Liquid Biopsy Cancer Detection

1. Liquid Biopsy Cancer Detection Liquid Biopsy Cancer Detection 1. What does Zepto Life technology do? Or one of the areas Zepto works Key words: GMR bio sensing platform, microfluidic technology, liquid biopsy assays, early cancer detection devise Point Of Care Lab Testing By combining the GMR biosensing platform with microfluidic technology Zepto Life technology has developed series of liquid biopsy assays for early cancer detection. This innovative liquid biopsy assay allows for the early detection cancer re-occurrence months before a tumor would be visible by CT scan. Concurrently, the liquid biopsy assay is also able to eliminate false positive results offering an assay that is highly sensitive and highly accurate. 2. The introduction of Zepto Life (1) Key words: immunoassay testing device, point-of-care, highly sensitive and multiplexed immunoassays, point of care handheld device, Giant Magnetoresistance (GMR), GMR biosensing technology, Magnetoresustance, microfluidic technology, D-Dimer product, immunoassay, biosensor array device, liquid biopsy cancer detection. Zepto Life Technology is building an immunoassay testing device for Point Of Care Diagnostics in hospitals. Currently headquartered in St. Paul, Minnesota, the company has been a privately held since 2014.Zepto Life Technology’s mission is to deliver quality results at point-of-care. Development of new innovative highly sensitive and multiplexed immunoassays that are both fast while also being reliable is another part of Zepto Life Technology’s mission. The simplistic yet versatile design allows Zepto Life’s platform to be used for many types of assays and biomarkers. This point of care handheld device sensor utilizes Giant Magnetoresistance (GMR) which won the 2007 Nobel Prize in Physics.The GMR biosensing technology is the core of all Zepto Life’s projects. When a magnetic field is induced across the GMR sensor it produces an impedance. Using the impedance, the Magnetoresistance can be calculated through an equation. This technology is being licensed from the University of Minnesota. The assembly of the GMR cartridges starts with the GMR sensor being married to a printed circuit board (PCB). Another key component of Zepto Life’s platform is its microfluidic technology, which greatly reduces sample volume requirements. These fluidic bodies combine with the GMR sensor PCB to form a cartridge.Together the GMR biosensor, PCB, microfluidic technology combines to deliver a high sensitivity assay. However not only is it highly sensitive but it also has high specificity testing functions.One of Zepto Life’s first products is focused on the biomarker D-Dimer, which is associated with testing for blood clots and can help diagnose venous thromboembolism (VTE). The D-Dimer product can also help rule out other serious conditions such as deep vein thrombosis (DVT) and

2. pulmonary embolism (PE).The immunoassay is built using the tradition antibody sandwich method. The capture antibody is coated on the GMR sensor, then antigen is flowed over the surface which will bind to its specific antibody, after all unbound antigen is washed away the detection antibody will then also bind to the antigen, this creates the ‘sandwich’. All these steps of the assay are being done inside one of Zepto Life’s specially design cartridges to deliver a ‘Lab on chip’ test. This on chip bio surface technology could use a single cartridge to perform a variety of test because each antigen is unique to its antibody pair, that’s what make multiplexing possible. This point of care testing is also currently being developed for liquid biopsy of cancer. This process takes a blood sample and analyzes it for cancer cells or fragmented DNA from a tumor that are circulating in the blood of a patient, allowing for detection of cancers at an early stage. The best part of this intelligent biosensor array device is that it can be developed for a wide range of different types of protein or DNA assays, not just liquid biopsy cancer detection. All these features come compact into one portable, handheld design that makes it very easy to move from one place to the next, instead of other conventional bulky benchtop immunoassays. Point Of Care Labs 3.What does Zepto Life do? Key words: Our immunoassay, liquid biopsy platform technology, cardiac panel tests , minimal disease monitoring, Alzheimer’s disease early diagnosis , fast fungi detection We are excited to bring the superior diagnostic results to where patients really need them. Zepto integrated GMR biosensor, microfluidic, disposable cartridge, and high-sensitive reader system to detect very small change of targeted analyses. Our portable immunoassay platform and liquid biopsy platform will transform how standard care is delivered by providing accurate and fast diagnostic results at point of need. Our immunoassay and liquid biopsy platform technology help to address the most challenging health care problems: Provide accurate cardiac panel tests results to detect the presence of acute coronary syndrome and cardiac ischemia and decide appropriate therapy as soon as possible. Provide gene mutation test for cancer early detection, drug treatment selection, drug treatment resistance monitoring, and minimal disease monitoring. Provide decentralized and easy to use blood test for Alzheimer’s disease early diagnosisto enable early intervention and delay in the early stage of the disease. Provide fast fungi detection test results that can save patient lives from deadly fungi infection. Provide accurate diagnostic results to manage challenging diseases such as Type 1 diabetes screen, stroke, concussion, thyroid, and sepsis.

3. 4. Key words explainations Key words: Magnetic sensors, GMR biosensing technology, GMR biosensors/ biosensors array, Early disease detection, GMR-based immunoassay technology, A portable diagnostic platform, GMR biosensing What Is Lab On A Chip Magnetic sensors are a common technological application for magnetic materials and can be used to detect quantities such as speed, displacement, magnetic fields, and temperature. The sensors are often based on magnetoresistive devices, which typically consist of a trilayer sandwich structure in which two ferromagnetic layers are separated by a non-magnetic layer. The non-magnetic layer could be metallic (if the sensor relies on the giant magnetoresistance effect) or insulating (if the sensor relies on the tunnelling magnetoresistance effect). With such structures, changes in the relative angle between the magnetization of the two ferromagnetic layers will result in a change in resistance. To rotate or switch the magnetization of the magnetic materials, an external magnetic field is usually applied. GMR biosensing technology has advantages such as high sensitivity, low cost, real-time signal readout, and compatibility with the modern integrated circuits (IC). This technology has been applied for the detection of nucleic acids, protein biomarkers. By combining GMR biosensors with high moment magnetic nanoparticles (MNPs) such as FeCo, higher sensitivities are observed. GMR biosensors/ biosensors array operate in a similar sandwich structure to ELISA where instead of using a fluorescent molecule as the tag, magnetic particles tag is introduced. The advantages such as low background level and lower detection limit of blood sample can be obtained due to a lack of detectable magnetic content from blood sample naturally. Since the sensors are fabricated as an array, multiplex of multiple assays can be gained within one chip. Besides, the sensors are IC- compatible and microfabrication-compatible so that a high throughput and cost-effective sensor production can be achieved. Microfluidics Manufacturing Early disease detection is an important factor in effective medical treatment and better patient outcomes. Technologies that facilitate early detection of disease provide a path toward less expensive, more effective, and sustainable healthcare. Protein biomarker quantification has been proposed as a potential method of screening for many diseases but stringent requirements must be met for clinical screening and subsequent early detection. To be useful for wide-spread screening, the optimal technology is required to be small, affordable,fully automated, and with the sensitivity and precision of central laboratory equipment while measuring concentrations of multiple biomarkers simultaneously (i.e., multiplex).

4. GMR-based immunoassay technology has been successfully applied for the detection of different biomarkers and pathogens, such as mycotoxins, aspergillus fumigatus allergen, Escherichia coli, chloramphenicol, human interleukin 6 (IL-6), and so forth. However, all of these immunoassays are limited to lab-based testing. A portable diagnostic platform that is capable of performing on-site testing by nontechnicians instead of complicated laboratory tests by technicians is crucial in the field of animal disease surveillance and control. Zepto’s GMR handheld platform will provide exactly the need for point of care detection. Liquid Biopsy Cancer Detection In relation to optical biosensing technologies, GMR biosensing has some inherent advantages including matrix insensitivity and the potential for high sensitivity multiplex assays in a portable instrument. Multiplexing is quite natural in GMR biosensing simply because the magnetic dipole fields remain localized near each sensor surface and therefore do not interfere with neighboring sensors. These attributes make GMR biosensors versatile and strong candidates for future high performance molecular diagnostic applications. 5. The Introduction of Zepto Life (2) Key words: point-of-care, immunoassay, biosensor array device, high-sensitivity assays, point- of-care testing Zepto Life Technology is developing a variety of innovative and technologically superior products to revolutionize point-of-careservices in the healthcare industry. Zepto’s technology utilizes Giant Magnetoresistance (GMR) to analyze human blood samples. Product development at Zepto requires a considerable amount of research and development, as GMR has only recently been implemented as biosensing technology. However, our team of highly trained scientists and engineers are always finding new ways to maximize the efficiency and efficacy of our products. Zepto’s immunoassay platform, which focuses on the detection of cardiac biomarkers, is poised to be our first product through the FDA approval process. Its handheld design allows for ease of use in a wide range of point-of-care settings. The platform exists as two halves: a disposable microfluidic cartridge that houses the detection assay and a handheld cartridge controller that processes and displays results. Patient sample is introduced to the microfluidic cartridge, which is then inserted into the cartridge controller. The sample is pneumatically directed through a complex array of channels in the cartridge and across our patented bio- surface technology. Patient sample reacts with the bio-surface within the cartridge, which the cartridge controller interprets and presents to the user as a diagnostic result. Another device that is close behind Zepto’s immunoassay platform is our liquid biopsy platform. This system focuses on the detection of various DNA strands within patient sample for the identification of various diseases or conditions. The liquid biopsy platform is a tabletop system that functions as a combined DNA extraction device, thermocycler, and GMR detection system. The microfluidics and GMR telemetry processing and presentation is similar to the immunoassay platform, but the liquid biopsy platform covers a wide range of DNA-based detection assays.

5. The mechanical engineering department at Zepto is constantly developing innovative methods for overcoming new and exciting challenges. The point-of-care testing market is vast, and to successfully compete in the industry, the mechanical engineering team must support Zepto’s high-sensitivity assaysto the furthest extent. Zepto’s biosensor array device includes microfluidics, high precision mechanical components, and state-of-the-art electrical-mechanical interfaces. With this level of complexity comes many challenges, but at Zepto, we have the skills and determination to develop a panel of point-of-care diagnostic platforms that revolutionize the medical industry. 6. The Introduction of Zepto Life (3) Key words: immunoassay platform, immunoassays, giant magnetic resistance (GMR) technology, GMR biosensor, GMR sensor, GMR platform, GMR system Zepto Life Technology is a diagnostic testing company developing a point of care immunoassay testing device. The testing platform is based on the giant magnetic resistance (GMR) technology, the same technology that is used in computer hard disk drives. The GMR biosensor has many attributes that make it advantageous for diagnostic testing. These attributes include the small size of the sensor, the small sample size required and the rapid signal generation. The GMR biosensor at the core of the testing platform is coated with capture molecules, like antibodies to make the tests highly specific for the analytic target. The GMR signal which is proportional to the concentration of the analyte can generate the signal in real time, meaning that the signal is not dependent on a separate chemiluminescent or fluorescent reaction, thus providing the clinician with results more quickly. The Zepto Life Technologyimmunoassay platform is capable of performing the same diagnostic tests currently conducted in a central lab in the clinician’s office. The GMR sensor is built into the microfluidic cartridge which contains all of the reagents necessary to conduct the diagnostic test, as well as instructions to the reader for how to handle the reagents, the timing of the reaction(s), the flow rates through the microfluidics and the information to be reportedwithout further intervention by the operator. The microfluidic cartridge is capable of handling plasma, serum, whole blood or other body fluids including urine, saliva and cerebrospinal fluid. The GMR technology for the immunoassays has a large dynamic range and high sensitivity. The large dynamic range means that it is capable of quantifying an analyte over a broad range of concentrations, which is useful for quantifying biomarkers that may be present at a wide range of concentrations in the population or fluctuate over a range within an individual. The high sensitivity of GMR biosensors means that the system is capable of conducting high sensitivity assays for analytes present at very low concentrations, like troponin in healthy adults. With the lab on a chip technology the biosensor can be multiplexed and used to detect and report the results from multiple analytes simultaneously from a single sample. This capability can provide the clinician with the results for several biomarkers that when combined give a more accurate or complete diagnosis or improve the monitoring of a previously diagnosed disease in a patient. This includes diagnosing acute diseases like many of the infectious diseases, the monitoring of chronic diseases like diabetes and rheumatoid arthritis or to assess the effectiveness of ongoing treatments like those for cancer or cardiac disease. In addition to the utility of the Zepto Life Technology platform in the medical field, the high specificity and versatility of the GMR sensors makes it adaptable to be implemented in other fields. The GMR platform could be used in environmental monitoring for the assessment of toxins in water or nutrient levels in soil. It could also be used for the monitoring of the food supply to assess the quality and safety of food and beverage products. The multiple analyte capabilities, small size and speed of the result delivery of the GMR system makes it very adaptable and capable of being used across many fields to improve the lives and quality of life for many people.

6. 7. Zepto Life integrated system Key words: molecular diagnostic integrated system, microfluidic technology, Giant magnetoresistive (GMR) biosensor, GMR biosensors, all-in-one microfluidic chips The Zepto LB integrated system is a molecular diagnostic integrated systemfor use in point-of- care settings. The Zepto LB integratedsystemhouses a miniaturized sample-in-answer-out platform for the applications of liquid biopsy such as cancer detection or fungal detection. The Zepto LB integrated system incorporates microfluidic technology which can operate multiple tasks from sample preparation, DNA amplification, detection and verification. Human plasma is loaded to the disposable all in one cartridge. Cell free DNA (cfDNA) is extracted as the first step in the cartridge. Zepto’s on-chip DNA extraction method allows Zepto to reach more than 90% cfDNA extraction efficiency. The extracted cfDNA is then amplified by PCR. This molecular approach provides higher sensitivity and faster turn around time than blood culture for fungal detection. Next, the amplified DNA is hybridized to the DNA probes attached on Giant magnetoresistive (GMR) biosensor. Unlike fluorescent based detection platform suffers high background noise due to inherent properties of biological molecules, GMR biosensors rely on magnetic tag which is absent in biological molecules. Furthermore, qPCR is limitedin multiplexing capabilities, as only a certain amount of filters arepresent to detect different fluorophore fluorescence. Hence GMR biosensor is an excellent platform to provide high sensitivity and multiplexing capabilities. Finally, the signal is verified using melting curve analysis which further enhances the assay specificity. This verification step significantly reduces any false positive results. The tasks are fully automated which reduces human error and contamination, as well as ensures accuracy every step of the way. The all-in-one microfluidic chips along with the fully automated system makes the Zepto LB integrated system highly sensitive and specific, achieving a limit of detection in 0.1% cancer mutation. 8. The introduction of Zepto Life (4) Key words: biotechnology company, GMR (giant magneto resistance), GMR biosensor, lab on a chip technology, immunoassay testing devices, high specificity testing, microfluidic devices Zepto Life Technology is a biotechnology company committed to advancing the field of medicine through cutting edge research and the application of novel technologies towards revolutionizing the diagnostics industry. From its inception in 2014, Zepto Life Technology has committed itself to expanding on the 2007 Nobel Prize winning GMR (giant magneto resistance) breakthrough by delivering groundbreaking in vitro diagnostic applications targeting a vast array of disease states. From the beginning, Zepto Life Technology has invested in exploring the myriad applications for this technology and has been successful in prototyping a wide range of products addressing diverse markets requiring early stage detection options like heavy metal, heart attack, and cancer. Zepto Life Technology’s development of novel GMR biosensor designs coupled with advanced lab on a chip technology has resulted in the creation of powerful immunoassay testing devices tailor-made for the fast-paced medical field. These platforms package the high specificity testing capabilities of a central lab into proprietary microfluidic devices with rapid analysis delivered by a portable point of care testing device. This product line empowers healthcare providers to deploy the latest advances in microfluidic technology and point of care diagnostics towards improving patient outcomes worldwide. All Zepto Life

7. Technology products incorporate unique microfluidic design strategies aimed at mitigating cost and error potential, with special attention paid to material engineering and pneumatic control. With a large team of dedicated microfluidic engineers working to optimize these assay platforms, ever-greater progress is made towards delivering these products to the medical market. With several exciting assay products exiting feasibility, Zepto Life Technology looks forward to delivering a powerful new tool into the hands of clinicians. A core technology utilized in all Zepto Life Technology products are the customized lab on a chip designs for each assay. Each unique assay requires sample processing and preparation unique to that test, and these elements drive cartridge design. These cartridges have pneumatic actuated valves, mixing features, heating zones, and many other processes, all automated and packaged into the small plastic disposables. Zepto Life Technology has committed to delivering the precision of a laboratory technician into the hands of clinicians, and this goal has been realized through years of product development and testing. Now, Zepto Life Technology stands ready to demonstrate its vast diagnostic product line.