Continued Development

The TECAN is a widely used laboratory automation solution, often employed for various tasks in molecular biology, including data analysis. A future team will utilize the TECAN to further test the riboswitches and create new switches. These new riboswitches can be within the original 70 base pair limit, or longer given the foundation from this year’s team.

1. Testing New Riboswitches:
- Fluorescence Measurements: Utilize the TECAN's fluorescence detection capabilities to monitor riboswitch activity. Fluorescent markers can indicate the switch's state (activated or deactivated).
- Real-Time Monitoring: Automate the process of monitoring riboswitch behavior over time, capturing real-time data to assess their responsiveness.

2. Creating More Riboswitches Within the 70 Base Pairs Limit:
- Library Generation: Design and synthesize a library of short oligonucleotides within the 70 base pairs limit using Aptagen Aptamer Index.

3. Creating Riboswitches with Longer Lengths:
- Library Generation: Design and synthesize a riboswitch using Aptagen or literature to find new medically relevant switches.

4. Functional Testing:
- Use the TECAN for high-throughput functional testing of the longer riboswitches, evaluating their performance and responsiveness to specific ligands or environmental conditions.

To ensure accurate quantification of the developed riboswitches, it is imperative for future teams to conduct a more exhaustive and thorough characterization.
In addition to measuring fluorescence, the trehalase production must be measured more accurately. Using larger test volumes would allow for more glucose measurements over time to develop a more reliable predictive curve. This additional characterization data will help SensoREX more accurately calculate proportional concentrations of biomarkers in samples.
We will also need to test at varying concentrations. Presently, we are examining our riboswitches at specified oversaturated concentrations to enure maximum binding. It is recommended that future teams explore both higher and lower concentration ranges to assess their influence on the riboswitch's affinity to biomarkers. Changing concentrations will also allow us to gather more data to more accurately mimic and predict human blood/serum tests.

The team currently relies on the Sigma70 master mix provided by Daicel Arbor Biosciences’ myTXTL protein expression kit. A potential progression for the team involves formulating an in-house mix, enhancing precision in the implementation of our riboswitches. Producing cell-free systems in-house would greatly reduce costs for bulk production.

As a part of the Human Practices segment of the project, our team engaged in discussions with the Penn State Clinical Trials and Translational Science Institute (CTSI). To progress to the next stage involving clinical trials, the team will comprehensively characterize the chosen riboswitches and establish a clear connection to the use of a medical device. Completing necessary paperwork is crucial, and obtaining approval could potentially take anywhere from six months to a year.
According to the CTSI, the team would need the following for a clinical trial:

1. Define Your Research Question:
- Clearly define the purpose of your study. What do you want to investigate? What is your hypothesis?

2. Design Your Clinical Trial:
- Study Protocol: Develop a detailed study protocol outlining the objectives, methodology, participant eligibility criteria, and the overall study plan.
- Ethical Approval: Submit your study protocol to an Institutional Review Board (IRB) or Ethics Committee for ethical review and approval.
- We would submit directly to the ethics board in CTSI if we conducted trials at Penn State.
- Clinical Trial Registration: Register your trial in a public clinical trials registry, as required by regulations.

3. Recruit Participants:
- Informed Consent: Design an informed consent form explaining the study, risks, and benefits. Participants must provide informed consent before participating.
- Recruitment Plan: Develop a strategy to recruit eligible participants. This can involve collaboration with healthcare providers, advertising, or outreach programs.

4. Implement the Trial:
- Randomization and Blinding: If applicable, randomize participants into different groups and decide whether the study will be single-blind, double-blind, or open-label.
- Intervention: Administer the experimental treatment according to the study protocol.
- Data Collection: Collect data as outlined in the protocol. This can include medical tests, surveys, or other forms of data.
- We met with the biostatistics department who would assist in data collection and analysis.

5. Monitor and Ensure Quality:
- Monitoring: Implement a monitoring plan to ensure the trial is conducted, recorded, and reported according to the protocol, Good Clinical Practice (GCP), and regulatory requirements.

6. Data Analysis and Reporting:
- Data Analysis: Analyze the collected data using appropriate statistical methods.
- Results: Summarize the results and their implications. Determine if the results support your hypothesis.
- Clinical Study Report: Prepare a comprehensive clinical study report detailing the methods, results, and conclusions.

The steps can vary based on the nature of the clinical trial and the specific requirements of the regulatory authorities overseeing the trial.

In the industry, target product profiles are used in development to plan out the intended uses and effects of a product in compliance with FDA guidelines. The TPP would become especially useful in the case of approaching commercialization of the product. The team developed a Target Product Profile for SensoREX: