responds well to is found. Repeated trips to the patient’s healthcare provider also become necessary, adding to the load of the cancer patient’s therapeutic process of recovery.
An approach that allows cancer patients to self-administer a test for monitoring response to cancer treatment can substantially eliminate these accumulated costs of repeated diagnostic tests, and reduce the frequency of trial and error of various cancer treatment drugs. While such diagnostic devices exist for monitoring diseases like diabetes and blood pressure, there are currently no convenient at-home diagnostic tests that a patient can use to monitor their therapeutic progress. The WIT proposes to address this gap through introduction of a non-invasive, portable device paired with a special biomaterial that can regularly track the response of patients to cancer therapy in real-time, or to be used as maintenance detection for patients in remission. Once developed, the Integrated Circuit Chromatography Device (I3C) can be used at home by patients to accurately and sensitively measure the presence of specific cancer markers in their blood. The data obtained from this device will then be automatically transmitted to a database for immediate access by physicians. Collectively, regular use of the device will provide personalized data of real-time response to treatment, highlighting drug response trends that can impact prescribing decisions by physicians.
The I3C device contains a unique Near Infrared Transceiver that has been developed by the WIT. This transceiver measures the signal generated from porous silicon, a non-toxic derivative of elemental silicon with optical conductance properties that is naturally present in the human body, conjugated to a selective agent that is recognized by cancer antigens or biomarkers. The amount of cancer antigens present clouds up the signal generated by porous silicon, and this is detected by the transceiver. The change in signal is directly proportional to the amount of antigen present in the sample, in turn indicating the disease state of the individual.
Importantly, the I3C is non-invasive, and once fully developed, it will be administered by holding the device against the surface of the skin. The novel near infrared transceiver is designed to bypass the limitations of skin penetration to ensure that signal is not lost during its application, reducing the possibility of error. The novel technology of the I3C paired with porous silicon mirrors can be used for diagnosis, monitoring treatment response, and detecting recurrence of cancers. On a broad impact level, the technology is designed to be adaptable for the detection of any proteins in the blood, so its application can be expanded to the detection of other diseases for which non-invasive testing methods are not available, such as Alzheimer’s disease.
II. Significance of Problem
The human body is made up of millions of building blocks called cells. Cancer occurs when one or more of these cells breaks free from its normal restraints and starts to multiply in an abnormal uncontrolled way. This can happen to more or less any type of cell (blood, bone, skin, etc). The environment we live in, our diets, genetics, smoking, and drinking all play a major role in the growth and spread of these abnormalities. Unfortunately for us, these diseases all have different symptoms and often respond differently to treatment.
Each cell in our body has a control (nucleus) center, which gives the cell instructions as to how it should behave. The instructions tell the cell what type it should be, where it should live in the body, and when it should make copies of itself. This control center contains a blueprint for all these instructions written in special code. Each coded instruction is called a gene.
In cancer, cells go wrong as a result of faults in their genes, which mean that there was a mistake in the cell instruction. Genetic faults can either be inherited from our parents, or more often they are the result of damage by common carcinogens. Carcinogens such as tobacco, alcohol, viruses, sunlight, industrial factors and the dietary factors that we encounter day to day are just a few of the myriad reasons we accumulate genetic mistakes. It takes more than one fault to turn a cell into cancer, but as we got through our lives, the more susceptible we become to these accumulated mistakes turning into cancer.
Cancer is not infectious and does not spread from person to person. However, once it advances beyond a certain point, it has the power to spread throughout the body. A tumor that has the potential to spread is said to be malignant because it has more serious implications on the patient. Cancers spread by invading the surrounding tissues until they reach a blood or lymph vessel. Smaller groups of these cells break off from the original structure or organ. These are then carried through the bloodstream to other parts of the body where they may settle and grow. This process of travel and the cancers are known as secondary cancers due to metastatic spread.
The Problem: Self-administered non-invasive methods of cancer detection to monitor progress of treatment currently do not exist. Currently, cancer patients must rely on physical blood tests that require extraction of blood at a laboratory facility to monitor how they respond to treatment, resulting in lag-time of days to weeks to monitor how they respond to treatment. Other diagnostic tests that use heavy equipment and in-clinic procedures have even longer wait times. Unlike, other diseases for which self-monitoring portable devices exist, such as glucometers for diabetes, similar devices do not exist for monitoring the progress of cancer treatment. Therefore, cancer patients are forced to undergo repeated invasive blood tests in a laboratory facility, which adds to the financial and physical burden that they must face throughout their treatment regimen. Given the high prevalence of cancer globally, and the existence of reliable biomarkers as cancer indicators, the cancer diagnostics market is in crucial need of addressing the lack of self-monitored tests that allow patients to track how they respond to treatment. Such a test can lessen the number of cancer drugs that a patient must try throughout their treatment plan by providing real-time data of the personalized performance of the drug. The large-scale data obtained from such a test can also provide crucial information about response trends to certain types of therapeutic drugs.
The Innovation: A non-invasive device to measure cancer antigens
The Integrated Circuit Chromatography (I3C) device is a small, handheld and portable device that contains a unique near infrared (NIR) transceiver. This transceiver can emit light at 800 nm in the near infrared range up to a depth of 10 mm on a surface. The I3C was patented by the WIT in 2014 and operates according to the block diagram in Figure 1. The integrated circuit includes an application-specific integrated circuit (ASIC) that is customized for a particular use, and a light source. Also included is a transducer containing analog to digital conversion, two data acquisition microcontrollers that perform arithmetic functions for input and output, read only memory (ROM) for data storage, and a WAN interface for the transfer of data that is stored in the device. The device is designed such that a sensor that is incorporated into the ASIC can report an analog signal that is proportional to a protein marker level to a transducer, which is then converted into a digital value by the transducer. The ROM stores the data in analog or digital form until it is transferred wirelessly.
III. Rationale for Research
Cancer is a group of diseases that is growing at an alarmingly rapid rate globally, with an estimated one in six deaths caused by cancer worldwide (2).The disease can affect almost any part of the body, and to date, there are over 200 types of cancer marked by uncontrolled cell proliferation that leads to tumor formation. As cancer is a very diverse disease at both the cellular and tissue level, there is currently no single cure that is equally effective in all patients. Varying cellular abnormalities from one cancer to another in different individuals have proven to be challenging in underlying a solitary molecular signature that applies to all cancers. However, certain biomarkers have been established that are cancer-type specific, and these biomarkers have been utilized as the basis for cancer detection tests that monitor how a patient responds to treatment.
Early detection of cancer greatly impacts the outlook for cancer patients and is associated with a much better prognosis for most types of cancer. However, too often patients are diagnosed with cancer after the disease has progressed considerably due to lack of free cancer screening in many health insurance plans, and because many cancers do not manifest symptoms in the early stages. Thus, many patients must resort to increasingly aggressive cancer treatment plans that include trial and error with multiple therapeutic drugs. Current detection methods for cancer mostly involve the use of heavy imaging equipment combined with blood tests, physical examination and biopsy for confirmation of a diagnosis. Once a diagnosis is made, methods to treat and monitor progress are mostly invasive and can cost a patient up to $250,000 a year in healthcare costs depending on the type of cancer (3). Patients with cancer types that are notoriously difficult to treat, such as pancreatic and ovarian cancers, have limited options to mitigate this hefty cost. The WIT proposes to address the needs of these patients by providing access to a non-invasive, portable wireless device that can lessen the burden that cancer patients face during their treatment process.
Other rationale for support of such a facility extends beyond the purely pedagogic. Research dedicated to a project of this magnitude supports an enhancement of nanotechnology, information engineering, medicine, and oncology. These are people who will enhance the interconnections of information engineering sources and information users. Industries, Hospitals, Vocational schools, Universities, and Government institutions will respond to this facility due to the demands placed on the research of terminal illnesses. Our field experiences in our domestic and international work sites indicate an ever-increasing demand for this type of research. Since the majority of the personnel required for this project come from the science and medical fields, it follows that an investment in this type of facility is an investment in the United States economy. By creating and enhancing this facility, WIT will add to the element of its role in political and local economic support. Finally, anything that strengthens the competitiveness of our industry constituents and the economy is a win-win investment.
IV. Scope
The central role of this research is to facilitate a technical approach to cancer management. Since phase II is the full R & D effort, Phase III being the Clinical trial and FDA approval cycle, and commercialization being last, we will first concentrate on the technical content and feasibility for this phase I scope.
The I3C in its entirety will be approximately the size of a commercially available PDA. It will consist of a Customized Integrated Circuit (FPGA/ Light Source), a transducer (with analog to digital conversion), two data acquisition microcontrollers (microprocessors: arithmetic functions and I/O), Read Only Memory (information and data storage), and a WAN interface (wide area network/data transfer). When these electronic components are integrated into a system, WIT will have created a wireless device which from the patient’s blood allows a sensor (as incorporated into the FPGA) to report an analog voltage proportional to the protein marker concentration to a transducer. The transducer will convert this voltage to a digital value (analog to digital conversion) for storage in ROM. The ROM receives the information and stores it along with the time and date.
Porous Silicon Mirror (PSM) particles are biodegradable surfaces that dissolve harmlessly over a period of time in the body. These mirrors consist of particles of silicon etched with nano-scale patterns of pores making them extremely efficient light reflectors. PSMs have been designed for early detection of initial incidence and recurrence of cancers. This is achieved by an application of a selective process of a tumor antibody (CEA: Colorectal Cancer Antigen). The mirrors, which will have chemicals (Immunoassay) placed into etchings, will be implanted just below the skin and will be approximately 5mm wide and 0.5mm thick. The PSM particles will bind to the CEA molecules. The porous silicon used in the mirrors is biocompatible and biodegradable, which means they cause no side effects while in the body or after they disintegrate. It differs from silicon because it has been treated under special acid conditions which make it porous. Once the silicon becomes porous, it acts like a highly reflective mirror. The mirrors eventually breakdown into silicic acid which scientists say is harmless to the body. (http://news.bbc.co.uk/1/hi/health/1272330.stm)
The I3C monitors this process with its optimized Custom Integrated Circuit sensor and light source (laser/infrared). The patient will place a finger on the sensor and the light source will activate causing the PSM in the blood to fluoresce. The reflected light from the mirrors (an analog signal) will then be detected by a transducer for analog to digital conversion. This number is then transferred to a data acquisition equipped microcontroller. The microcontroller will calculate how much antigen concentration is present in the blood.
The actual mathematical equivalent for this in calculus terms is Antigen/Time Elapsed and in medical terms is called the Slope of Patient Response. Another microcontroller will calculate the difference in level of CEA. This is the most important function of the I3C because this calculation determines the exact amount of CEA in the blood, which is the determining factor in cancer recurrence. The mathematical equivalent of this process is expressed in the calculus formula known as the Discrete Approximation of Time Differential (Delta Antigen/Delta Time) and in medical terminology as differential amount of CEA in time allotted. These two key quantities are then transferred to the physician’s database via email (WAN wireless interface capabilities). Once the physician receives the two key quantities, he/she will take the difference of the two to determine the Patient’s Rate of Change. It is this number that tells the physician if the patient’s CEA level is at a low, moderate, or recurring amount.
V. Growth Potential
The initial scope of the project and facility is limited strictly to professional use only, yet it is not without growth potential. As a recruiting tool, the facility should trigger an upturn in potential internships for engineering and medical students. This will help in credit hour and fee generation which should allow some degree of expansion. At the very least, additional revenue would be key in maintaining this whole process. Placing an optimistic future for this type of research, the facility will serve as the basis for the enhanced study of the fields’ of science, medicine, and engineering toughest questions. Adding a full time staff from each field to the facility would add a level of robustness to the facility. This could exhibit itself in adding certification options similar to those offered by technical conglomerates Oracle and Microsoft. These, in turn, could be yet additional recruitment features for students. The facility will also eventually support graduate education as the full time staff increases.
Recognizing the potential economic impact the I3C device will have at every level of society, WIT takes pride in partnerships with institutions of higher education for accreditation, scholarships, and grant monies. Our five year economic forecast correlates directly with the collegiate educational system after the fourth year with the creation of additional positions for Pharmacology research, Oncology, Engineers, Scientist, Professors, Health maintenance Organization professionals (HMO), FDA trial cycles, and Insurance company representatives. The representatives from the HMO and Insurance backgrounds will be added for more robust consultations on future industry needs. This will allow WIT to work in conjunction with universities to stimulate the ‘Think Tank”. The Think Tank will work as an incubator for innovation, where students are able to collaborate with industry and education professionals to create a constant environment of “Technical Innovation” with the emphasis remaining on facilitating a technical approach to cancer management. With this type of “Innovative Thought” course added to the engineering curriculum, this will stimulate grant funding, accreditation, and recruiting due to the level of innovation that could come from a facility of this magnitude. The fifth year will see the I3C as a global standard in cancer management with a host of jobs and educational opportunities ranging from education, scholarship and grant services, pharmaceutical research and sales, engineering, oncology, information technology, and manufacturing creating a solid financial impact on Florida’s state and educational economy. WIT projects an estimated $5-10 million dollars in additional annual revenue for the state of Florida.
VI. Implementation (Phase I Work Plan)
Technical Description of Objectives
Specific Objective 1: Optimization of the Near Infrared Transceiver to detect porous silicon in simulated body fluid.
Rationale: This aim tests the stability of porous silicon as a base material for detection by the near infrared transceiver of the I3C device. Porous silicon will be incubated in human simulated body fluid (SBF), an artificial solution that mimics the ionic composition of human plasma, the part of the blood that carries proteins. The stability and safety of porous silicon within SBF will be assessed, along with a test of its ability to reflect light back to the I3C when NIR light from the device is applied. This objective will provide data on whether porous silicon will functionally behave as a ‘mirror’ for the base of cancer antigen detection.
Proposed Digital Work
Programming of the I3C device for detection of porous silicon
As per the block diagram in Figure 1, next in line to NIR transceiver is the (OP AMP/ 8 bit resolution, 0-256 numerical inputs) operational amplifier for signal conditioning purposes so the human epidermis differences will not mask the integrity of the signal. Fixed inputs and output bias currents will be programmed here for highest and lowest readings possible in SHP, with and without polymer coating. The transducer is set in place for analog to digital conversion (16 bit resolution, 0-512 numerical inputs). All arithmetic functions will be programmed into one data acquisition microcontroller such that the slope of patient response (antigen/time elapsed (days between treatment) and discrete approximation of time differential (change in amount of antigen with respect to time) (▵antigen/▵time) can be calculated. This final number will represent the actual patient antigen amount and will then be stored in ROM (read only, storage, think of caller ID # storage). The ROM will be (16 bit resolution, 0-512 numerical inputs EEPROM) electronically erased after prescribed sample amounts have been transferred to physicians database via WAN portal. Various Integrated circuits will be examined for arithmetic and logic functions, including data transfer speeds. HIPPA compliance and encryption will also be addressed. Data Transfer integrated circuits will be programmed with the encrypted data transfer information in binary to a database that can be accessed only by authorized individuals.
Proposed Experimental Plan
1. Optimization of the biochemical properties of porous silicon in simulated body fluid (SBF) for detection by the I3C.
The stability, biodegradability and potential bioactivity of porous silicon will be assessed using simulated body fluid (SBF), which has an ionic strength that mimics human plasma in vitro. Since the ultimate goal of the technology is to introduce porous silicon as a carrier and signaling substance for a substance that attracts cancer antigens, this experimental approach will provide data on how porous silicon will behave inside the bloodstream. SBF is a well-characterized and standard medium to test the bioactivity and stability of biomaterials for their potential application inside the human body (6). A readout of the dissolution rate can be measured by periodically weighing the porous silicon layers after different periods of incubation in SBF and factoring in layer thickness and surface area (7). As porous silicon is also subject to potential oxidation, this will be measured as previously described (8). Following this, NIR light will be applied from the I3C device onto porous silicon soaked in SBF. Both dynamic and static solutions of SBF containing porous silicon will be examined to mimic the natural environment of the bloodstream. The light reflected back from the optical conductance of porous silicon will be measured by the device and analyzed for stability and porosity of the silicon particles (9).
2. Testing the ability of the NIR transceiver to detect signal from porous silicon in human cell culture in vitro.
The porous silicon will serve as a substratum for the attachment of chemical or biological selective agents that can attract cancer antigens. Therefore, a necessary initial step is a test to ensure no loss of signal when NIR light is applied from the I3C to human cells that contain porous silicon. SBF does not contain amino acids and proteins normally secreted by cells, so this experimental approach will test whether these normal constituents of human cells affect the properties of porous silicon. Porous silicon that is optimized for stability and biocompatibility in the first experimental approach will be applied to in vitro cell culture of human epithelial and ovarian cancer cell lines. The rationale for using these cell lines is to assess whether proteins present in non-cancerous versus cancer cell lines can ‘cloud’ up the porous silicon non-specifically, leading to a false positive reduction in the signal that is transmitted to the NIR transceiver. Light emitted back to the I3C will be measured and stored, and results will be compared to the signal obtained by porous silicon in SBF.
3. In vitro assessment of an immune response to porous silicon.
An important component of introducing porous silicon into the bloodstream of patients is to assess whether porous silicon can elicit an innate or adaptive immune response in the human body. In vitro tests of immune response are a standard approach for generating data on the immunotoxicity of a chemical or biological substance (10). To investigate this question, in vitro immune response assays will be performed with porous silicon. These assays will include examination of T-cell activity as measured by cytotoxicity, cytokine production and proliferation, and effects on human immune cell culture of monocytes, dendritic cells, etc. Results from these tests will indicate whether the porous silicon itself induces an immune response and whether it can be safely used as a substratum to capture cancer antigens by the I3C.
Pitfalls and Alternatives to Objective 1
Initial designs of porous silicon may not display ideal stability and half-life characteristics within SBF or cell culture. The porous silicon design can be altered to address this potential pitfall to develop a silicon model with the desired properties. Additionally, the porous silicon design may also be altered to generate optimal signal that can be received by the I3C without the drawbacks of signal saturation, but providing the flexibility for a large number of cancer antigens to bind to the porous silicon, providing a large limit of detection. Previous studies have demonstrated that porous silicon can be safely administered in humans and safely excreted as silicic acid (11). Therefore, an immune response is not expected. However, if an immune cell response is observed, the porous silicon can be redesigned to modify the porosity and layers to modulate its immunomodulatory properties and ensure safety for application purposes.
Specific Objective 2: Specific detection of cancer antigens with porous silicon mirrors in vitro.
Rationale: Porous silicon will act as a substratum for the attachment of selective agents for cancer antigens. In this objective, porous silicon will be conjugated to a selective agent for CA-125, one of the major biomarkers for ovarian cancer. The porous silicon will serve as the host for an infrared mirror surface. The amount of decrease in reflective signal captured by the device will be mathematically converted into the levels of CA-125 present, thus providing a proof-of-concept for the use of the I3C to detect the levels of cancer antigens.
Proposed Experimental Plan
1. Testing candidate selective agents for CA-125 for conjugation to porous silicon.
A selective agent for CA125 will be screened for conjugation to the porous silicon. Screening for
selective agents that can bind to CA-125 will be performed by Dr Yuri Mackeyev..
Figure 2. Porous silicon as a carrier and signaling component for the I3C.
Immobilization of the selective agent on the porous silicon may be conducted through various methods such as site-specific covalent attachment for porous silicon (12), direct covalent attachment to carboxylic acid terminated surfaces of porous silicon through amide bonding (13), or immobilization to hydrogen terminated silicon through 1-undecenylaldehyde (14). The stability, bioactivity and biodegradability of the porous silicon attached to selective agent will be assessed as described in the first experimental approach in Objective 1. Additionally, the ability of the I3C to detect the porous silicon conjugated to a selective agent will be tested by transmitting NIR from the device and measuring the reflectance that returns to the transceiver.
2. Determination of the range and limit of detection of CA-125 by the I3C.
Once a selective agent for CA-125 is conjugated to the porous silicon, the limit of detection and range of detection that can be achieved with the NIR transceiver of the I3C will be determined. Briefly, a quantitative antigen response curve of CA-125 binding will be constructed using known amounts of purified CA-125 bound to porous silicon in SBF. Quantification will be done using the NIR transceiver of the I3C and compared to detection with an enzyme-linked immunosorbent (ELISA) assay using antibodies against CA-125. Since the gold standard for detection of CA-125 is through ELISA tests, this data will provide a comparison between the innovative technology of the I3C and the current standard assay. Using purified CA-125 commercially obtained (an alternative is by expressing recombinant CA-125 in house), different known amounts of CA-125 will be titrated into the SBF to produce a response curve of the porous silicon mirrors to the presence of CA-125. Lower and upper limits of detection will be determined. The lower limit of detection for CA-125 using ELISA assays is reported to be 35 kU/L (15). Detection by the I3C will be optimized for a sensitive limit of detection comparable to the ELISA. This approach is made feasible through the availability of purified CA-125, anti-CA-125 antibodies and/or CA-125 ELISA kits (Acrobiosystems, San Jose, CA; Abcam, Cambridge, MA).
3. Testing the ability of the I3C to measure CA-125 levels produced in ovarian cancer cells in vitro.
After determining the antigen-response curve using purified CA-125, the I3C will be tested for its ability to accurately quantify the levels of unknown amounts of CA-125 in ovarian cancer cells. NIH-OVCAR3 cells are human ovarian cancer cell lines that secrete the mature form of CA-125 in culture between 1-4 hours after culture (16). The ability of the I3C device to accurately measure the secreted amounts of CA-125 in these cells will be measured by administration of porous silicon mirrors conjugated to the selective agent for CA-125 and compared to the standard ELISA test for CA-125.
Since CA-125 can exist in many isoforms, standard ELISA tests usually rely on the use of one antibody as a capture antibody and a second, distinct antibody as a tracer antibody (17). Applying the I3C device to NIH-OVCAR3 cells that endogenously produce CA-125 isoforms, in contrast to full-length CA-125 as with the purified protein, will provide crucial data on how well the I3C can perform compared to standard ELISA assays in the detection of CA-125. Since other proteins are present in ovarian cancer cells, this experiment will test the specificity of the assay in the presence of other endogenous human proteins to indicate the likelihood of false positive results that can be caused by the reduction of IR reflectance back to the transceiver by non-specific binding of other secreted cellular proteins to the porous silicon conjugated to the selective agent.
4. In vitro assessment of an immune response to porous silicon conjugated to a selective agent for CA-125.
As described in Objective 1, after conjugation of the selective agent for CA-125 to the porous silicon, a test of immune response in vitro will be conducted to assess whether the newly conjugated selective agent to porous silicon has the capacity to produce an immune response in cells.
Pitfalls and Alternatives Approaches to Objective 2
The biggest challenge of this objective is the identification of a compound or biological molecule that can specifically attract CA-125 without triggering an immune response by cells. To address this, several types of selective agents will be tested including carbohydrates known to bind to CA-125, necessary segments of the binding domain of proteins known to interact with CA-125, and synthesized compounds. Additionally, another technical challenge is programming the I3C device to accurately calculate the slope of the antigen response curve to accurately represent the levels of an unknown amount of antigen when the device is applied. Several modifications and trials may be necessary to correctly program the device, and to address this we will program the FPGA’s and Data Acquisition Microcontrollers with these specific arithmetic instructions.
Timeline for Success of Phase I project
We will monitor the progress of the project with optimization of the porous silicon detection by the NIR transceiver in Objective 1 in the first few months following the project start date. We expect rapid progress to occur on remaining goals of Objective 1 once the device is programmed to detect this base constituent of the assay. Selection and optimization of a selective agent to detect CA-125, and fine-tuning of the device for quantitative detection of the antigen levels, will require the bulk of the time period for the proposed project. We aim to complete this Phase I feasibility study within 18-24 months of receiving funding.
Milestones for commercial feasibility
Several benchmarks will be used to track the progress of this Phase I project for the development of a commercially feasible non-invasive device that can measure cancer antigens. These benchmarks span both research objectives and include programming of the transceiver to detect signal reflected from porous silicon, no significant immune response examined by in vitro tests of immune cell activity, and specific and highly sensitive limit of detection of CA-125 through correct programming of the NIR transceiver. A major indicator of the future commercial feasibility of the device will be a range and lower limit of detection of CA-125 that is comparable to the standard ELISA test.
VII. Proposed budget
Upon Request
VIII. Research Facilities
WIT has access to world-class facilities and equipment at the University of Texas Health Science Center at Houston, as outlined below.
Equipment
Major equipment (departmental): Overall, the core laboratory space within the cancer biology wing at UTH is equipped with the following: 3 Nuare tissue culture BSL-2 hoods, 4 incubators with automatic CO2 tank control, several Eppendorf table top Centrifuges (5810R), Neon Transfection system, BioRad TC20 Automated Cell Counter, inverted microscope, hemacytometer and coulter counter, liquid nitrogen storage container, 4 VWR digital adjustable temperature heat blocks, real time PCR system, 96-well thermocyclers, MilliQ water system by Millipore, bacterial incubators, multiple horizontal and upright gel electrophoresis apparatus, Kodak Image Station 4000R, temperature-controlled table-top centrifuge, Invitrogen iBlot, Beckman Coulter DU730 UV/Vis spectrophotometer, ELISA plate reader, fluorescent microplate reader, VWR temperature-controlled water bath, electronic multichannel pipettes, autoradiograph cassettes, Sanyo ultralow -150°C VIP+ freezer, 2 -80°C Freezers (Thermo Scientific and Sanyo), several explosion-proof refrigerators (4°C) and freezers (–20°C), tissue homogenizer, chemical fume hood, flammable storage cabinets, microwave oven, light microscope, Mettler-Toledo AB54-FACT analytical balance, and digital balance.
Major equipment (departmental): Overall, the core laboratory space within the cancer biology wing at UTH is equipped with the following: 3 Nuare tissue culture BSL-2 hoods, 4 incubators with automatic CO2 tank control, several Eppendorf table top Centrifuges (5810R), Neon Transfection system, BioRad TC20 Automated Cell Counter, inverted microscope, hemacytometer and coulter counter, liquid nitrogen storage container, 4 VWR digital adjustable temperature heat blocks, real time PCR system, 96-well thermocyclers, MilliQ water system by Millipore, bacterial incubators, multiple horizontal and upright gel electrophoresis apparatus, Kodak Image Station 4000R, temperature-controlled table-top centrifuge, Invitrogen iBlot, Beckman Coulter DU730 UV/Vis spectrophotometer, ELISA plate reader, fluorescent microplate reader, VWR temperature-controlled water bath, electronic multichannel pipettes, autoradiograph cassettes, Sanyo ultralow -150°C VIP+ freezer, 2 -80°C Freezers (Thermo Scientific and Sanyo), several explosion-proof refrigerators (4°C) and freezers (–20°C), tissue homogenizer, chemical fume hood, flammable storage cabinets, microwave oven, light microscope, Mettler-Toledo AB54-FACT analytical balance, and digital balance. Major equipment (institutional): Shared equipment is also available in the core laboratories in the UT medical school building, which is close to the principal investigator’s lab space. The following equipment is at WIT’s disposal: BD-Calibur Flow cytometry, Bio-plex luminescence detector, Top Count Chemiluminescence detector, Cell harvester, 2-D gel system, Cryostat, UV transillumination box, Gel image analysis system, Alpha Innotech Inc, with computer recording system, speedvac system, gel drying system, Revco -135°C freezer, beta and gamma scintillation counters, ultracentrifuge, high speed centrifuge, shaking incubators, autoclaves, coulter counters, PHD cell harvester, hybridization incubator, UV Stratalinker, gel dryer, pH meter, speedvac, dark room with X-ray film developer. UTH also has confocal microscopy and electron microscopy core facilities. The inverted confocal microscope (LSM 410, Carl Zeiss, Inc., Thornwood, NY) for immunocytochemistry and the laser capture microdissection microscope system (Pix-Cell) are located in the DeBakey Building on the Texas Medical Center Campus, a short walk from the PI’s laboratory. Other resources available to the PI include a medical illustration department, which can meet all audiovisual or printed material needs, and the Houston Academy of Medicine/Texas Medical Center Library. Computer networks permit access to national and international databases such as GenBank, MedLine, and TexSearch.
Major equipment (institutional): Shared equipment is also available in the core laboratories in the UT medical school building, which is close to the principal investigator’s lab space. The following equipment is at WIT’s disposal: BD-Calibur Flow cytometry, Bio-plex luminescence detector, Top Count Chemiluminescence detector, Cell harvester, 2-D gel system, Cryostat, UV transillumination box, Gel image analysis system, Alpha Innotech Inc, with computer recording system, speedvac system, gel drying system, Revco -135°C freezer, beta and gamma scintillation counters, ultracentrifuge, high speed centrifuge, shaking incubators, autoclaves, coulter counters, PHD cell harvester, hybridization incubator, UV Stratalinker, gel dryer, pH meter, speedvac, dark room with X-ray film developer. UTH also has confocal microscopy and electron microscopy core facilities. The inverted confocal microscope (LSM 410, Carl Zeiss, Inc., Thornwood, NY) for immunocytochemistry and the laser capture microdissection microscope system (Pix-Cell) are located in the DeBakey Building on the Texas Medical Center Campus, a short walk from the PI’s laboratory. Other resources available to the PI include a medical illustration department, which can meet all audiovisual or printed material needs, and the Houston Academy of Medicine/Texas Medical Center Library. Computer networks permit access to national and international databases such as GenBank, MedLine, and TexSearch.Facilities and Other Resources
The University of Texas Health Science Center at Houston (UTHealth) is Houston’s Health University and Texas’ resource for health care education, innovation, scientific discovery and excellence in patient care. The most comprehensive academic health center in The UT System and the U.S. Gulf Coast region, UTHealth is home to schools of biomedical informatics, biomedical sciences, dentistry, nursing and public health and the John P. and Kathrine G. McGovern Medical School. UTHealth includes The University of Texas Harris County Psychiatric Center and a growing network of clinics throughout the region. The university’s primary teaching hospitals include Memorial Hermann-Texas Medical Center, Children’s Memorial Hermann Hospital and Harris Health Lyndon B. Johnson Hospital. The University harbors several iLab cores including: Quantitative genomic and microarray services, proteomic service center, magnetic resonance imaging core, human genetics center laboratory, flow cytometry service center, advanced microscopy, biomedical informatics group, etc.
Laboratory: Dr. Krishnan’s laboratory is in an open laboratory space of 15,000 sq. ft. adjacent to other labs in the Institute of Molecular Medicine in the Center for Translational Cancer Research, facilitating frequent interactions to enrich the scientific environment. His laboratory is equipped with a separate space for tissue culture room equipped with three biosafety hoods, 4 incubators with automatic CO2 tank control. He has access to shared freezer space which harbors 4 (-80) and 2 (-20) freezers. Within the laboratory are six desks, with each researcher desk equipped with an Ethernet connection. In addition, on the same floor, his laboratory can access a shared room where all freezers are placed, a shared autoclave room, a large walk-in cold room, a large walk-in warm room, and a shared conference room where the group meeting takes place. The laboratory is fully equipped for competitive nanomaterials, biochemical, molecular and cell biology research, and assay development. For tissue culture work, the lab has 3 tissue culture hoods, 5 CO2 incubators, dry glove boxes, a fume chemical hood, and two inverted microscopes. Nanoparticle characterization equipment includes a Cary 300 UV-Vis spectrophotometer, an FTIR instrument and microscope, an Agilent 1200 HPLC, a Malvern zetasizer, a Combi FLASH chromatography system, a rotary evaporator with chiller and pump, a Perkin Elmer inductively coupled plasma – mass spectrometer, and an Applied Biosystems MDS Sciex API5000 Mass Spectrometer. A Shared Centre for IMM equipment includes top-of-the-line flow cytometers (FACSAria, LSR-II and FACSVantage), a Leica TCS SP5 confocal microscope with LAS AF image processor, upright and inverted Olympus fluorescence microscopes. Other shared equipment includes a Luminex workstation for Multiplex Immunoassay (Millipore), Beckman DU spectrophotometer for various calorimetric assays, a Labconco Console Freezone freeze-dry system, Millipore Elix water purification systems, Konica SRX-101A film processors, Amersham Biosciences Storm imaging system, as well as Beckmann L8-70 and Sorvall RC5B ultracentrifuges. Available for shared use is a RadSource2000 160kVp irradiator for cell and animal irradiation at variable dose rates 1-4Gy/min typically with custom shielding for partial body or thigh alone irradiation. This is serviced every 6 months with NIST-traceable standards used for calibration.
Laboratory: Dr. Krishnan’s laboratory is in an open laboratory space of 15,000 sq. ft. adjacent to other labs in the Institute of Molecular Medicine in the Center for Translational Cancer Research, facilitating frequent interactions to enrich the scientific environment. His laboratory is equipped with a separate space for tissue culture room equipped with three biosafety hoods, 4 incubators with automatic CO2 tank control. He has access to shared freezer space which harbors 4 (-80) and 2 (-20) freezers. Within the laboratory are six desks, with each researcher desk equipped with an Ethernet connection. In addition, on the same floor, his laboratory can access a shared room where all freezers are placed, a shared autoclave room, a large walk-in cold room, a large walk-in warm room, and a shared conference room where the group meeting takes place. The laboratory is fully equipped for competitive nanomaterials, biochemical, molecular and cell biology research, and assay development. For tissue culture work, the lab has 3 tissue culture hoods, 5 CO2 incubators, dry glove boxes, a fume chemical hood, and two inverted microscopes. Nanoparticle characterization equipment includes a Cary 300 UV-Vis spectrophotometer, an FTIR instrument and microscope, an Agilent 1200 HPLC, a Malvern zetasizer, a Combi FLASH chromatography system, a rotary evaporator with chiller and pump, a Perkin Elmer inductively coupled plasma – mass spectrometer, and an Applied Biosystems MDS Sciex API5000 Mass Spectrometer. A Shared Centre for IMM equipment includes top-of-the-line flow cytometers (FACSAria, LSR-II and FACSVantage), a Leica TCS SP5 confocal microscope with LAS AF image processor, upright and inverted Olympus fluorescence microscopes. Other shared equipment includes a Luminex workstation for Multiplex Immunoassay (Millipore), Beckman DU spectrophotometer for various calorimetric assays, a Labconco Console Freezone freeze-dry system, Millipore Elix water purification systems, Konica SRX-101A film processors, Amersham Biosciences Storm imaging system, as well as Beckmann L8-70 and Sorvall RC5B ultracentrifuges. Available for shared use is a RadSource2000 160kVp irradiator for cell and animal irradiation at variable dose rates 1-4Gy/min typically with custom shielding for partial body or thigh alone irradiation. This is serviced every 6 months with NIST-traceable standards used for calibration. Office: The Krishnan laboratory has office space for 6 postdoctoral fellows, research scientists, technicians, or graduate students. Secretarial assistance is available through the resources of the Departments. Dr. Krishnan’s office space is located close to the laboratory space and is equipped with a computer, printer, scanner, and telephone. Individual desks and computers are provided to all research fellows in the department. All research personnel have access to a shared copier and fax machine.
Office: The Krishnan laboratory has office space for 6 postdoctoral fellows, research scientists, technicians, or graduate students. Secretarial assistance is available through the resources of the Departments. Dr. Krishnan’s office space is located close to the laboratory space and is equipped with a computer, printer, scanner, and telephone. Individual desks and computers are provided to all research fellows in the department. All research personnel have access to a shared copier and fax machine. IX. Key Personnel
Marlon Williams, CEO
Marlon Williams is a biomedical engineer with over 20 years of experience in systems and network analysis. He is an expert in laser systems for the CT, MRI, PET, Linac, and Cyberknife imaging systems. Mr. Williams routinely interacts with oncologists and hospital directors to resolve quality control issues and meet critical cancer center/hospital targets, showcasing the necessary experience in the oncology field that is required for the proposed project.
Additionally, Mr. Williams has expertise in Embedded Integrated Circuit design, and programming and analysis of application-specific integrated circuits with optical sensing capabilities using VmWorks 6.9 for use in wireless diagnostics devices. He utilized this expertise to program arithmetic functions of 16-bit data acquisition microcontrollers and 8-bit transducers for arithmetic functions and A-D conversion using Microsoft parallax data acquisition tool contained within the I3C and will continue to utilize these skills in the execution of this Phase I proposal.
Dr. Sunil Krishnan, MD, PhD, Mbbs
Dr. Sunil Krishnan of the Mayo Clinic Jacksonville will also join WIT for the Phase I project as senior personnel, with his blend of expertise in management of esophagogastric, hepatobiliary, pancreatic, and anorectal tumors with radiation therapy. His expertise also includes stereotactic body radiation therapy, proton radiation, intraoperative radiation, and brachytherapy. In addition to his clinical activities, Dr. Krishnan is active in patient-centered research via clinical trials, laboratory research focusing on identification of compounds and nanoparticles that sensitize tumors to radiation therapy, translational research aimed at defining biomarkers of response to treatment, and education and training of residents and fellows in the clinic and the laboratory.
Will Blake, Jr., Senior Engineer
Will Blake, Electrical Engineer and Founder of EMSYS, Inc. (Embedded & Mobile Systems, Inc.) was instrumental in patenting the I3C device. Mr. Blake will resume the role of Senior Engineer for the execution of the Phase I project. Mr. Blake will utilize his 20+ years of experience in embedded systems and firmware to condition the I3C for the accurate detection of cancer antigens and the remote transfer of data to a comprehensive database. Mr. Blake specializes in retrieving data from devices to route to decision-making and analysis platforms, and his expertise includes diagnostic devices.
X. Conclusion
The Phase I study will provide crucial data of predictive measures of how porous silicon can be paired with the I3C device for the long-term goal of applying it for in vivo detection of cancer antigens within the bloodstream. The completion of this feasibility study will provide the proof-of-concept for the ability of the novel NIR transceiver to accurately detect, measure, and store a signal that is directly proportional to the amount of cancer antigens present. Future studies in a Phase II project will bring the parameters defined in this Phase I study closer to the market through in vivo tests of the device to accurately and specifically detect CA-125 after injection or ingestion of the porous silicon mirrors conjugated to a selective agent for CA-125 in mouse models of ovarian cancer that have varying levels of CA-125 in their bloodstream. The in vivo safety, stability and elimination rates of the bioconjugated porous silicon will also be assessed in the Phase II study. Several clinical studies have previously demonstrated the safety of ingesting silicon that is converted to silicic acid within the body (11). Therefore, these in vivo tests will indicate whether cancer antigens can be detected with the I3C device and tests for implementation of the device with human cancer patients under strict Institutional Review Board (IRB) approval will be conducted. Ultimately, the WIT aims to expand the technology to other diseases for which non-invasive in vivo monitoring tests are not available, such as Alzheimer’s Disease.
