Paramagnetic particles for immobilization of metallothionein – promising biomarker of head and neck cancer

Metallothioneins (MTs) are a group of low-molecular weight (~6 -10kDa), cysteine-rich, evolutionarily conserved proteins, found in generally all life forms. The MTs are multi-functional and play crucial roles in the transport of essential trace elements as well as in the detoxification of harmful metallic elements ¹. Four major MT isoforms, MT-1, MT-2, MT-3 and MT-4, have been identified in mammals ². Specifically, MT-3 has been reported to be secreted mainly in neural cells. Recent reports established that this isoform play an important protective role in brain injury and metal-linked neurodegenerative diseases ³. In general, MT is known to modulate three basic processes: the release of hydroxyl radical or nitric oxide ⁴, apoptosis ⁵ and the binding and exchange of heavy metals such as zinc ⁶, cadmium ⁷ or copper ⁸. Previous studies have shown a positive correlation between the expression of MT with invasion, metastasis and poor prognosis in various cancers, including head and neck cancers ⁹, where increasing levels of MT concentration in tumor (vs. healthy tissues) was observed ¹⁰. Moreover; due to high affinity towards heavy metals, MT can be employed for modification of electrodes in development of electrochemical biosensors ¹¹. In view of these facts, immobilization of MT on paramagnetic particles (PMPs) may provide many possibilites, such as simplification of biosensing of low levels of MT, through its pre-concentration. The present study demonstrates immobilization of MT on a surface of paramagnetic particles (gold modified). For confirmation of MT presence two detection techniques were employed: matrix-assisted laser desorption/ionization - time of flight mass spectrometry and differential pulse voltammetry with adsorptive transfer technique utilizing Brdicka solution as supporting electrolyte. For binding capacity evaluation MT isolated from rabbit liver was employed and ideal particles for its immobilization were chosen, simultaneously with optimization of immobilization conditions (pH and temperature).

Figure 1. MALDI-TOF/TOF mass spectra of MT fractions after the fast protein liquid chromatography purification (concentration of MT - 2.0 μM). In analyses the linear positive mode, (A) HCCA and (B) DHB matrixes were employed.

Figure 2. MALDI-TOF/TOF mass spectra showing the results of screening of various types of paramagnetic particles (MANX) for their ability to bind MT (1 µM) on their surface. MS measurements were carried out in linear positive ion mode, with DHB matrix and laser power established to 80%.

Figure 3. (A) Typical voltammogram, obtained by using AdT DPV with Brdicka electrolyte, where (a) stays for Brdicka electrolyte (1 mM Co(NH3)6Cl3 and 1 M ammonia buffer (NH3(aq) and NH4Cl, pH = 9.6), (b) for bare MAN53, (c) for MT standard (500 nM) and (d) for MAN53 + MT conjugate. (B) Optimization of ideal binding conditions – pH of binding borate buffer (5.0 - 7.0). (C) Influence of temperature (10 – 37oC) on binding of MT on MAN53. All optimization conditions were evaluated by using AdT DPV. Values are means of three independent replicates (n = 3). Vertical bars indicate standard error.

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