Metodología propuesta

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the spirit of biosensing experiments are presented. Here, detection of chemical parameters like the pH value of a medium or ionic concentrations of buffers can be utilized to identify metabolism of living microorganisms and to track the kinetics of a chemical reaction leading both leading to time-dependent alterations of the ionic composition of the medium. The utilization of the pH value and ionic concentrations in sensing experiments will be discussed in detail in the following paragraph. Additionally, a compact overview of one family of leukocytes are presented and why their detection and accompanied composition analysis has a high impact in the fields of biomedicine and medical assays.

2.5.1 Peripheral blood mononuclear cells (PBMCs)

Every higher-developed living organism has a developed system, namely the immune system, for defense against pathogenic intruders which enter an organism through its interactions with the environment. In humans, this system consists of two different branches: The static innate non-specific immune system and the adaptive immune system having high specificity and memory against pathogens. In both branches, all cells (also called leukocytes) of the immune system develop from a shared omnipotent stem cell in the bone marrow144_.

During development, two ways of cell differentiation can be observed: the myeloid development of e.g. granulocytes and monocytes, which main task is phagocytosis of pathogenic intruders. Second, T- and B-lymphocyte maturation, responsible for antigen recognition, can be found in the lymphoid development. In the following, the focus will be set on one family of immune cells, the peripheral blood mononuclear cells (PBMCs), which act in the adaptive immune response branch and consist of lymphocytes and monocytes. Lymphocytes consist of three populations: B- T- and natural killer (NK) cells. Here, the nomination is based on the spatial origin of differentiation, namely Thymus for T cells and Bursa Fabricii for B cells (only found in birds, located in the bone marrow in humans). The main task of B cells is the secretion of immune globulins upon recognition of the antigen thereby maturing in plasma cells. On the other hand, T cells are divided into several sub-groups fulfilling several tasks in the adaptive immune response, including maturation of B cells, activation of cytotoxic T cells, destruction of infected cells, immune response memory and many more. Further, NK cells have the task to activate cells of the phagocytosis system upon infection as well as destruction of virus-infected cells. Finally, monocytes are progenitors of

25 macrophages, responsible for destruction of foreign pathogens as well as presentation of protein-antigens upon digestion145_{. Here, analysis of transcription}

and translation levels of PBMCs as well as their percentage composition of its subfamilies grant insight into the detection and identification of diseases, e.g. several types of cancer146–148_{, infections}149,150 _{and various auto-immune}

diseases151_.

For instance, several types of leukemia, a type of cancer located in the bone marrow, can be detected by analysis of the PBMC subpopulations. The disease affects the composition, appearance and functioning of white blood cells causing a malfunctioning and weakened immune system with an increased risk of infections of the diseased person152_{. Over 350,000 new leukemia cases are}

estimated worldwide and it represents the 10th deadliest cancer type with over 265,000 deaths worldwide, most dominantly in developed countries153_{. The}

general origin of leukemia is mostly unknown and only few hypotheses try to point out the multiple factors, e.g. ionizing radiation and exposure to benzene and anti- cancer drugs154_{. Within the four main types of leukemia, acute myeloid leukemia}

(AML) represents the most abundant type with around 80% of cases155 _{and is}

classified by a heterogeneous disorder caused by the loss of differentiation ability of haemopoietic progenitor cells156 _{and accompanied reduced white blood cells}

count and performance157_{. Symptoms include anaemia, leukopenia und}

accompanied higher rate of infections. AML patients only have a five-year survival rate of 20-30%, strongly reduced for aged patients over 65 years even with clinical treatment154,155_.

2.5.2 Physical parameters

Next to detection of nano- and microscopic biological and molecular species, detection and determination of ionic concentrations, i.e. molar concentrations of ions and molecules as well as the pH value, represents a substantial liquid parameter with high biological relevance. In this, biological living and accompanied correct functioning of chemical reactions, metabolism, protein folding and interactions and enzyme function depend on the ionic concentrations of the surroundings158,159_{. Thus, precise determination of these liquid parameters}

plays an important role in biotechnology, biomedicine, pharmaceutics and biochemistry. Here, the pH value is used for determining the acidity or basicity of aqueous solutions, ranging from pH 1 (acidic) to pH 14 (basic) and is defined by

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the negative decimal logarithm of the hydrogen ion activity160_:

𝑝𝐻 = −𝑙𝑜𝑔10(𝑎𝐻+) (5)

With respect to biological systems, the pH value both acts as a regulator and parameter in various fields. During development, maturation and metabolism of both eukaryotic and prokaryotic cells, chemical parameters of their surroundings play an important role in case of correct information pathways, induced changes of the surroundings during development or metabolism of the cells. For instance, the extracellular pH value, or the concentration of H+ _{ions in a medium, has a}

dramatic effect on bacterial and fungi growth rates161,162_{, metabolite production}

and consumption161,163 _{and correct maturation of stem cells}164_{. Next to the}

importance of the aqueous pH values, the molar concentration, or molarity, plays an important role for correct function of chemical reactions as well as survival conditions for biospecies in solution. It represents the concentration of a chemical in solution per unit volume (𝑚𝑜𝑙

𝐿 ) and is defined by:

𝑐 = 𝑚

𝑉𝑀_𝑊 (6)

Where m is the mass and MW the molecular weight of the solute and V the volume

of the solution. It is the driving parameter for the solution’s compatibility to be suitable for cell incubation since it defines the osmotic pressure acting on suspended biospecies. Thus, if the ionic concentration of the solution is too high or low compared to the inner cell ionic concentration, bursting or shrinking of the suspended cell will follow165_{. Thus, precise determination of the molar}

concentration has to be done for correct incubation of prokaryotic and eukaryotic cells. Further, the molar concentrations of chemicals have to be precisely set in order to guarantee correct environment conditions for many chemical reactions. In conclusion, detection of above mentioned liquid parameters represents an important technique to guarantee the successful operation of various laboratory assays in biomedicine, pharmaceutics and biochemistry. Additionally, these parameters define the survivability of microorganisms in suspension and thus efficiency of cell survival in biological assays as. Thus, determination of liquid parameters is substantial for a wide variety of scientific and industrial protocols, where their values have to be precisely set.

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