Evaluating Viscoelastic Properties and Membrane Electrical Charges of Red Blood Cells with Optical Tweezers and Cationic Quantum Dots – Applications to β-Thalassemia Intermedia Hemoglobinopathy

Carinna N. Lima, Diogenes S. Moura, Yandilla S.S. Silva, Tiago H. Souza, Fabiano A.P. Crisafuli, Diego C.N. Silva, Jaqueline C. Peres, Carlos L. Cesar, R.E. de Araujo, Adriana Fontes

Biomechanical and electrical properties are important to the performance and survival of red blood cells (RBCs) in the microcirculation. This study proposed and explored methodologies based on optical tweezers and cationic quantum dots (QDs) as biophotonic tools to characterize, in a complementary way, viscoelastic properties and membrane electrical charges of RBCs. The methodologies were applied to normal (HbA) and β-thalassemia intermedia (Hbβ) RBCs. The β-thalassemia intermedia disease is a hereditary hemoglobinopathy characterized by a reduction (or absence) of β-globin chains, which leads to α-globin chains precipitation. The apparent elasticity (μ) and membrane viscosity (ηm) of RBCs captured by optical tweezers were obtained in just a single experiment. Besides, the membrane electrical charges were evaluated by flow cytometry, exploring electrostatic interactions between cationic QDs, stabilized with cysteamine, with the negatively charged RBC surfaces. Results showed that Hbβ RBCs are considerably less elastic, have a higher ηm, and presented a reduction in membrane electrical charges, when compared to HbA RBCs. Moreover, the methodologies based on optical tweezers and QDs, here proposed, showed to be capable of providing a deeper and integrated comprehension on RBC rheological and electrical changes, resulting from diverse biological conditions, such as the β-thalassemia intermedia hemoglobinopathy.

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Study of non-covalent interactions on dendriplex formation: Influence of hydrophobic, electrostatic and hydrogen bonds interactions

María Sánchez-Milla, Isabel Pastor, Marek Maly, M. Jesús Serramía, Rafael Gómez, Javier Sánchez-Nieves, Félix Ritort, M. Ángeles Muñoz-Fernández, F. Javierde la Mata

The interaction of a double stranded small interference RNA (siRNA Nef) with cationic carbosilane dendrimers of generations 1–3 with two different ammonium functions at the periphery ([−NMe2R]+, R = Me, (CH2)2OH) has been studied by experimental techniques (zeta potential, electrophoresis, single molecule pulling experiments) and molecular dynamic calculations. These studies state the presence of different forces on dendriplex formation, depending on generation and type of ammonium group. Whilst for higher dendrimers electrostatic forces mainly drive the stability of dendriplexes, first generation compounds can penetrate into siRNA strands due to the establishment of hydrophobic interactions. Finally, in the particular case of first generation dendrimer [G1O3(NMe2(CH2)2OH))6]6+; the presence of hydroxyl groups reinforces dendriplex stability by hydrogen bonds formation. However, since these small dendrimers do not cover the RNA, only higher generation derivatives protect RNA from degradation.

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Surface charge and hydrodynamic coefficient measurements ofBacillus subtilis spore by Optical Tweezers

Giuseppe Pesce, Giulia Rusciano, Antonio Sasso, Rachele Isticato, Teja Sirec, Ezio Ricca

In this work we report on the simultaneous measurement of the hydrodynamic coefficient and the electric charge of single Bacillus subtilis spores. The latter has great importance in protein binding to spores and in the adhesion of spores onto surfaces. The charge and the hydrodynamic coefficient were measured by an accurate procedure based on the analysis of the motion of single spores confined by an optical trap. The technique has been validated using charged spherical polystyrene beads. The excellent agreement of our results with the expected values demonstrates the quality of our procedure. We measured the charge of spores of B. subtilis purified from a wild type strain and from two isogenic mutants characterized by an altered spore surface. Our technique is able to discriminate the three spore types used, by their charge and by their hydrodynamic coefficient which is related to the hydrophobic properties of the spore surface.

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A new method for the covalent attachment of DNA to a surface for single-molecule studies

Daniel J. Schlingman, Andrew H. Mack, Simon G.J. Mochrie and Lynne Regan

Attachments between DNA and a surface or bead are often necessary for single-molecule studies of DNA and DNA–protein interactions. In single-molecule mechanical studies using optical or magnetic tweezers, such attachments must be able to withstand the applied forces. Here we present a new method for covalently attaching DNA to a glass surface, which uses N-hydroxysuccinimide (NHS) modified PEG that is suitable for high-force single-molecule mechanical studies. A glass surface is coated with silane-PEG-NHS and DNA is covalently linked through a reaction between the NHS group and an amine modified nucleotide that has been incorporated into the DNA. After DNA attachment, non-reacted NHS groups are hydrolyzed leaving a PEG-covered surface which has the added benefit of reducing non-specific surface interactions. This method permits specific binding of the DNA to the surface through a covalent bond. At the DNA end not attached to the surface, we attach a streptavidin-coated polystyrene bead and measure force-versus-extension using an optical trap. We show that our method allows a tethered DNA molecule to be pulled through its overstretching transition (>60 pN) multiple times. We anticipate this simple yet powerful method will be useful for many researchers.

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Mechanical property analysis of stored red blood cell using optical tweezers

Yanjie Li, Cheng Wen, Huimin Xie, Anpei Ye and Yajun Yin

The deformation of human red blood cells subjected to direct stretching by optical tweezers was analyzed. The maximum force exerted by optical tweezers on the cell via a polystyrene microbead 5 μm in diameter was 315 pN. Digital image correlation (DIC) method was introduced to calculate the force and the deformation of the cell for the first time. Force–extension relation curves of the biconcave cell were quantitatively assessed when erythrocytes were stored in Alsever's Solution for 2 days, 5 days, 7 days and 14 days respectively. Experiment results demonstrated that the deformability of red blood cells was impaired with the stored time.

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