Electrochemical Measurement of Cytochrome c Release from Microdissected Cancer Tissues

Introduction:: Cancer is a major global health issue, and advancing effective cancer drug treatment is an ongoing challenge. A primary reason drugs fail in clinical trials is the poor predictive power of existing preclinical models.¹ To address this limitation, our lab has developed a multifluidic platform for drug development based on regularly sized, cuboidal-shaped microdissected tumors (“cuboids”).² Because the tissues are never dissociated and retain much of the native tumor microenvironment (TME), the platform has the potential to be an effective tool for preclinical screening in drug development and for cancer precision therapy.³ Currently, the platform relies on fluorescent labeling, a semi-quantitative method best suited to be a single-time-point terminal assay. Thus, in this study, we developed an aptamer-based electrochemical sensor that can be integrated into our cuboid platform to monitor the dynamic response of cytochrome c (Cyt c), a soluble cell death indicator, precisely and periodically (Fig. 1a).

We fabricated an electrode array using standard dry film resist (DFR) techniques and a gold sputtering process. Next, we immobilized a hairpin structure aptamer for Cyt c. The aptamer was thiolated and conjugated with a redox reporter methylene blue (MB). To correct for baseline drift critical for measurement over time in culture and accurate calibration, enhanced signal-to-noise, and reduced sensor-to-sensor variability, we utilized the Kinetic Differential Measurement (KDM) approach.^4,5 Finally, we demonstrated that our platform could measure Cyt c from intact cuboids treated with a drug.


Materials and Methods:: We used the DFR technique to create a patterned mask for gold electrode deposition on a poly (methyl methacrylate) (PMMA) surface using a lamination process combined with standard photolithography technology.^6,7 After developing the exposed DFR, we sputtered a thin (50 nm) layer of gold. We then peeled off the DFR layers and cleaned the gold surface using hydrogen peroxide and sodium hydroxide as previously reported (Fig. 1b-c).⁸ In the next step, the 40-mer-Cyt c aptamer sequence was synthesized by IDT Technologies as follows: 5’-Thio-C6-Disulfide-CCGTG TCT GGG GCC GAC CGG CGC ATT GGG TAC GTT GTTGC-NH2-3’.⁹ We modified that aptamer at a 3-terminus with an amide group for redox probe (methylene blue, MB) conjugation through succinimide ester (NHS) coupling and a 5-terminus with a C6-disulfide linker. We then conjugated MB-NHS with aptamer and immobilized them on the electrode surface following the previously reported protocol.¹⁰

We employed the KDM approach that used the difference between signal-ON (increase current) and OFF (decrease current) curves at different frequencies.¹¹ To characterize the behavior of our sensors, we generated a signal map by probing a range of square-wave frequencies and amplitude. We identified amplitude-frequency pairs that achieve the largest signal difference between no target and saturation.¹¹ In this case, we picked 50 Hz (signal-OFF) and 1000 Hz (signal-ON). Lastly, we cultured live cuboids inside Cellink bio-ink matrix. We then measured Cyt c secretion periodically from cuboids treated with Mocetinostat (MOC) (10 μM) in dimethylsulfoxide (DMSO) and cuboids without the drug in DMSO only as a control.


Results, Conclusions, and Discussions:: This work aimed to develop an aptamer-based electrochemical sensor for Cyt c. In the strategy outlined above, aptamer against Cyt c was tagged with a redox reporter (methylene blue, MB) and immobilized on the Au electrode. The appearance of the target Cyt c at the electrode surface caused the aptamer to undergo a conformational change upon target binding. The occurrence of signal-ON and signal-OFF behavior in the sensor provides an opportunity to employ KDM. KDM works by measuring two electron transfer mechanisms: fast, signal-ON where the target binding increases the electrochemical signal (Fig. 1f), and slow, signal-OFF, where the target binding decreases the electrochemical signal at different frequencies (Fig. 1e).⁵ We generated KDM values by subtracting the normalized peak current at signal-ON and signal-OFF frequencies and dividing by the average. We then used KDM values collected over a range of target concentrations to create a calibration curve fitting the Hill-Langmuir isotherm (Fig. 1g).¹² After the calibration, we washed the sensors with 6 M urea to regenerate the aptamer before culturing them in vitro.

To demonstrate the ability to measure Cyt c periodically from live cuboids, we loaded cuboids in the Celllink bio-ink matrix to keep them in place. We then pipetted them on the working electrode (Fig. 2a). We cultured the cuboids on our sensor platform (Fig. 2b) with the treatment of MOC in DMSO compared to the control culture treated with DMSO only. We measured the sensor periodically at 12h, 24h, 36h, 48h, 60h, and 84h. The results showed that the cuboids treated with the drug secreted a higher Cyt c concentration than the control over time (Fig. 3c). Our results suggest we can use our platform to quantify the temporal dynamic release of Cyt c in intact tissue.




Conclusion

Using the platform, we demonstrated the ability to measure Cyt c over time from live cuboids cultured with different drug conditions on our platform. Consequently, the platform could help us understand the temporal dynamics of Cyt c response to treatments in intact tissue and thereby shed light on predicting and determining the response of cancer to different treatments.


Acknowledgements (Optional): : This work was partially supported by a Brotman Baty Institute Fellowship (T.N.H.N) and by National Cancer Institute awards R01CA181445 and R01CA272677.


References (Optional): : 1. Harrison, R. K. Phase II and phase III failures: 2013–2015. Nat. Rev. Drug Discov. 15, 817–818 (2016).

2. Horowitz, L. F., Rodriguez, A. D., Au-Yeung, A., Bishop, K. W., Barner, L. A., Mishra, G., Raman, A., Delgado, P., Liu, J. T. C., Gujral, T. S., Mehrabi, M., Yang, M., Pierce, R. H. & Folch, A. Microdissected ‘cuboids’ for microfluidic drug testing of intact tissues. Lab Chip 21, 122–142 (2021).

3. Letai, A. Functional precision cancer medicine—moving beyond pure genomics. Nat. Med. 23, 1028–1035 (2017).

4. Ferguson, B. S., Hoggarth, D. A., Maliniak, D., Ploense, K., White, R. J., Woodward, N., Hsieh, K., Bonham, A. J., Eisenstein, M., Kippin, T. E., Plaxco, K. W. & Soh, H. T. Real-time, aptamer-based tracking of circulating therapeutic agents in living animals. Sci. Transl. Med. 5, (2013).

5. Dauphin-Ducharme, P., Yang, K., Arroyo-Currás, N., Ploense, K. L., Zhang, Y., Gerson, J., Kurnik, M., Kippin, T. E., Stojanovic, M. N. & Plaxco, K. W. Electrochemical Aptamer-Based Sensors for Improved Therapeutic Drug Monitoring and High-Precision, Feedback-Controlled Drug Delivery. ACS Sensors 4, 2832–2837 (2019).

6. Bruch, R., Kling, A., Urban, G. A. & Dincer, C. Dry film photoresist-based electrochemical microfluidic biosensor platform: Device fabrication, on-chip assay preparation, and system operation. J. Vis. Exp. 2017, 1–7 (2017).

7. El Hasni, A., Pfirrmann, S., Kolander, A., Yacoub-George, E., König, M., Landesberger, C., Voigt, A., Grützner, G. & Schnakenberg, U. Six-layer lamination of a new dry film negative-tone photoresist for fabricating complex 3D microfluidic devices. Microfluid. Nanofluidics 21, 1–9 (2017).

8. Fischer, L. M., Tenje, M., Heiskanen, A. R., Masuda, N., Castillo, J., Bentien, A., Émneus, J., Jakobsen, M. H. & Boisen, A. Gold cleaning methods for electrochemical detection applications. Microelectron. Eng. 86, 1282–1285 (2009).

9. Shafaat, A., Faridbod, F. & Ganjali, M. R. Label-free detection of cytochrome: C by a conducting polymer-based impedimetric screen-printed aptasensor. New J. Chem. 42, 6034–6039 (2018).

10. Xiao, Y., Lai, R. Y. & Plaxco, K. W. Preparation of electrode-immobilized, redox-modified oligonucleotides for electrochemical DNA and aptamer-based sensing. Nat. Protoc. 2, 2875–2880 (2007).

11. Arroyo-Currás, N., Dauphin-Ducharme, P., Scida, K. & Chávez, J. L. From the beaker to the body: Translational challenges for electrochemical, aptamer-based sensors. Anal. Methods 12, 1288–1310 (2020).

12. Downs, A. M., Gerson, J., Leung, K. K., Honeywell, K. M., Kippin, T. & Plaxco, K. W. Improved calibration of electrochemical aptamer-based sensors. Sci. Rep. 12, 1–10 (2022).