Week 1 HW: 🧬 Automated Cell-Free Biomanufacturing of an Insulin Aptamer
Automated Cell-Free Biomanufacturing of an Insulin Aptamer for Screening Insulin After Refrigeration Failure
Core idea: Produce an insulin-binding DNA aptamer using a cell-free system and use it in a disposable electrochemical cartridge to rapidly test insulin samples after a refrigerator or cold chain has failed.
Figure 1. Three-dimensional cartoon representation of insulin (PDB 1TRZ). Image by Diego Mariano, Wikimedia Commons, licensed under CC BY-SA 4.0.
🎯 1. What would you like to biomanufacture and why?
Target product: an insulin-binding DNA aptamer, to be used as the recognition element in an electrochemical sensor.
The main purpose of the device would be screening insulin after refrigeration failure. Insulin requires controlled storage, so a refrigerator malfunction, power outage, or other cold-chain failure can create uncertainty about whether stored insulin should still be used. This idea came to me as a result of my personal experience living in Egypt, where during the summer, frequent blackouts occur to conserve energy. If these blackouts are prolonged, refrigerated medicines are at risk of going bad, and there’s no way to identify what went wrong. I chose insulin as it’s a medication that many of my friends and loved ones rely on for their well-being.
I therefore propose a small, disposable cartridge that rapidly measures insulin in a stored sample after suspected temperature change. The result would be used as a screening step to identify samples that may require further investigation or confirmatory testing.
The device would not initially replace validated pharmaceutical quality-control or potency testing. Instead, it would be designed as a rapid screening tool that could help identify potentially compromised medication for further analysis, and to avoid using them until confirmation of their potency is obtained.
Why an aptamer?
Aptamers are short nucleic-acid molecules that bind specific targets. In this project, the aptamer would recognize insulin, while the electrode converts the binding event into a measurable electrical signal.
🧪 2. What living system would you use?
I would use a cell-free system.
A cell-free system makes sense because the product is a DNA aptamer, so living cells are not required to produce it. The reaction could contain:
- a circular DNA template containing the aptamer sequence;
- a primer;
- DNA polymerase;
- dNTPs; and
- reaction buffer.
The DNA polymerase amplification reaction would generate many copies of the aptamer sequence, which could then be processed and purified.
The purified aptamer would be attached to a gold electrode in a disposable cartridge. Insulin binding changes the aptamer structure, which can be converted into an electrochemical signal. Published insulin aptasensors have used this general approach. The aptamer would be manufactured in bulk before cartridge assembly, rather than produced during every individual test.
Proposed cartridge architecture
🤖 3. How would you use automation?
I would use an Opentrons OT-2 to automate the DNA-polymerase amplification system part of the cycle and produce large amounts of aptamer.
The robot could:
- dispense DNA templates and reagents;
- prepare different reaction conditions;
- run replicates and controls; and
- transfer samples for purification and testing.
I believe it would be useful during the development steps, however, I’m unsure about how efficient it would be if this experiment was conducted on a larger scale, perhaps a larger robot?
📏 4. How much would you need to make, and how long would it take?
For a small pilot, I would aim to make enough aptamer for 1,000 disposable screening cartridges.
1. Parameters & Assumptions
- Concentration Ratio: 250 nM aptamer to 10 mircoM insulin (10,000 nM insulin).
- Source: ScienceDirect Article
- Ratio: 1 mole of aptamer per 40 moles of insulin (1:40 molar ratio).
- Standard Pen Cartridge Volume: 3.0 mL(0.003 L) per cartridge.
- Standard Insulin Concentration (U-100): 100 units/mL.
- 1 unit of insulin approx 0.0347 mg.
- 100 units/mL = 3.47 mg/mL = 3.47 g/L.
- Molar Mass of Human Insulin: approx 5808 g/mol.
- Standard Insulin Concentration in Molarity: Concentration = 3.47g/L over 5808 g/mol = 5.974 x 10^(-4) M = 597.4 microM
2. Aptamer Calculations per Cartridge
Using the 1:40 molar ratio of Aptamer to Insulin:
Aptamer Concentration = Insulin Concentration divided by 40= 597.4 microM/ 40 = 14.935 microM = 1.4935 x 10^(-5) mol/L
For 1 cartridge 3.0 mL = 0.003L:
Moles of Aptamer per Cartridge = 1.4935x 10^(-5) mol/L x 0.003L} = 4.48 x 10^(-8) moles (44.8 nmol)
3. Total Requirement for 1,000 Cartridges
Total Moles of Aptamer = 44.8 nmol/cartridge x 1000 cartridges = 44.8 micromol (4.48 x 10^(-5) moles)
Mass Calculation (Assuming average DNA/RNA aptamer molecular weight)
- Assuming an average 40-mer DNA aptamer mass of 12,500 g/mol:
Total Mass of Aptamer = 4.48 x 10^(-5) mol x 12,500 g/mol = 0.56 grams (560 mg)
The exact yield would need to be established experimentally because the amount of DNA produced does not directly indicate how much functional, purified aptamer will be available for the sensor.
Estimated time
| Stage | Estimated time |
|---|---|
| Automated reaction setup | ~0.5–1 h |
| DNA amplification | ~3 h |
| Processing / purification | ~1–2 h |
| QC | ~1 h, partly in parallel |
| Total | ~5.5–7 h |
⚠️ 5. What don’t you know yet?
1. Can the cartridge distinguish usable from slightly compromised insulin?
This is the most important question for the cold-chain screening application. A sensor could show that insulin is present, but this does not automatically prove that the insulin still has full biological potency. Therefore, I would compare samples stored under acceptable conditions with samples exposed to controlled temperature excursions and compare the sensor results with established analytical or potency measurements.
2. Can the test work with real samples?
Blood or pharmaceutical formulations can contain other substances that may affect the sensor. I would therefore test selectivity, calibration, storage stability, and batch-to-batch variation.
📚 References
Yoshida, W., et al. (2009). Selection of DNA aptamers against insulin and construction of an aptameric enzyme subunit for insulin sensing. Biosensors and Bioelectronics, 24, 1116–1120. https://doi.org/10.1016/j.bios.2008.06.016
Wu, Y., Midinov, B., & White, R. J. (2019). Electrochemical Aptamer-Based Sensor for Real-Time Monitoring of Insulin. ACS Sensors, 4, 498–503. https://doi.org/10.1021/acssensors.8b01573
Zhou, L., et al. (2024). Ratiometric electrochemical aptasensor with strand displacement for insulin detection in blood samples. Analytica Chimica Acta, 1317, 342823. https://doi.org/10.1016/j.aca.2024.342823
Karim, A. S., & Jewett, M. C. (2021). Toward sustainable, cell-free biomanufacturing. Current Opinion in Biotechnology, 69, 136–144. https://doi.org/10.1016/j.copbio.2020.12.012
Petzold, C. J., & Mukhopadhyay, A. (2025). From bench to biofactory: High-throughput technologies and automated workflows to accelerate biomanufacturing. Current Opinion in Biotechnology, 94, 103320. https://doi.org/10.1016/j.copbio.2025.103320
🤖 AI disclosure
AI client: OpenAI ChatGPT (GPT-5.6 Luna)
Date accessed: September 21, 2026
Prompt used:
“Provide me with the relevant scientific papers for an insulin-detecting aptamer biosensor cartridge, selecting an appropriate microbial, mammalian, or cell-free manufacturing system, describing automation, calculating manufacturing scale and time, and identifying technical unknowns. Write this out as a Markdown file with relevant emojis, Font Awesome Free icons, and a free/openly licensed image relevant to the topic.”