A Disposable Amperometric Immunosensor for the Monitoring of Granzyme B in Blood Plasma Samples of Cancer Patients
Authors: Saputra Heru Agung , Seo Kyeong-Deok , Chung Jae Heun , Park Deog-Su , Shim Yoon-Bo 
Published: December 2020

Granzyme B (GzmB) is a cytotoxic protease found in the granules of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), which participates in inducing apoptosis of target cells for NK cells and cytotoxic CD8+ lymphocytes [1]. GzmB is the major effector of the CTLs and NK cells in its killing attack on cancer cells, although the level of GzmB expression and its cytotoxic potential decrease significantly in the presence of cancer. This decrement is due to a ‘pro-cancer’ environment, where the cancer cells secrete soluble mediators that could inhibit and thus contribute to the decreased GzmB by CD8+ T cells and a reduction in their ability to cause apoptosis of the cancer cells. Once GzmB is secreted from cytotoxic cells, it can be discovered in two different host compartments, namely inside the target cell or extracellularly [2]. Due to this, the change of GzmB level in blood plasma may serve as indication of the patient’s cancer cure progress. Hence, we developed a disposable amperometric GzmB sensor for the blood plasma samples.

In developing a robust electrochemical sensing platform, an efficient device is required. To enhance the electrochemical response, catalytic nanoparticles can be used as the electrode material, particularly nanocomposites composed of functionalized conducting polymers. They have been well-thought-of as the candidate of electrode substrate materials for the electrochemical sensing platform due to their ability to augment the electrical conductivity, electron-transfer rate, mechanical strength, surface area, and binding affinity to a specific molecule [3]. Therefore, 3’-(2-aminopyrimidyl)-2,2’:5’,2’’-terthiophene (PATT) bearing an amine group was electro-polymerized onto gold nanoparticles (AuNPs) modified electrode. Then, the stabilized immobilization of antibody on the SPCE/AuNPs/pPATT layer was accomplished through the formation of an amide bond between amine groups of the polymer and carboxylic acid groups of the antibody.

In this study, a disposable amperometric immunosensor composed of a sensing probe and a bioconjugate was developed for the detection of GzmB. The sensing probe was fabricated by immobilizing the GzmB monoclonal antibody (Ab1) on the polyPATT/AuNPs layer. The bioconjugate particle was synthesized by self-assembling the monomer mixture of 2,2:5,2-terthiophene-3-(p-benzoic acid) (TBA) and PATT onto AuNPs, then covalently bonding brilliant cresyl blue (BCB) on pTBA and GzmB polyclonal antibody (Ab2) on pPATT layer, respectively. The sensing layers were characterized using the surface analysis, cyclic voltammetry, impedance spectroscopy, and X-ray photoelectron spectroscopy.

A redox peak of the bioconjugate (Ab2-SAM-BCB) in the presence of GzmB in the sample was observed at -0.35/-0.37 V, corresponding to the redox reaction of the redox indicator BCB. Optimization of experimental parameters were carried out in terms of temperature (35°C), pH (7.4), the concentration of antibody (300 μg/ml), applied potential (-0.38 (reduction)), and binding time (20 min). At the optimized conditions, the developed immunosensor showed a dynamic range from 3.0 to 50.0 pg/ml and from 50.0 to 1000.0 pg/ml in two slops, with a detection limit of 1.75 ± 0.14 pg/ml. Interference effect, stability, and reproducibility were also evaluated. Reliability of the proposed immunosensor was evaluated through comparison of the results among GzmB concentration of lung cancer patients before and after medicine treatment, and healthy volunteer using human blood plasma samples, where the low level of GzmB was present in the blood plasma of lung cancer patients before medicine treatment (10.50 ± 1.02 pg/ml), which slightly increased level in patients after medicine treatment (15.85 ± 1.99 pg/ml), and the significantly higher level was present in the normal healthy individual (39.62 ± 0.79 pg/ml). Conclusively, the progress of cancer before and after the cure of the patients has been evaluated through monitoring GzmB concentration in the blood plasma.

[1] Kurschus, F.C., Jenne, D.E., 2010. Immuno. Rev. 235, 159-171.

[2] Boivin, W.A., Cooper, D.M., Granville, D.J., 2009. Lab. Invest. 89, 1195-1220.

[3] Naveen, M.H., Gurudatt, N.G., Shim, Y.-B., 2017. Appl. Mater. Today 9, 419–433.