EmpireSpit's project AptaStress was only possible with the input of numerous stakeholders and professionals throughout every step of development. We designed aptamers that can detect salivary biomarker alpha-amylase, correlated with acute stress, in response to the rising mental health crisis in the world and the market's lack of precise, cost-effective mental health evaluations, thanks to everything we learned from our interviews with psychology and biology experts. Throughout the course of our study, we sought for ways to make stress level monitoring more widely available and to broaden the scope of the objectives for maintaining mental health.

We contacted numerous mental health specialists who could give a clinical viewpoint on diagnosis and therapy, knowing that the interpretation of stress level detection data was best performed by skilled medical professionals. Some of the experts we spoke with were unsure of using salivary biomarkers to diagnose broad mental health issues, emphasizing the significance of differentiating between different illnesses, such as anxiety and depression, due to their varied diagnostic characteristics. In response, we contacted prominent salivary biomarker experts to examine what might be done to improve alpha amylase's potential for use as a biomarker in clinical settings, and narrow our scope down to identifying stress levels as a proactive measure against mental issues. Finally, we contacted specialists who work with stress-related variables, such as those who work with PFA chemicals, and talked with potential stakeholders in accessing the mental health of those unable to advocate for themselves easily, such as teachers and livestock farmers to find AptaStress’ ideal niche market.


Dr. Tony Buchanan

In our interview with Dr. Tony Buchanan, we discussed the purpose of stress tests and their difficulties. First, we went over how stress is linked to the development of various chronic diseases, including cardiovascular diseases, hypertension, and cerebral vascular problems. Additionally, those who face chronic stress are more susceptible to anxiety and depression. Dr. Buchanan strongly emphasized the importance of distinguishing between anxiety and depression due to their distinct diagnostic profiles, while also highlighting the unfortunate lack of a specific diagnostic marker for these conditions. As for methods of quantifying and detecting stress, Dr. Buchanan spoke of the utilization of radioimmunoassays and competitive binding assays to measure levels of stress-related proteins. One of these proteins, alpha-amylase, is particularly responsive to the flight or fight response and exhibits rapid changes in stressful situations, while cortisol, a different stress-related protein, responds much more slowly.

Dr. Buchanan told us that scientists often use saliva samples because they are minimally invasive, but they come with a caveat: they are not directly from the source. Another challenge with measuring cortisol lies in its cyclical fluctuation throughout the day, with the highest levels observed in the morning and lower levels as the day progresses. To accurately understand an individual's regular cortisol patterns, multiple samples must be tested over a lengthy period of time, which is inefficient and costly. As a result, commercially available testing can be expensive. There are some more affordable options for self-testing, like Salimetrics. Both alpha-amylase and cortisol are crucial in stress assessment, as they serve different purposes and respond differently to stressors. Alpha-amylase, due to its rapid response, is particularly useful in detecting sudden stress events like panic attacks. However, careful preparation is still necessary for accurate results, which includes hydration (without diluting the sample) and refraining from eating and consuming caffeine directly before testing.

Dr. Buchanan also highlighted how cortisol responsiveness varies significantly based on factors such as age and gender, with younger individuals typically exhibiting larger stress responses, making it challenging to pinpoint specific thresholds. One hindrance in the progression of stress research is ensuring that studies are ethical, which involves obtaining numerous necessary permissions from university boards - a time-consuming process involving documentation and feedback. The conclusion reached from our discussion with Dr. Buchanan was that while self-diagnosis is discouraged, tracking stress fluctuations over time and in response to events can be informative and can help individuals gauge the effectiveness of lifestyle changes on stress levels.

Dr. Tony Buchanan
Figure 1: Our Meeting with Dr. Tony Buchanan

Dr. Hedyeh Ahmadi

During our interview with Dr. Ahmadi, we gained valuable insights into the field of salivary research, statistical analysis, and their applications to our potential project. Dr. Ahmadi initially began her career in a salivary lab, where she was hired as a statistician, working with two professors on salivary bioresearch. She told us about how she underwent a comprehensive two-year training program, which directly coincided with the outbreak of the pandemic. COVID-19 brought a large shift in Dr. Ahmadi’s professional role, leading to her appointment as a neuroscience statistician. Despite the difficult timing of this transition, Dr. Ahmadi said that the pandemic-driven challenges fully enriched her knowledge of bioresearch. Notably, she delved into the examination of various types of saliva and explored optimal approaches for interpreting salivary data. For more information, Dr. Ahmadi directed us to the Salimetrics website, which was interesting because we had already heard about the company from our most recent interview with Dr. Buchanan.

Ahmadi emphasized her specialization in data analysis, specifically post-salivary data collection. She emphasized her ability to effectively analyze her teams’ extensive data, and informed us on how we could apply statistical analysis to our project. Dr. Ahmadi elaborated on her recent projects, citing two notable instances. The first project involved analyzing cortisol levels as a biomarker of stress among a population of transgender women. This analysis also encompassed an exploration of how different risk factors such as diseases and societal judgment could impact cortisol levels, while her second project focused on longitudinal data and regression analysis. Dr. Ahmadi’s models frequently incorporated age as a temporal factor, alongside other variables including time of day, ethnicity, and sex. She stressed the significance of considering factors like age, sex, race, ethnicity, living situation, socioeconomic status, and education level within the household in their analyses.

Finally, to assist in our further research, Dr. Ahmadi provided a range of valuable websites to guide us. These included Dagitty which can be useful in identifying correlations and regressions between cortisol/protein levels and various variables we intend to study; links to her own work and ongoing research, such as Megan Herting and her research on air pollution exposure and prefrontal connectivity. These sources provided valuable insights into identifying relevant variables, and informed us on a tool named "gpower" for determining sample sizes within specific populations. She recommended a minimum sample size of 100 for reliable results.

Additionally, Dr. Ahmadi referred to a notable paper by Riis et al. (2020) that outlined best practices for managing factors such as time, temperature, and testing equipment to minimize fluctuations in salivary analytes like cortisol. These considerations are crucial to ensure accurate sample collection. Furthermore, Dr. Ahmadi highlighted the importance of obtaining Institutional Review Board (IRB) approval from NYC for any research involving human subjects. In essence, the meeting with Dr. Ahmadi provided a deep understanding of her expertise in salivary research and statistical analysis, along with invaluable resources to guide our future endeavors in this field.

Dr. Ahmadi
Figure 2: Our Meeting with Dr. Hedyeh Ahmadi

Dr. Andrew Savelgsburgh

During EmpireSpit’s meeting with Dr. Andrew Savelgsburgh, we focused on the specific correlation between microRNA quantity and acute psychological stress. The project that Dr. Savelgshbergh studied with his research team aimed to investigate this relationship using salivary biomarkers, specifically cortisol and alpha-amylase. We discussed the potential stress induced by the blood drawing process, leading them to opt for non-invasive salivary samples so as to not artificially inflate stress levels. Cortisol, a stress-related hormone, was identified as a key biomarker in the study. The measurement of cortisol was relatively easy due to the availability of the enzyme-linked immunosorbent assay (ELISA) test, which is efficient and suitable for pipetting. In contrast, alpha-amylase, another stress indicator, required an additional step: dilution before testing. The team consistently collected 5 mL of saliva samples from participants to analyze. For cortisol analysis, 10 microliters of saliva were used, while alpha-amylase was diluted.

Our discussion then transitioned to the methods for analyzing microRNA, with Dr.Savelgsburgh mentioning Salivette, a test tube company based in Sarstedt,Germany, and mass spectrometry as potential approaches. The use of the enzyme test (ELISA) was favored, despite the likelihood of requiring manual pipetting. This choice was supported by the test's efficiency and reasonable cost. We then asked Dr. Savelsbergh about the advantages and disadvantages of using aptamers, the alternative molecular recognition tools. We discovered that aptamers were less expensive and potentially more time-efficient than ELISA, so we decided to conduct a side-by-side comparison of ELISA and aptamers. Furthermore, Dr. Savelgsbergh introduced miR21 as a point of interest, emphasizing its potential link to reactive oxygen species within cells. Potentially, there could be a connection between the presence of miR21 and stress-induced heart rate elevation, which could result in reduced oxygen supply to the bloodstream of a stressed individual.

In summary, our meeting with Dr. Savelgsburgh highlighted EmpireSpit’s focus on studying the relationship between microRNA and acute psychological stress using salivary biomarkers. Cortisol and alpha-amylase were identified as key indicators, with the ELISA test chosen for cortisol analysis due to its efficiency. EmpireSpit contemplated various methods for microRNA analysis and discussed the potential significance of miR21 in relation to cellular response to stress. We also touched on the use of aptamers as an alternative to ELISA, with potential advantages and challenges outlined.

Dr. Savelgsburgh
Figure 3: Our Meeting with Dr. Andrew Savelsburgh

Dr. Allan Tasman

EmpireSpit reached out with University of Louisville Psychiatry Professor Dr. Allan Tasman to learn more about the factors influencing mental health, both positively and negatively. Dr. Tasman told us that there were countless factors affecting mental health, and often too many to take into whole consideration. He pointed out that it is very common for patients to be misdiagnosed, citing one patient who was diagnosed with psychosis due to her paranoia, when it was really just her own stress and anxieties manifesting as intrusive thoughts. As a result, he highlighted the importance of being able to assess stress levels accurately, to ensure that informed diagnoses are occurring.

Dr. Tasman was very optimistic about the future of mental health diagnoses and their accuracy. He was very enthusiastic about younger generations’ willingness to discuss mental health and share their personal struggles, as opposed to older generations with more stigma related to mental illness. Unfortunately, Dr. Tasman mentioned that depression was especially prevalent in young people after the pandemic, as a result of social isolation. We were intrigued, and considered that youth would make a good target for a stress-detection system, as younger generations would presumably be more open to using the product, and be more affected by its benefits.

Dr. Tasman
Figure 4: Our Meeting with Dr. Allan Tasman

Alvin Xu

EmpireSpit held an interview with entrepreneurial University of Pennsylvania Life Sciences and Management student Alvin Xu to learn more about successfully marketing AptaStress. Since Alvin is a college student with multifaceted interests and professional experience, we were able to ask him for advice regarding the practical, biological, and business components of our project. We told him about our goal of preventing mental health crises through proactive diagnoses using AptaStress, and Alvin informed us that at many elite universities, students struggle with reaching out for mental health diagnoses and recognizing when their stress levels are unsustainable. He emphasized that youth mental health is a delicate topic and that more effort needed to be put into resolving, or at least reducing, the detriments of academic stress and resulting mental health issues.

Alvin also highlighted the importance of finding a niche market, not necessarily the largest market, but one that would allow AptaStress to succeed. We discussed the various people who would benefit from AptaStress, and reached the conclusion that narrowing down our intended market by looking for similar products already successful in their own niche. He mentioned that if we wanted to make our project a pharmaceutical product, our next step would be seeking FDA approval, determining the ideal cost, and displaying the benefits of using AptaStress over other stress-detection methods currently in the market, such as its faster production of results and non-invasive, user-friendly nature.

Figure 5: Our Meeting with LSM student Alvin

Shailesh Senthil

We addressed our project's goal of assessing stress using a low-cost PCR machine with Shailesh Senthi Kumar, a molecular biologist at UPENN's LSM Program and a Stanford researcher. He informed us, through his study in nucleic acid identification in brain tumors, that room temperature amplification PCR is currently the "gold standard" for current miRNA detection methods, a variable we had hoped to investigate initially. The detection efficiency appears to be dependent on the combination of the protein and the aptamer. Validation of aptamers is currently considered as time consuming and unneeded by some in the scientific sector, as AI predictions of functional groups and 3D structure are already assisting, in addition to good pre-existing methodologies. He also knew that once an aptamer was validated, it was cheaper to use.

Figure 6: Our Meeting with LSM student Shailesh

Bianca Brandon

EmpireSpit held an interview with forensics expert Bianca Brandon to learn more about the nature of saliva analysis. Ms. Brandon earned her M.S. in molecular biology and spent much of her time in graduate school doing forensics research. Currently, she teaches the elective courses “Forensic Science” and “Science & Engineering Research” at Staten Island Technical High School in New York City. As a forensics teacher and expert, she has a tremendous amount of experience with saliva-based science as saliva is often used as forensic evidence.

We began the interview discussing the effects acute stress has on mental health, and how it can lead to long term stress and other problems. We then talked about acute stress and its relationship with alpha-amylase, sparking further discussion of our research on aptamers and its applicability to salivary biomarkers. As we talked about our engineering process of the artificial alpha-amylase with Mrs. Brandon, we discussed the problems we encountered with the restriction digest of the PET-28a plasmid backbone and restriction enzymes. She proposed two potential reasons for the failure of the restriction digest: a flaw in the design of the homemade plasmid (linearized plasmid backbone and human alpha-amylase gene insert) or a problem with the restriction enzymes, XhoI and XbaI. Ms. Brandon also noted our plasmid backbone wasn’t as established in the scientific community as other backbones. As a result, when we encountered problems with our specific plasmid backbone sequence, we couldn’t turn to literature or previous research for guidance.

Figure 7: Our Meeting with Mrs.Brandon

Dr. Benjamin Renquist

Our meeting with Dr. Benjamin Renquist, an associate professor at the University of Arizona for Animal and Comparative Biomedical Sciences, tackled an alternative method of measuring alpha-amylase without using aptamers. Due to his experience in researching both animal stress and obesity, Dr. Renquist was familiar with researching enzyme activity and was using enzyme activity assays to detect enzymes in the liver for diabetes. Dr. Renquist gave us valuable information on using enzyme activity assays to measure alpha-amylase, suggesting that we could detect the amount of alpha-amylase in saliva by testing how starch broke down over a period of time in saliva, as alpha-amylase is a digestive enzyme that breaks down starches and converts them into glucose.

Dr. Renquist gave us avenues to pursue using enzyme activity assays with, such as a homemade kit and a commercially available kit that could be used to measure alpha-amylase. We also discussed the benefits and drawbacks of using enzyme activity assays over aptamers. Dr. Renquist said that enzyme activity assays are easier and cheaper to use compared to aptamers. He discussed how enzyme activity assays are used for proteins that have enzyme activity, but aptamers were used to measure levels of proteins that did not have enzyme activity to measure.

Finally, we addressed using PCR to measure mRNA to detect stress. Dr. Renquist talked about how there are two different ways to do PCR: quantitative polymerase chain reaction and inpoint polymerase chain reaction. He informed us that quantitative PCR was the more common method as it is more sensitive to changes in mRNA levels, but still had difficulty with finer change that was less than a doubling. A drawback in measuring mRNA is that mRNA degrades quickly and needs to be preserved right away. RNA is traditionally unstable and sample quality affects the result of the amount of mRNA we measure, which would be a problem we would need to overcome in order to create easily accessible tests to detect acute stress.

Figure 8: Our Meeting with Dr Benjamin Renquist

Potential Stakeholders

Alice Melendez

In a discussion with Kentucky farmer Alice Melendez, EmpireSpit gained valuable insight into the adverse effects of stress on animal health and weight gain, which can lead to reduced profitability for farmers due to insufficient meat production. Ms. Melendez strongly emphasized the challenge of stress, as it often actually induces stress in animals, which could lead to inflated levels recorded. Additionally, most stressors for animals are mainly physical, aside from pecking order, and Ms. Melendez worried that the potential of stress-testing technology would raise concerns among organic farmers who fear devaluing animals to machines and statistics. Since animals are naturally calm, and it’s very obvious when they aren’t, Ms. Melendez doubted that our stress test would be very useful for farmers to help their own animals. However, Ms. Melendez told us that a livestock stress-test could be very useful for farmers to verify the treatment of their animals; many consumers desire organic and humanely-treated produce, but worry that companies don’t actually treat their animals well. If there were a simple way to assess the quality of care animals received, it would allow farmers to quantify the quality of treatment animals received.

Ms. Melendez
Figure 9: Our Meeting with farmer Alice Melendez

Zach Cahill

Our interview with Zach Cahill revealed that some farmers would not find our stress detection kits useful. He echoed some of Ms. Melendez’s concerns for private farmer use, but disagreed with her about the usefulness of them for humane treatment verification. Unfortunately, he was relatively pessimistic about the applications of stress detection kits in the livestock industry. He mentioned that other professions dealing with subjects often in or under-capable of self-advocacy, such as teaching, may find that stress detection kits are more useful to determine the stress levels of their students.

Figure 10: Our Meeting with farmer Zach Cahill

Carol Díaz

In EmpireSpit’s interview with Puerto Rican teacher Ms. Carol Díaz, we discussed the different problems teachers have with helping support their students’ mental health. Ms. Díaz told us that oftentimes her students struggled with balancing their homework, tests, sports, family and friends, but were unaware of the levels of stress that they were under. She also mentioned that many students were reluctant to reach out for help, as mental health is a very stigmatized topic within some families and students are sometimes unable to realize that receiving external support is okay. Ms. Díaz informed us that a quantitative indicator of stress levels would be very helpful for teachers to recognize the duress that their students are under, allowing them to intervene, as many struggling students go under teachers’ radar and can even lash out, hurting others as a result.

Figure 11: Our meeting with potential stakeholder Carol Díaz

Dr. Alfred Leong

EmpireSpit held a virtual interview with Dr. Alfred Leong, a practicing medical doctor at the NewYork-Presbyterian hospital, concerning the real world implementation of AptaStress within hospitals. First, we asked him if many of his patients experience stress and if so, to what degree it detriments their health. Dr. Leong explained that it is common for stress to be experienced by and negatively affect his patients, but there are no current simple and affordable ways to detect stress. As a result, the question of the existence and impact of pure stress is often overlooked during check-ups or questions. Dr. Leong mentioned the existence of a Patient Health Questionnaire-9 (PHQ-9), which is a nine-item, self-administered diagnostic screening that doctors use to screen their patients for depression and anxiety. However, he agrees that there is no current non-invasive and cheap way of objectively assessing stress levels.

We introduced our project: AptaStress, utilizing aptamers to detect alpha amylase levels, indirectly quantifying stress. Dr. Leong was enthusiastic about our idea, but also pointed out potential ethical concerns with testing on people and advised us to first use animals to avoid backlash. Towards the end of our interview, Dr. Leong inquired about our knowledge of the relationship between alpha amylase and acute stress. After we shared our current sources indicating the connection of alpha-amylase production and stress, Dr. Leong wished us luck with the development of our project and we concluded our interview.

Figure 12: Our meeting with potential stakeholder Dr. Alfred Leong

Frances Wong

EmpireSpit spoke with Ms. Frances Wong, the Healthcare Director of the Institute at NewYork-Presbyterian Brooklyn Methodist Hospital. She handles the bulk of business operations, and has a strong expertise in strategic planning. In her time as Director, she has enhanced patient care by developing programs and services to improve the hospital's responsiveness and sensitivity to the needs of their patients. She is also a registered clinician and psychotherapist.

Given Ms. Wong’s background in business, our team wanted to interview her about the attractiveness of AptaStress in hospitals and ascertain if there exists a specific target audience that our product would benefit most. She told us that stress kits may be especially beneficial for students, who are more prone to stress as a result of the demands of their studies and expectations. However, with her background as a psychotherapist, Wong mentioned that there are other ways that are non-quantifiable but still useful that currently exist for detecting stress. For example, she mentioned that a good way to gauge how someone’s stress level is to look at their speech patterns, like the rate at which someone is talking or the presence of perspiration. In the absence of a kit, a medical professional would still be able to gauge the seriousness of stress. However, she conceded that there were definitely certain user convenience advantages to a stress test like AptaStress.

Figure 13: Our Meeting with CEO Frances Wong

Parthiv Patel

We met with potential investor Parthiv Patel, an undergraduate student with professional investment experience, to gauge interest in AptaStress from an investor’s perspective. Patel told us that our idea was promising, but outlined several criteria that make certain companies look promising from an investor’s point of view that we could improve upon. First, he told us that we needed to highlight what made our product stand out within our chosen market. Patel told us to go in depth with our research, and to make sure that we were able to identify specific strengths of AptaStress compared to competitors within our niche: the pre-existing salivary stress tests. Our final proposal should include thorough explanations for every single design choice we made while creating AptaStress, such as why we chose to make it a non-invasive salivary test - for user comfort and easier use. Patel also mentioned that it would be beneficial to not only prove why one strength of our project, but to then do an in-depth market analysis of all salivary based stress tests and further indicate the additional benefits of our product, like our choice to use aptamers and smaller nucleotides.

Figure 14: Our Meeting with potential investor Parthiv Patel

Dr. Hanja Brandl

In a meeting with Dr. Hanja Brandl, an animal behavior researcher, EmpireSpit discovered that noninvasive saliva testing could be extremely useful to researchers who work with wild animals. Researchers analyze animal stress for a variety of reasons, such as conservationists observing the impact human-caused environmental changes have on animals. Right now, blood samples are the most common way researchers measure the animal stress hormones cortisol and corticosterone. However, blood sampling often ends up stressing animals more while researchers collect the blood and researchers have less than two minutes to collect the sample before stress hormones are released into the bloodstream and affect data. At the moment, it is challenging for researchers to measure animal stress levels due to the large required quantity of samples needed for accurate testing, and resulting difficulty in obtaining samples from animals, while still doing so quickly to minimize stress on the animal. Alternative methods of quantifying cortisol are testing animal urine and feces, but they only indicate acute stress stemming from events hours later, making them less accurate indicators than saliva or blood.

Dr. Brandl emphasized that she saw the usefulness of a saliva based testing kit for researchers like herself. While animals and humans respond to stressors in a similar fashion physiologically, animals cannot simply tell humans how they feel, thus leading to a niche for a noninvasive way to measure stress in animals for scientists that AptaStress can fill.

Figure 15: Our Meeting with animal behavior research Dr. Hanja Brandl