key: cord-0691216-ayg1byl5
authors: Yıldırım, Mehtap; Baydemir Peşint, Gözde
title: Molecularly imprinted spongy columns for Angiotensin(II) recognition from human serum
date: 2020-12-30
journal: Biotechnol Prog
DOI: 10.1002/btpr.3112
sha: 801f72e809f9a3ea4dd395ffdd75034319cd6a12
doc_id: 691216
cord_uid: ayg1byl5

Angiotensin II (AngII), the effector peptide of the renin angiotensin system and has an important role in regulating cardiovascular hemodynamics and structure. AngII is an important biomarker for certain diseases that are associated with cardiovascular disorders, i.e., influenza, SARS‐CoV‐2, tumors, hypertension, etc. However, AngII presents in blood in very low concentrations and they are not stable due to their reactivity, therefore spontaneous detection of AngII is a big challenge. In this study, AngII‐imprinted spongy columns (AngII‐misc) synthesized for AngII detection from human serum, and characterized by surface area measurements (BET), swelling tests, scanning electron microscopy (SEM), FTIR studies. AngII binding studies were achieved from aqueous environment and maximum binding capacity was found as 0.667 mg/g. It was calculated that the AngII‐miscs recognized AngII 8.27 and 14.25 times more selectively than competitor Angiotensin I and Vasopressin molecules. Newly produced AngII‐misc binds 60.5 pg/g AngII from crude human serum selectively. It has a great potential for spontaneous detection of AngII from human serum for direct and critical measurements in serious diseases, that is, heart attacks, SARS‐CoV‐2, etc.

AngiotensinII (AngII) is an active hormone of the Renin angiotensin System (RAS) and has an important role within the complex network of other endocrine, paracrine systems, where RAS affects many tissue and organ systems. [1] [2] [3] AngII effects include vasoconstriction, aldosterone release, antidiuretic hormone synthesis, sympathetic activation, and salt absorption from kidney tubules. 4 However, when the RAS system disorders occur, all these effects could lead to the development of hypertension. The most important effect in hypertension is the direct contraction effect of AngII on arterial smooth muscles. It also causes irregularity in endothelial cells, medial hypertrophy and increase in connective tissue, causing atherosclerosis, growth of myositis in the heart muscle, development of left ventricular hypertrophy and development of heart failure. 3, 5, 6 Moreover, current studies showed that AngII levels increase, due to the Severe Acute Syndrome Coronavirus-2 (SARS-CoV-2) triggers the down regulation of angiotensin converting enzyme 2 (ACE-2). 7, 8 That is why AngII is an important biomarker for certain diseases, i.e., heart attacks, SARS-CoV-2, influenza infections, tumors, etc., that are associated with hypertension, hypotension. 9 However, AngII presents in blood in very low concentrations and they are not stable due to their reactivity, therefore spontaneous detection of AngII is a big challenge. [10] [11] AngII is mostly detected in biological fluids using HPLCradioimmunoassay (RIA), 12 enzyme-linked immunosorbent assay (ELISA) 13 and LC-MS/MS 14 methods. Although these methods detect AngII for diagnosis, they have important drawbacks, i.e., time consuming, laborious, includes hazardous radioactive materials (for RIA), limited storage stability (for ELISA). That is why there is still a vital need to develop alternative methods for direct detection of AngII from body fluids. 10, 11 Molecular imprinting is a technique, which is used for recognizing a target molecule (e.g., ion, protein, peptides, etc.) from a complex media, in a single step with high selectivity. Basically molecularly imprinted polymers designed in three steps; An interaction occurs between the functional monomers and the template molecule. The functional monomer-template complex is polymerized in presence of crosslinkers and monomers. And the target molecule is removed from the polymer to get target specific cavities with the shape and chemical structure memory towards target. [15] [16] [17] The fact that they can be stored for several years without any change in their performance, they are reusable materials. Their advantages enabled them to be used as synthetic recognition elements in many application fields, i.e., diagnosis, separation, purification, in health, food, environment, etc. [18] [19] [20] Although imprinting of many small molecules is successfully applied in MIPs, there are still difficulties in the printing of large molecules such as protein. So far, polymerization methods such as bulk (3D), surface (2D) or partial (epitope) printing has been used for proteins. 21, 22 Cryogels are soft materials which are synthesized at subzero temperatures (between 0 and −20), with three-dimensional sponge-like elastic morphology and pore sizes from a few microns to several hundred microns. 23, 24 Interconnected macrospores provide low flow resistance property to the structure and thus the spongy structure allows to work without difficulty even with viscous liquids. 25, 26 When these properties of spongy columns are combined with the molecular printing technique, it is possible to obtain advantageous materials with high selectivity that can provide ease of working with viscous liquids such as blood. 27, 28 In this study we offer an alternative method for direct AngII detection from Human serum in a single step with high selectivity. For this purpose we prepared PHEMA based molecularly AngII imprinted spongy columns.

The selectivity studies were performed against AngI and VASP, and AngII detection was achieved from human serum as well. 

AngII-molecularly imprinted spongy column (AngII-misc) was prepared as follows, AngII-VIM complex was prepared by dissolving AngII and functional monomer VIM (31 mg) in 1/10 n/n mol ratios in 1 ml of water and stored at +4 C for 12 hr to complete complexation. One milliliter of HEMA and 0.25 g MBAA was dissolved in 5 and 6 ml of water, respectively, to obtain 10% monomer concentrations. Then prepared pre-complex was added into that mixture. Finally 150 μl of APS solution (10% w/w) and 10 μl of TEMED were added to the polymerization mixture as initiator and mixed in the ice bath. Then the prepared mixture was equally divided into 3 ml syringes and frozen in a cryostat at −16 C for 10 hr to complete the polymerization process.

Polymers taken from the cryostate and left to room temperature for melting ice crystals in frozen polymeric columns and let them form interconnected macropores to get spongy columns. Non-imprinted spongy columns (Nisc) were prepared in the same way without using AngII, these columns functioned as a control group. The prepared columns were washed with ethanol (30:70, v/v) for 30 min and water for 10 min, respectively, at room temperature to remove unreacted monomers and impurities.

AngII molecules removed from the AngII-misc to get AngII specific cavities. Twenty milliliters of desorption buffer PBS buffer (10 mM, pH 7.4) was passed through the column for 2 hr continuously. At the end of this cycle a washing step was applied by passing 20 ml of water through the column. All measurements were done at 283 nm, using UV-spectrophotometer (Genesys 150, Thermo Fisher Scientific).

These steps repeated until no AngII detected in elution solution, spectrophometrically. The template removal amount was calculated using;

In here, Q TR (mg/g) is removal amount of template from unit mass of dry polymer, C i and C f are AngII concentrations before and after treatment of desorption agent with AngII-misc, V (ml) is volume of desorption agent and m (g) is mass of dry polymer. Removal ratio is calculated using:

AngII Removal Ratio % ð Þ = Removed AngII amount from AngII AngII amount in unit mass of AngII−misc polymerization mixture ð2Þ

Template-removed AngII-misc were washed with water using a peristaltic pump for 30 min at room temperature and stored in water at +4 C until use.

Water uptake ratio of AngII-misc and Nisc were determined by swelling tests. Briefly; the AngII-misc and Nisc were dried in lyophilizer and weighed with an accuracy of ±0.0001 and placed in a beaker containing 20 ml of water for 2 hr at 25 ± 0.5 C at constant temperature. Then it was removed from the beaker and weighed by removing excess water from the surface. Dry and wet weights are averaged of three repeated operations, the water uptake ratio of the material is determined with the following equation:

W 0 and W s are the weights of the AngII-misc/Nisc before and after swelling in g, respectively.

In order to determine the macropore amount of AngII-misc/Nisc, they were immersed in water and swollen samples weighed (W 1 ) in g. Then, swollen cryogel sample was squeezed delicately to remove water in macropores of the cryogels and weighed (W 2 ) in g. The macropore amount of AngII-misc and Nisc was calculated using the equation below.

The polymerization efficiency of the prepared cryogels is calculated using the dry weight of produced cryogels (W p ) and the weight of total reactant monomers (W m ) used for polymerization. Polymerization yield calculated as follows:

Synthesized AngII-misc and Nisc morphologies were character- 

AngII binding onto AngII-misc and Nisc from aqueous solutions was performed by a continuous system using a peristaltic pump, and all experiments were repeated three times and average of obtained results were reported. The effects of the equilibrium AngII concentration onto AngII binding was investigated in the range of 0.01-0.2 mg/ml AngII concentrations. The effects of binding time onto

AngII binding capacity was investigated in the range of 0-120 min and the effect of flow rate was studied 0.5-4 ml/min. In each step, columns were equilibrated with PBS buffer by applying 5 ml of PBS to the column. All AngII solutions were prepared in PBS buffer (10 mM, pH 7.4). All parameters were evaluated using UVspectrophotometer at 283 nm and the binding capacities were calculated using:

Here, Q is binding capacity (mg/g), C o and C f are AngII concentrations of the AngII solutions before and after the interaction with AngII-misc and Nisc columns (mg/ml), V is volume of the solution (ml), m is the dried mass of the column (g).

The selectivity of AngII-misc against AngII molecules was examined by selectivity tests. In this study, the binding behavior of the AngII molecule was compared against competitor AngI and VASP molecules. 

After each desorption step, excess of water was pumped through the AngII-misc and Nisc for washing and columns were equilibrated with PBS buffer at pH 7.4 for 10 min before reuse.

Swelling tests were performed for analyzing water uptake ratio, and macropore amount. The surface morphology of the prepared Ang-misc and Nisc samples were examined by scanning electron microscopy (SEM) that provides high magnification. In this study, Figure 1a C N stretching band was also shifted left after complexation and C N C aromatic ring stretching peak shifted higher frequencies and intensity of that peak increased. These changes showed that the precomplex was formed between AngII and VIM. According to FTIR results of AngII-MIP; common bands from HEMA monomers are seen at around 3400 cm −1 ( OH stretch band) and 1700 cm −1 ( C O stretch band) clearly. When the spectra examined, characteristic imidazole ring stretching vibration band at around 1530 cm -1, ring vibration at around 1249, 1153, and 1076 cm −1 C H bending in plane ring can be seen clearly. These data confirmed the presence of VIM in AngII-misc.

Template removal ratio was calculated as 85% AngII from the AngIImisc and removal amount found as 680 μg AngII/g AngII-misc.

The effect of equilibrium AngII solution concentration on maximum 

Q is the capacity of molecules that bind to the material (mg/ml), The Freundlich isotherm assumes that adsorption occurs physically and reversibly on heterogeneous surfaces. According to this isotherm model, adsorption is multi-layer adsorption, the surface is heterogeneous and binding sites are not equal energetically. The

Freundlich isotherm model is an exponential equation and assumes that adsorption occurs through multiple layers instead of a single layer. 31, 33 Linearized Freundlich equation can be given as:

In this equation, Q eq is the amount of adsorption (mg/g) and C e is the equilibrium concentration in the solution (mg/L). K f and 1/n are Freundlich constants, which are indicating adsorption capacity and adsorption intensity. Experimental data were adapted to the Freundlich model and lnC eq was plotted against lnQ eq . Adsorption constants were calculated from the cut-off point and slope. 33 values showed that the adsorption data is more compatible with Langmuir isotherm, and it can be concluded that the monolayer adsorption is favorable for AngII-misc. These results also in agreement with 

In order to determine the effect of interaction time on maximum AngII binding amount of the column, 3 ml of 0.05 mg/ml AngII solution was interacted with the column with a flow rate of 0.5 ml/min at pH 7.4.

Samples were taken for 2 hr with certain time intervals and the maximum binding time interval was determined. It was observed that the maximum binding was reached at 60th min. and binding time was taken as a basis in all subsequent experiments ( Figure 5 ).

Time-binding relationship used in kinetic model calculations to understand adsorption controlling mechanisms. In here pseudo-first order kinetic and pseudo second-order kinetic equations were used for the adsorption of an analyte from its aqueous solution. The pseudo first-order kinetic model calculated using Lagergren's equation; Δq t /dt = k 1 (q eq − q t ) and linearized as log(q eq − q t ) = log (q eq ) -(k 1 t)/2.303. In here, k 1 is first order adsorption rate constant (min −1 ); q eq and q t are AngII adsorption amounts at equilibrium and at time t (mg/g), respectively. The pseudo-second order equation based on adsorption equilibrium capacity expressed as; Δq t /dt = k 2 (q eq − q t ) and can be linearized as (t/q t ) = (1/k 2 q eq 2 ) + (1/q eq ) t. Here k 2 is pseudo-second order adsorption rate constant (g mg −1 /min). The rate constants k 1 , k 2 , and equilibrium adsorption amounts q eq obtained F I G U R E 3 Effect of equilibrium initial AngII concentration on AngII binding amount. intercepts and slopes of log(q eq − q t ) vs t plot for first order ( Figure 5b ) and (t/q t ) vs t plots for second order (Figure 5c ), respectively and summarized in Table 3 .

According to results shown in Table 3 the adsorption process can be expressed via second order mechanism. It is obvious that the R 2 of pseudo second order is 0.99 greater than that of pseudo first order; moreover the theoretical pseudo second order q e (0.688 mg/g) is closer to experimental q e (0.667 mg/g) than that of pseudo first order (2.024 mg/g). As a result, pseudo-second order mechanisms control the adsorption process via chemisorption rather than diffusion, obtained results support the specific interactions occur between AngII and AngII-misc via size and chemical structure. 31,34

The effect of flow rate on AngII binding was investigated. 0.05 mg/ml AngII in PBS (10 mM, pH 7.4) solution was applied to the column at different flow rates; 0.1, 0.5, 1.0, and 2 ml/min. As shown in Figure 6 , adsorbed AngII amount was decreased by increasing flow rate, due to limited interaction between column and AngII molecules. The maximum

AngII binding capacity of the appropriate flow rate was determined as 0.5 ml/min, and the rest of the study was performed at this point.

Selectivity experiments were done for demonstrating the selectivity of AngII-misc towards Ang II and competitors; AngI and VASP. Figure 7 summarized the selective binding amounts of all molecules onto AngII-misc and Nisc. This figure showed that the AngII binding amount is higher for AngII-misc than competitors. This is an evidence for selective binding and obtained data also used for calculation of distribution coefficient K d , imprinting factor If and selectivity coefficients k, for evaluating selectivity in detail. 34, 35 The distribution coefficient (K d , ml/g) calculated according to following equation:

Here C i is initial solution concentration (mg/ml); C f is concentration of the solution after interaction with the column (mg/ml); V is volume of solution (ml) and m is dry column weight (g). 

In order to examine the reusability of the produced columns, the AngII solution in a certain concentration was applied to the same column 10 times and the results obtained after 10 binding desorption cycles are shown in Figure 8 . The decrease in capacity of AngII binding of the columns is negligible (3%) and obtained results showed that the synthesized AngII-misc can be used several times without any capacity decrease or any structural change.

AngII, an important biomarker in human blood for cardiovascular diseases, SARS-CoV-2, influenza infections, tumors, etc. AngII-misc was synthesized for the detection of AngII from human serum for the T A B L E 3 Adsorption kinetic model constants and adsorption amounts

Exp. Pseudo-first-order-kinetic Pseudo-second-order-kinetic Initial conc. (mg/ml) q eq (mg/g) k 1 (1/min) q eq (mg/g) R 2 k 2 (1/min) q eq (mg/g) 

The peer review history for this article is available at https://publons. com/publon/10.1002/btpr.3112.

Data available on request from the authors.

Gözde Baydemir Peşint https://orcid.org/0000-0001-8668-8296

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How to cite this article: Yıldırım M, Baydemir Peşint G. Molecularly imprinted spongy columns for Angiotensin(II) recognition from human serum