Week 1 HW: Final Project Ideas

Biomanufacturing an anti-troponin Fab fragment for point-of-care heart diagnostics

What would I like to biomanufacture and why?

Target product: a recombinant Fab (antibody fragment) that binds to cardiac troponin I (cTnI — a specialized protein found exclusively in heart muscle cells) to be used as the detection reagent in a point-of-care lateral flow immunoassay test strip for diagnosing myocardial infarctions. The end product would work the same as a pregnancy test — for sensitive, rapid, and user-friendly quantification.

Motivation: Troponin I is located inside the heart’s muscle cells, where it normally should be, but damage to the cells causes troponin to leak into the blood. This is why cTnI is invaluable for cardiac diagnosis and assessing heart attack severity. When I was first introduced to the study “AI-driven lateral flow immunoassay for point-of-care detection of cardiac biomarkers in acute myocardial infarction” (Lu et al., 2026), I quickly realized how important point-of-care technology is becoming these days, and how migrating from slower turnaround times to instant results could speed up the decision to treat. According to the study, the prototype showed quantitative results in about 8 minutes, a roughly 47% improvement in detection efficiency over conventional methods (Lu et al., 2026). The antibody reagent itself is usually the expensive, hard-to-scale component of these devices, which is exactly the piece a biomanufacturing perspective can address. As a biomedical engineer living in a country where underdeveloped areas are common (Honduras), I firmly believe in the phrase “time is muscle,” and in how reliable and low-cost technologies can save countless lives.

Target audience: rural and low-resource clinics, and emergency department triage — basically any setting where lab-based troponin testing is too slow or unavailable.

What living system would I use?

Chassis: E. coli strain BL21(DE3), which is widely used for the expression of recombinant proteins in biomanufacturing. This bacterium would be engineered to express and secrete the anti-cTnI Fab fragment into the periplasm of the cell (its oxidizing environment promotes the bond between troponin I and the anti-cTnI fragment).

Why a microbial cell? This project proposes a diagnostic reagent, where the sole focus is high-affinity binding to ensure its functioning. Fab fragments lack the Fc region and can fold correctly without glycosylation, so bacterial expression is sufficient. This is a well-established approach for diagnostic-grade antibody fragments and is far cheaper and faster to scale than mammalian cell culture.

  1. In: glucose/rich growth medium for high-density growth, an inducer (IPTG) to turn on protein production and make it constant, pH, and controlled temperature.
  2. Process: The specialized bacterial strain is grown to high density through fed-batch fermentation, then expression is induced after glucose depletion. Heavy and light chains are co-expressed with a periplasmic signal peptide so the Fab folds correctly in the oxidizing periplasmic environment needed for its disulfide bonds.
  3. Out: harvested cells/media → periplasmic extraction or cell lysis → affinity purification using Protein L → purified Fab ready for conjugation and application onto a lateral flow membrane. Functionalization follows citrate-reduction synthesis of gold nanoparticles, surface functionalization, and covalent antibody conjugation via EDC/NHS coupling chemistry, as stated in the study for the lateral flow test strip.

Assumptions: the anti-cTnI Fab fragment is completely compatible with bacterial expression, and periplasmic secretion gives an acceptable fraction of correctly folded, active protein.

To consider: a cell-free protein synthesis (CFPS) system using E. coli lysate/extract directly in a reaction, rather than growing live cells, as an alternative chassis. Addition of whey permeate to E. coli-based cell-free extracts has been reported to enhance cell-free protein synthesis yield by about 50% (Rasor et al., 2021).

Automation

Technology: a desktop liquid-handling robot (Opentrons OT-2) paired with a plate reader to corroborate binding, applied to the antibody screening and expression stage of the workflow.

What I would automate: the first step, and one where automation would be ideal, is to screen anti-cTnI Fab fragments and growth conditions — also to ensure expression conditions have been met to find the combination that gives the best yield and the best-folded, highest-affinity antibody. These steps include colony picking, small-culture growth, induction, lysis, and ELISA binding assays, which a liquid-handling robot is perfect for. At the manufacturing stage, automation would shift toward downstream processing with an automated FPLC system for purification runs.

For the cell-free alternative, a programmable digital microfluidics platform (Nuclera) would be the best fit, since cell-free reactions don’t require cell growth or an induction step, so each test could be run and screened on-chip directly.

How much would I need to make, and how long would it take?

Scale definition: enough Fab reagent to supply point-of-care troponin strips for every emergency department chest-pain evaluation in Massachusetts in one year. Nationally, chest pain is the second most common reason for an ED visit in the U.S. — 6.5 million visits per year out of a U.S. population of roughly 335 million. Massachusetts has a population of about 7.15 million (2025). Applying the national rate to Massachusetts gives an estimated demand:

National chest-pain-visit rate:
6,500,000 ÷ 335,000,000 ≈ 1.94% of population/year

Applied to Massachusetts:
7,150,000 x 1.94% ≈ 138,800 strips/year

For the antibody loading per strip, published lateral flow assay development protocols typically formulate the solution at about 1 mg/mL, with a separate amount used to functionalize the test strip. Based on these figures, I estimate roughly 1 µg of total Fab (capture + detection) per strip.

138,800 strips/year x 1 µg Fab/strip
= 138,800 µg ≈ 0.139 g (139 mg) of Fab fragment per year

How long? Fed-batch E. coli periplasmic Fab expression has been demonstrated at titers around 100 mg/L of culture in bench-scale (10 L) and pilot-scale (300 L) fermenters.

Volume of culture needed:
139 mg ÷ 100 mg/L ≈ 1.4 L of culture

This is designed to fit in a 2- to 10-L benchtop bioreactor; no parallel reactors needed. At ~3 days/batch (growth + induction + harvest + purification), Massachusetts’s full annual demand could be produced in a single ~3-day run.

Honduras

Honduras has the highest ischemic heart disease (IHD) and cardiovascular mortality rate in Central America, with an average death rate of 82.7 per 100,000 people, yet Honduran Ministry of Health (SESAL) figures report just over 100 heart attack deaths per year nationwide. This gap aligns with a well-known problem in resource-limited settings: underdiagnosis of acute MI, often driven by the same access and lab-turnaround barriers this project targets.

Suspected chest-pain evaluations/year:
1,300,000 x 1.94% ≈ 25,200 strips/year

Fab needed: 25,200 x 1 µg/strip ≈ 0.025 g (25 mg)/year

Culture volume: 0.025 g ÷ 100 mg/L ≈ 0.25 L (250 mL)
→ producible in a single small shake-flask or benchtop
  fermenter run, well under 1 batch, ~2-3 days total

What don’t I know yet?

  • Antibody required per strip: my ~1 µg/strip figure is inferred from published lateral flow formulation concentrations, not a number specific to a troponin assay. I’d need to prototype actual strips and titrate the true capture/detection antibody load required for a clinically useful sensitivity cutoff.
  • Correctly folded, active fraction from bacterial expression: periplasmic Fab expression can partition unpredictably between periplasm, culture medium, and insoluble inclusion bodies. I’d need to experimentally measure what fraction of expressed protein is soluble and competent for antigen binding, since this could substantially change the effective fermentation volume needed.
  • Scale-up fidelity: fermentation titers measured in small pilot runs (10 L) don’t always hold at larger production scale. I’d need to run and validate titer at the actual production bioreactor scale before trusting the timeline.

References

Lu, Y., Peng, X., Yin, Z., Fan, X., Fan, J., Mi, Y., & Li, G. (2026). AI-driven lateral flow immunoassay for point-of-care detection of cardiac biomarkers in acute myocardial infarction. Mikrochimica Acta, 193(5), 323. https://doi.org/10.1007/s00604-026-08054-y

Cleveland Clinic. (2022, marzo 17). Troponin test. Cleveland Clinic. https://my.clevelandclinic.org/health/diagnostics/22770-troponin-test

Rasor, B., Vögeli, B., Landwehr, G., Bogart, J., Karim, A., & Jewett, M. (2021). Toward sustainable, cell-free biomanufacturing. Current Opinion in Biotechnology. https://doi.org/10.1016/j.copbio.2020.12.012