Folic acid conjugated Prussian blue nanoparticles: Synthesis, physicochemical characterization and targeted cancer cell sensing

Journal Pre-proof Folic acid conjugated Prussian blue nanoparticles: synthesis, physicochemical characterization and targeted cancer cell sensing Oznur Akbal, Gulcin Bolat, Yesim Tugce Yaman, Serdar Abaci

PII:

S0927-7765(19)30799-4

DOI:

https://doi.org/10.1016/j.colsurfb.2019.110655

Reference:

COLSUB 110655

To appear in:

Colloids and Surfaces B: Biointerfaces

Received Date:

17 August 2019

Revised Date:

30 October 2019

Accepted Date:

21 November 2019

Please cite this article as: Akbal O, Bolat G, Yaman YT, Abaci S, Folic acid conjugated Prussian blue nanoparticles: synthesis, physicochemical characterization and targeted cancer cell sensing, Colloids and Surfaces B: Biointerfaces (2019), doi: https://doi.org/10.1016/j.colsurfb.2019.110655

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Folic acid conjugated Prussian blue nanoparticles: synthesis, physicochemical characterization and targeted cancer cell sensing

Oznur Akbala†, Gulcin Bolatb†, Yesim Tugce Yamana, Serdar Abacia,b*

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Advanced Technologies Application and Research Center, Hacettepe University, Ankara,

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Turkey b*

These authors contributed equally.

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Corresponding Author: Serdar Abaci

e-mail:*[email protected]

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Fax: +90-312-299 2163

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Phone: +90-312-780 7919

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Department of Chemistry, Faculty of Science, Hacettepe University, Ankara, Turkey

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Graphical Abstract

Highlights    

Folic acid doped Prussian blue nanoparticles (FA-PB NPs) was fabricated by one route co-precipitation technique The stability and cell affinity of Prussian blue nanoparticles were maintained by folic acid coating Pencil graphite electrodes (PGEs) were modified with fabricated FA-PB NPs to achieve electrochemical cancer cell (DLD-1) sensing FA-PB NPs coated PGEs is a promising material for cancer cell sensing application and demonstrated good analytical performance.

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Abstract In the study, folic acid doped Prussian blue nanoparticles (FA-PB NPs) for theranostic applications were synthesized for the first time. Folic acid was chosen for maintaining nanoparticle stability and also to increase its binding affinity especially for cancer cells.

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Multifunctional PB NPs were fabricated by one route co-precipitation method to synthesize

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biocompatible NPs without any further process. Then, FA was doped on the surface of PB NPs. The characterization studies demonstrated that the FA-PB NPs modified sensor surface

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had large surface area with biocompatible and hydrophilic properties where cancer cells can easily bind. The FA-PB NPs were used for the modification of pencil graphite electrode

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(PGE) for electrochemical detection of colon cancer cell (DLD-1). The detection was based on the specific interaction between FA groups on the nanoparticle and FA receptors

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overexpressed on cancer cells. The voltammetric and impedimetric results showed that the

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FA-PB NPs based electrode had good sensing performance for the immobilized DLD-1 cells.

Key words: Prussian blue nanoparticle; folic acid; colon cancer cell; modified electrode; electrochemical impedance.

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1. Introduction Prussian blue (PB) is an ancient blue color pigment, is one of the metal hexacyanoferrates compound that are often used in especially biomedical and electrochemical research areas as drug delivery systems, imaging and molecular sensing agents [1]. With the help of

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nanotechnology, nanosized PB achieved unique physicochemical properties that cannot be

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obtained in their bulk analogs. PB nanoparticles (NPs), one of these nanostructures, are biocompatible, biodegradable, affordable and can be effortlessly synthesized that make them a

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good candidate for theranostic applications [2]. On the other hand, these nanoparticles are approved by food and drug administration (FDA) for use in the treatment of radioactive

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exposure and poisoning [3]. PB based nanomaterials with different size and shape can be synthesized by different techniques such as matrix assisted assembly, hydrothermal, co-

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deposition and co-precipitation [4]. The co-precipitation method is a one-route technique which maintains a biosafe nanoparticle preparation that can be used specifically in biomedical

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areas.

The design of surface functionalization significantly influence the colloidal constancy

of PB NPs. Coating of PB compounds with biocompatible materials is very important for its fate which is critical in the body fluids as blood or serum [5]. Thus, folic acid (FA) was preferred to coat PB NPs for inducing the stability and also to facilitate targeting in cancer

cell diagnosis. Usually, cell receptors on the cancer cell surface that is used for targeting consists of lysophosphatidic acid receptor, transferrin, muscarinic receptor, tyrosine kinases and folate. FA can strongly and specifically adhere to folate receptors which are mostly overexpressed on the surface of cancerous cells, thus considered as efficient tumor targeting molecules. Based on this specific recognition and binding process between FA and folate, FAconjugated structures have commonly been exploited in drug delivery and imaging purposes. However their use in sensor technology is a still growing research area. [6–12]. Especially,

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cancer biomarkers and cell detection based FA modified sensor systems are becoming more prominent studies because the conventional methods which are based on tissue sampling as biopsy, imaging as magnetic resonance imaging or computed tomography require experts,

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high cost devices and long analysis time [8].

Electrochemical biosensors modified with nanomaterials are promising alternative

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system with unique advantages such as inherent miniaturization, portability, high sensitivity

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and rapid detection of cancer biomarkers or cells, have found a vital area in clinics for screening of cancer [13]. Nanostructured materials are employed as interesting transduction

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elements for monitoring biomolecular interactions on electrochemical sensors because they provide larger active surface area, remarkable stability, high sensitivity and good

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biocompatibility which are important prospects for recognition events. Various nanomaterials such as gold nanoparticles (AuNPs) [14], peptide nanotubes (PNT) [15], carbon nanospheres

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(CNSs) [16] and peptide nanoparticles PNPs [17] etc. have been applied for the development of electrochemical cytosensor platforms and recently some of these nanomaterials have been functionalized with cell targeting aptamers or folic acid [18–22]. Some of these nanoparticles require complicated synthesis method and show low-specific detection. Thus, synthesis of functional materials for cytosensing is still of great importance.

Up to now, due to their nanoscale sizes, large surface-to-volume ratios, high electrocatalytic activities, PB NPs and its analogues have been employed in efficient electrochemical sensing of important molecules such as glucose [23], hydrogen peroxide [24], vitamins [25], heavy metals [26], DNA [27,28] and antigens [29,30]. On the other hand, functionalization of PB NPs with FA and its characterization is a recent attractive topic. There is only one study incorporating PB NPs and FA together by loading an anticancer drug as chemo/photothermal therapeutic application [31]. To the best of our knowledge, no study about electrochemical

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cancer cell diagnosis strategy incorporating PB NPs has been reported. Considering the above-mentioned facts about FA-PBs (their nanosize, catalytic effect, facile synthesis, biocompatibility etc.) together with the strong binding property of FAs to cancer cells, we

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aimed to represent the advantages of these nanoparticles in cytosensing in terms of sensitivity, rapidity, bioactivity preservation and easy preparation. In the light of all this information, it

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was planned rapid, precise and cost-effective alternative sensing system based on FA-PB NPs

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for diagnosis of cancer cells.

This work involved the synthesized of FA doped PB NPs by co-precipitation method and

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their use for the modification of disposable single-used graphite surfaces for sensitive colon cancer cell (DLD-1) sensor application. The synthesized FA-PB NPs were investigated and

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elucidated by chemical and spectroscopic methods. By virtue of dispersed graphite layers on the surface of the pen tip, modification was accomplished by physical adsorption method.

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Different methodologies were performed to reveal the success of modification and cell immobilization onto the graphite surface based on microscopic, physiochemical, biocompatibility and electrochemical properties. The linear calibration curve was calculated by electrochemical impedance method (EIS) based on the measurement of resistance resulting from DLD-1 cells-surface interaction. Our research denoted that FA-PB NPs modified disposable sensor platform is highly promising for the sensitive cancer cell detection.

2. Experimental 2.1.Reagents Dulbecco’s Modified Eagle’s Medium (DMEM) F-12, fetal bovine serum (FBS) (Atlanta Biologicals, USA), penicillin–streptomycin, L-Glutamine (Gibco BRL, USA), trypsin/EDTA, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) were purchased from Biological

Industries

(Turkey).

Phosphate

buffered

saline

powder,

FeCl3·6H2O,

K4[Fe(CN)6]·3H2O, K3[Fe(CN)6]·3H2O, citric acid monohydrate (CAM), folic acid, 2-N-

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morpholinoethanesulfonic acid (MES), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) and acetone were gathered from Sigma (Germany). Fluorescein-5-isothiocyanate (FITC) and carbonate-bicarbonate buffer were purchased from

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Sigma (Germany). All the chemicals used in the experiments were employed without any modification process. Colorectal adenocarcinoma cell line (DLD-1) was provided by

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Hacettepe University Advanced Technologies Application and Research Center (HUNITEK),

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Turkey.

2.2.Fabrication of folic acid doped Prussian blue nanoparticles (FA-PB NPs)

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Folic acid doped Prussian blue nanoparticles (FA-PB NPs) were prepared with a procedure reported previously by Li et al. with minimal alteration [32]. In the procedure, 1 mM of

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FeCl3·6H2O was dissolved in 20 mL ultrapure water at 60 °C under stable stirring (600 rpm)

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after adding of 1 mM of CAM. K4 [Fe(CN)6] solution in ultrapure water (20 mL, 1 mM) containing 1 mM of CAM was then added dropwisely to the as-prepared solution. The reaction mixture turned to green color followed by bright blue colour, demonstrating the formation of PB NPs. After washing by centrifugation, the pellets were freeze-dried for further experiments. To attach the FA to the surface of the PB NPs, 0.01 M of MES buffer was prepared. 10 mg PB NPs was transferred to 10 mL MES buffer (pH 4.9) and a known amount of EDC/NHS was placed to the medium and preserved for 1 hour under constant

stirring. The particles were then precipitated at 12000 rpm and washed to remove unbounded EDC/NHS. Later on, an amount of FA was added to the nanoparticle solution and incubated nightlong at 4°C. The nanoparticles were again precipitated at 12000 rpm and stored for use in PGE surface modification. 2.3. Characterization of FA-PB NPs The morphology features of the FA-PB NPs was enlightened by scanning electron

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microscopy (SEM) (Tescan GAIA 3 FIB-SEM/Czech Republic) and EDS (Energy Dispersive X-Ray Spectroscopy). EDS was recorded to show the elemental composition of nanoparticles. To set the samples, 10 μL nanoparticle solutions were placed on a wafer and masked with 5 nm thick gold. Also, scanning transmission electron microscopy (STEM) images were

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collected by Tescan GAIA 3 FIB-SEM/Czech Republic operated at 20.0 kV, which was used

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to sight the nanostructure. To investigate the sample nanoparticle solution was placed on carbon double side adhesive on the STEM stab and analysed under 190 kx magnification. The

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average size distribution and zeta potential of the nanoparticles were assessed by Zetasizer Nano ZS instrument (Malvern Instruments, UK). The data were recorded at room temperature The chemical characterizations of the nanoparticles were

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and 173° backscatter angle.

recorded by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR)

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(Nicolet TM ISTM 50 spectrometer, Thermo Fisher Scientific, USA). The spectra were gathered from 4000 to 600 cm-1 with a resolution of 4. UV–vis–absorbance spectra of the PB

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and FA-PB NPs in a wavelength range of 190–800 nm was performed by UV-vis spectroscopy (Shimadzu, Japan). The thermal properties of the nanoparticles were performed by Thermogravimetric Analyzer (TGA) (Q600 STD TA® instruments Inc., New Castle). The NPs were heated in a platin pot under nitrogen gases at a flow rate of 100 mL/min. The analysis was performed between 30°C to 1000°C with a heating rate of 10oC/min. 2.4. Cell culture, viability assay and fluorescence microscopy analysis

The colorectal adenocarcinoma cell line (DLD-1) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) F-12 containing 10% fetal bovine serum (FBS), 1% penicillin– streptomycin and 1% L- Glutamine in an incubator at 37 °C containing 5% CO2 . The cells were cultivated in 75 cm2 flasks for 48 h and were collected with 0.25% trypsin/EDTA after reaching 80% confluency. Then, the cells were precipitated at 4000 rpm for 5 min and suspended. To measure the cytotoxicity of the PB and FA-PB nanoparticles together with FA-PB

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nanoparticles modified PGEs and bare PGEs were tested by MTT assay in which living cells convert the tetrazolium dye MTT to insoluble formazan crystals that is purple in color by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cellular oxidoreductase

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enzymes. To perform the test DLD-1 cells were treated with aforementioned materials with the concentration of 100 μg/mL and were grown in 12-well plates (n=3) at 100,000 cells/well

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for 48 h. Control cells were only treated with medium without any material. Then, MTT

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(0.5mg/mL) was added to the cells and cells were incubated for 4 h at 37 °C in a 5% CO 2. MTT solution that dissolves the formazan dye was added to each well and incubated for 30

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min. The absorbance values were attained at 570 nm with a microplate reader and the cell viability was calculated [33].

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To investigate the DLD-1 cells treated with nanoparticles, both PB NPs and FA-PB NPs were blotted with FITC, a fluorescent stain. To prepare the FITC-blotted NP samples, 0.1 mL

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of FITC solution (2 mg/mL in PBS, 7.4) was dropped to a known amount of NPs solution in carbonate buffer (0.01 M, pH 9.0) and then the mixture was conserved for 2 h in the dark at 4 °C. To diminish unbound FITC, FITC conjugated NPs were washed (12.000 rpm, 30 min) thrice and the pellet was suspended with cell medium. FITC/ NPs solution were applied on cells and the cells were conserved at 37 °C for 2 h in a 5% CO2 incubator. Then, cells were washed with PBS (pH 7.4), the cells were imaged with a fluorescence microscopy (Nikon,

Japan). Fluorescence signals were recorded at excitation/emission wavelengths of 488/530 nm for FITC [34]. 2.5.Preparation of cell sensor system The FA-PB NPs modified disposable cell sensing system was carried out by physical adsorption, which is a moderately easy method for modification. Thus, PGEs were immersed to FA-PB NPs solution for 1 h, unbound nanoparticles were removed by immersion in

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deionized water (DI water) for 2 s and electrodes were allowed to dry. The developed sensor surface was called as FA-PB NPs/PGE. To immobilize the cancer cells onto this electrode, FA-PB NPs/PGE surfaces were dipped in PBS containing different amount of cancer cells.

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After the immobilization step, graphite surfaces were immersed in DI water and dried. 2.6. Characterization-detection methods for FA-PB NPs/PGE

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The image of graphite surfaces after modification and cell immobilization were screened via

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SEM (Tescan GAIA 3 FIB-SEM/Czech Republic). The wettability of nanoparticles modified pencil graphite electrode (PGE) surfaces were evaluated with contact angle measurements in

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which the liquid nanoparticles droplet were deposited on a solid surface (PGE) with and without FA. A calibrated micro-syringe was used to place a drop of ultrapure water on the

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solid PGE surface modified with and without PB NPs and FA-PB NPs. Then, the images were taken by a camera attached to an optical microscope (Biolin Scientific, Attension Theta

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Modal, Sweden) that records the drop-substrate interface and gives an angle with the surface and water droplet.

All electrochemical analyses were taken via regular three electrode combination including

pencil graphite as a working electrode, Ag/AgCl and Pt wire as a reference and counter electrode, respectively. 0.5 HB graphite tips were purchased from a local stationer. Electrochemical characterization and detection methods were carried out by CHI Instruments.

The fabricated surfaces were electrochemically analysed by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in 5.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) medium (in pH 7.4 PBS). Conditions for this CV analysis: Einitial: -0.4 V, Efinal: 0.9 V (vs. Ag/AgCl), scan rate (sr): 100 mVs-1. Besides, PB NPs show electro-active properties so anodic/cathodic peak currents of iron groups in PB nanostructure bound to the electrode surface were proved by CV in the range of -0.4 V to 0.6 V (sr: 100 mVs-1) in pH 7.4 PBS. Electrochemical stabilization studies were carried out by CV in the range of -0.4 V to 0.9 V

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(sr: 100 mVs-1) at 50 cycles in pH 7.4 PBS. Also, cyclic voltammograms were recorded in pH 7.0 PBS with potential scanning from 0.0 V to 1.3 V (vs. Ag/AgCl) (sr: 100 mVs-1) to demonstrate the success of immobilization step by analysing of adenine and guanine electro-

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oxidation process. Electrochemical impedance spectra were measured in the frequency range 0.1 kHz to 1.0 MHz, with amplitude of 10 mV. After taking EIS measurements for cell

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detection at different concentrations, the calibration curve was obtained by using electron

calculated by using Rct values.

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transfer resistance (Rct) values. The linear working range and limit of detection values were

2.7. Detection of DLD-1 cells in peripheral blood medium

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DLD-1 cell is a kind of circulating tumor cell therefore they can be appear in peripheral blood that contains erythrocytes, leucocytes, and platelets that are found in the circulating blood

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pool and retained in the lymphatic system, spleen, liver or bone marrow [35]. Therefore as a

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real sample, peripheral blood medium was used to mimic the host organism. To prepare the sample, cells were collected by trypsinization with 0.25% trypsin/EDTA from the flask and were precipitated at 4000 rpm for 5 min. Then the cells were calculated with the amount of 103 and 104 cell/mL in peripheral blood medium.

3. Results and Discussion 3.1. Characterization of folic acid doped prussian blue nanoparticles (FA-PB NPs)

There are different preparation methods for PB nanoparticles (NPs) reported previously by different research groups including reverse micelle, aerosol co-deposition and templateassisted method using polymeric matrix, scaffold or any templates to interfere agglomeration and induce formation. However, some of the methods trigger the non-biocompatible nanoparticle formation with plurality of production steps. In this study, one route method (coprecipitation) was performed for the synthesis of nanoparticles including an agent, citric acid, both as nucleation destructive and as surface-capping agent to stabilize the nanoparticles, to

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prevent aggregation and to control the size [1,36]. Briefly, to synthesize PB NPs the aqueous solutions of FeCl3 and K4[Fe(CN)6] were mixed directly in the presence of citric acid. Then, folic acid was used to coat the nanoparticles to increase the stability and to induce targeting.

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After synthesis, the morphologic and elemental characterizations of synthesized FA-PB nanoparticles were performed with SEM-EDX microscopy. According to SEM image, the

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nanoparticles had monodisperse with a size of around 30 nm. To identify the elemental

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composition of FA-PB NPs, EDX analysis was employed. As it can be seen in the EDX spectra, the nanoparticles included carbon (C), oxygen (O), and nitrogen (N) arising from PB

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and FA and additionally potassium (K) and iron (Fe) from PB structure [37].

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Fig. 1. A) SEM image, B) EDX spectrum of FA-PB NPs. (Scale bar 200 nm and 100,000x

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nm.

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magnification) and STEM image of FA-PB NPs at different scale bar C) 100 nm and D) 50

STEM images demonstrated the morphology of the FA-PB NPs as cubic shape with a diameter of nearly 40 nm which was correlated with SEM results and literature [38]. According to DLS measurements, the average size of the PB and FA-PB NPs were recorded approximately as 12 nm and 25 nm, respectively, in accordance with the SEM results. Both nanoparticles had narrow size with good polydispersity index (PDI) as 0.257 and

0.450 for PB and FA-PB NPs, respectively. On the other hand, the zeta potential of the PB nanoparticles was recorded as −29.4 mV, because of the citric acid protection on the nanoparticle surface. After the FA was doped in PB NPs structure, the zeta potential was

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slenderly declined to −24.6 mV.

Fig. 2. A) Size distribution and B) Zeta potential of PB NPs and FA-PB NPs. C) FT-IR

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spectrum of a) Folic acid, b) PB NPs, c) FA-PB NPs. When the FTIR spectrum of PB NPs was analysed, the following information can be

deduced from the spectra. The absorption bands around 680 cm-1 was because of the Fe2+-CNFe3+ linkage of Prussian blue. Also, the strong band at 2065 cm-1 was the specific absorption peak of PB and was assigned to the stretching vibration of C≡N in potassium hexaferricyanide that proved Fe2+- CN-Fe3+ in PB. Also, the band at 1602.5 cm-1 was assigned

to the H‒O‒H bonding, which indicated the interstitial forces in PB NPs. With the conjugation of FA and PB NPs, the same absorption band was also observed in the FA doped PB NPs spectrum together with FA spectrum that demonstrated the N-H stretching with a small shift at 1603.4 cm-1 absorption frequency. In addition, besides the characteristic groups for PB NPs, the spectrum demonstrated all the characteristic groups for FA. Furthermore, the bands between 3600 and 3100 cm−1 are due to the hydroxyl stretching of L-glutamic acid residues and N-H stretching of pterin ring that could be seen both in FA alone and FA-PB

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NPs spectra. The bands at 2973, 2927 and 2840 cm−1 were attributed to –C-H stretching frequency while the band at 1639 cm−1 was attributed to C=O stretching of –CONH2 group. Also, the characteristic absorption peaks at 1684 and 1476.9 cm-1 observed in the spectrum of

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free FA and FA-PB NPs were assigned to the C=O stretching of the carboxyl group and absorption frequency of phenyl skeleton, respectively. The band at 1411 cm−1 of FA was

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assigned to O-H deformation band of the phenyl ring, whereas it appeared at 1406 cm−1 in the

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spectrum of FA-PB NPs with a small shift. As a result of all these experiments, it can be determined that FA interacted with PB NPs to form FA-BP NPs successfully.

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In nanoparticle based cell sensors, high surface affinity to cells as well as non-toxic effect are known as favorable factors to achieve cell viability. Therefore, MTT assay was

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applied to evaluate the percent cell viability. Toxic effect of the synthesized nanoparticles, electrode materials and modified electrodes were assessed according to cell viability with

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respect to control group in which the control cells were presumed to perform 100% viability. As a result, after 48 h treatment of cells, no significant toxic effect was observed on cells, also they displayed biocompatible feature. Cell viability percentages of nanoparticle-treated cells showed similar results to those of PGEs modified with these materials. For instance, while FA-PB NPs treated cells demonstrated 91.2% cell viability, the FA-PB NPs modified PGEs performed 93.9% (Fig. S1).

It is well known that nanoparticles assemble more expeditiously in tumor tissues or cells by enhanced permeability and retention (EPR) effect which results in passive targeting of nanostructures to tumor area and increase cancer cells targeting [39]. For this reason, to investigate the localization of nanoparticles, they were conjugated with FITC for imaging. After 2 h exposure of DLD-1 cells with FITC conjugated NPs, the images were taken. As it can be seen from the images, both nanoparticles modified with FITC (FITC/PB NPs and

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FITC/FA-PB NPs) were localized around cell membranes even after 2 h treatment (Fig. 3).

Fig. 3. A) Light microscopy image of DLD-1 cells. Fluorescence microscopy images of B)

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PB NPs treated DLD-1 cells and C) FA- PB NPs treated DLD-1 cells (20x magnification). The stimulation of surface plasmon vibrations in metal nanoparticles was carried out

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by collective stimulation of electrons in the conduction band close to the surface of nanoparticles, and the surface plasmon resonance of PB NPs was analysed by UV-Vis

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spectroscopy with a range of 190-800 nm. As it can be seen from the UV graph, the PB NPs

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possessed a broad absorption band with a strong absorption peak at ∼710 nm. Especially at 710 nm, a band formation due to charge transfer between Fe+2-CN-Fe+3 in the structure of PB NPs was observed. On the other hand, in the spectrum of folic acid there were two broad absorption peaks at 257 nm and 282 nm which were characteristic for folic acid that could also be seen in the spectrum of FA-PB NPs. A new peak observed at 282 nm was for FA whereas the peak at 257 nm was overlapped by FA-PB NPs [40–42].

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Fig. 4. UV spectrum of a) FA-PB NPs, b) PB NPs and c) FA. The thermal decomposition profile of FA-PB NPs under nitrogen atmosphere was performed

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by TGA from room temperature up to 1000°C. It is well known that PB is stable at high temperature but loses some of its mass as a result of three stages (Fig. S2). In first stage, the decrease was attributed to water loss from the PB structure. In the second and third

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stages, the mass decrease collected were due to cyanide (-CN) groups release from the PB

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structure. The remnant (37%) belonged to PB and TGA studies confirmed the potential interaction of PB according to mass change against temperature increase in time [43].

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3.2.Characterization of FA-PB NPs modified disposable graphite electrode

The FA-PB NPs was successfully synthesized and characterized by various methods. Then, the suitability of the synthesized material for the capture of colon cancer cells onto graphite surface was examined by SEM analysis (Fig. 5). As shown in Fig. 5A and inset, the SEM images obtained at higher and lower magnifications revealed that FA-PB NPs located on the

graphite surface with a homogeneous layer and an average diameter of NPs was 20 nm. The presence of FA-PBs nanostructures (Fig. 5A inset picture), which was intensely dispersed on PGE prior to cell immobilization was observed. DLD-1 cells were successfully immobilized on the surface after interaction with FA doped PB NPs (Fig. 5B). This result established that the FA-PB NPs modified PGEs provided an ideal environment for the adherence of colon

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cancer cells due to its excellent roughness and functionalization with FA.

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Fig. 5. SEM images of A) FA-PB NPs modified PGE (scale bar: 20 µM inset scale bar: 2 µM), B) 50,000 cells mL-1 DLD-1 immobilized onto FA-PB NPs/PGE (scale bar: 20 µM). C)

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Contact angles of a) PGE, b) PB NPs/PGE and c) FA-PB NPs/PGE.

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Previous studies showed that, when the developed cytosensor surfaces exhibited hydrophilic properties, the surface/cancer cells interaction increased [44]. Therefore, wettability properties of the fabricated surfaces were investigated by contact angle measurements and results were given in Fig. 5. Among the measured contact angles, FA-PB NPs/PGE surface had the lowest contact angle and most hydrophilic feature. This denoted that a suitable surface was established for cancer cells placement and immobilization.

Cyclic voltammetry method was carried out to reveal the alteration of sensor platform modified with FA-PB NPs (Fig. 6). Therefore, bare and modified electrodes were analysed in solution consisting of a redox pair. The physical adsorption of conductive PB NPs onto PGE surface caused the anodic/cathodic peak currents of redox pair to increase (Fig. 6A-b) when compared to bare PGE (Fig. 6A-a). Furthermore, anodic/cathodic peak potential values approached to each other which proved that the reversibility was better than the bare surface similar with the previous electrochemical characterization studies on PB nanostructures

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[28,29]. Functionalization of PB NPs with FA caused peak current values to decrease even lower values than the bare PGE surface (Fig. 6A-c), indicating that the electrical conductivity of the modified surface was reduced as an expression of the surface being successfully coated

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with FA-PB NPs.

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Fig. 6. Cyclic voltammograms of a) bare PGE, b) PB NPs/PGE, c) FA-PB NPs/PGE A) in 5 mM Fe(CN6)3−/4− (0.1 M KCl) redox probe solution, B) in pH 7.4 PBS. C) Cyclic voltammograms of FA-PB NPs/PGE swept for 50 cycles in redox probe. As shown in previous studies, PB NPs deposited on the electrode surface, presented electroactive properties [23,30]. Therefore, CV was taken to show the presence of one reversible peak of PB nanostructure in the buffer solution (Fig. 6B-b). As can be seen in the voltammogram, no peak formation was found at bare PGE (Fig. 6B-a). After FA doped in PB

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NPs structure, anodic/cathodic peak currents of the PB NPs decreased considerably and peak potentials were separated (Fig. 6B-c). This proved the presence of PB NPs and FA effectively on the surface. In addition, electrochemical stability was performed with CV method (Fig.

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6C). The redox pair peak currents did not change significantly after successive 50 cycles.

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Thus, FA doped PB NPs was shown to be electrochemically stable.

3.3.Electrochemical behaviour of cells adhered FA-PB NPs/PGE

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Cell binding affinity of the cytosensor was examined by CV in 0.1 M pH 7.4 PBS before and after cell immobilization (Fig. 7A). Two oxidation peaks were observed at FA-PB NPs/PGE

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in anodic scanning between 0.7 V and 1.0 V (vs. Ag/AgCl) due to the presence of FA, which exhibited electroactive properties, in the cytosensor structure. The absence of any reduction

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peaks indicated the irreversible electrode process in the reverse scan. The nature of the two-

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step irreversible oxidation of FA was also frequently indicated in the literature [45]. When DLD-1 cells immobilized onto FA-PB NPs/PGE, the peak at the +1.0 V (vs. Ag/AgCl) was blocked as a sign of efficient cell binding on the surface. This proved the formation of complex between FA on PBs and FA receptors on the cells. As can be seen in Fig. 7A-a, oxidation signal of guanine due to cell viability was monitored sharply at +0.75 V (vs. Ag/AgCl) [46]. Besides, a shoulder was observed at about +1.1 V (vs. Ag/AgCl) signed to oxidation signal of adenine (Fig. 7A-a). The FA-PB NPs modification accelerated the transfer

of electrons between the electro-active cell center and cytosensor platform, allowing the oxidation of adenine in the cell cytoplasm. Under the same conditions without cell immobilization at FA-PB NPs/PGE, no relevant peaks were observed as expected (Fig. 7A-b). DLD-1 cells were interacted with the bare electrode as a control group and cyclic voltammogram was recorded (Fig. 7A-inset). It was determined that DLD-1 cells interacted with the bare surface, but the voltammetric response of cells immobilized bare PGE was lower (Fig. 4.B). As a result of comparative experiments, FA doped PB NPs caused

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biospecific interaction with DLD-1 cells. Hence, the FA-PB NPs/PGE sensor showed

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sensitivity for the efficient adherence of cells, confirming the SEM results.

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Fig. 7. A) Cyclic voltammograms of a) 2.5 × 105 cells mL−1 immobilized FA-PB NPs/PGE,

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b) FA-PB NPs/PGE (Inset: a) 2,5 × 105 cells mL−1 immobilized PGE and b) PGE. B) Nyquist

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plots of a) PGE, b) FA-PB NPs/PGE and c) 5.0 × 104 cell mL−1 immobilized onto the FA-PB NPs/PGE.

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Although it was shown that the FA-PB NPs/PGE interacted more with DLD-1 cells by CV, this approach does not have adequate sensitivity for detection studies. Thus, electrochemical

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impedance spectroscopy (EIS), an impressive technique for monitoring the interface features of sensor surfaces, was carried out step by step analysis to investigate the effect of

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modification and cell binding on sensor response. Electron transfer resistance (Rct), calculated from the Nyquist plot semicircle diameter, recorded in the medium containing redox pair was shown in Fig. 7B. The Rct value of PGE was calculated to be 1462 Ω, while the Rct value of FA-PB NPs/PGE was greatly enhanced (2255 Ω) due to the presence of FA in the nanostructure. After immobilization of 5x104 mL-1 cancer cells onto FA-PB NPs/PGE (Fig. 7 B-c),

interfacial electron transfer and Fe (CN)

6

4-/3-

mass transfer were inhibited due to the attached

cells with insulating properties and high Rct value was recorded as 7802 Ω. It was seen that the obtained Rct values from the FA-PB NPs/PGE and the cell immobilized modified surface were well separated. Thus, EIS method was decided to be used, in which analytes can be detected even at very low concentrations, in the electrochemical diagnosis of DLD-1 cells because of the good separation of semicircle diameters.

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3.4. Detection principle of FA-PB NPs/PGEs for cancer cells EIS was utilized as a sensitive determination method for the electrochemical diagnosis of colon cancer cells. The FA-PB NPs/PGEs were incubated for 1 hour in solutions containing definite amount of cancer cells. Then, linear calibration range of fabricated sensor was

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practised using the Rct values obtained against increasing cell concentration. Rct values were

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recorded from the Nyquist curves which gradually increased up to 1.0x105 cells mL-1.

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Fig. 8. Nyquist plots of different concentration cells immobilized FA-PB NPs /PGE surfaces

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(5.0 × 102 to 1.0 × 105 cells mL−1). B) Linear calibration graph of developed sensor system.

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As it was seen from the Fig. 6, FA doped PB NPs modified cytosensor responded with a good linear impedimetric signal for DLD-1 cancer cells from 5.0 × 102 to 1.0 × 105 cells mL-1. The

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attained linear regression equation (Equation 1) as follows; 𝑹𝒄𝒕 = 𝟐𝟑𝟏𝟗. 𝟒 𝐥𝐨𝐠 𝑪 − 𝟑𝟎𝟏𝟑. 𝟖

𝑹𝟐 = 𝟎. 𝟗𝟖𝟓

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Limit of detection (LOD) and limit of quantification (LOQ) were estimated as 48 cell mL−1

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and 158 cell mL−1 by using slope of Equation 1. Circulating tumour cells usually occur in human blood system as 1- 3000 mL-1. Also the amount of erythrocytes is 109 [47]. From this point, the improved electrochemical detection strategy can be used in practical application in serum samples. The LOD value was comparable with the previously reported electrochemical cell sensors (Table S1). Also, 5 modified sensors were fabricated in the same way and reproducibility studies were carried out. As an indication of reproducibility, another critical

point of detection strategy, relative standard deviation (RSD) was determined as 3.77% (for n=5). Thus, this result proved that the impedimetric measurements were close to each other with a good precision. The experimental data occurred in this study, were comparable with previously published articles about diagnosis of various cancers of folic acid modified surfaces and electrochemical detection of DLD-1 cells (Table S1). 3.5.Detection of spiked DLD-1 from peripheral blood samples To mimic clinical samples, DLD-1 cells (1000 and 10000 cells mL−1) were spiked into

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peripheral blood medium. The spiked DLD-1 cells were immobilized at FA-PB NPs/PGE surfaces and then detected with EIS. The results in Table S2 showed that the recovery rate ranged from 97.6% to 108.4%, indicating that the developed FA-PB NPs based

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electrochemical cytosensor could distinguish DLD-1 cancer cells from the complex sample.

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4. Conclusion

This paper reports a one route co-precipitation method for the fabrication of folic acid doped blue

nanoparticles

(FA-PB

NPs).

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Prussian

Folic

acid

provided

a

prosperous

microenvironment to enhance the biocompatibility together with stability and cell binding

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affinity. To characterize the properties and electrochemical behaviours of the nanoparticles, SEM, EDX, ATR-FTIR, UV-Vis, Fluorescence imaging, contact angle and electrochemical

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(CV and EIS) techniques were performed. The low cost, one route fabrication technique, small size with good polydispersity index made folic acid doped Prussian blue nanoparticles

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can be a good candidate to be used in teragnostic applications. The fabricated FA-PB NPs exhibited excellent performance as cell sensor by using the electrochemical approach. The fabricated FA-PB NPs based sensor provided a fast response (∼ 1 min) with a wide linear DLD concentration range by simple preparation. To explore the feasibility of application in the complex blood sample, DLD-1 cells were added into the peripheral blood medium and quantified by FA-PB NPs based electrochemical cytosensor. The obtained results

demonstrated that the developed cytosensor could distinguish DLD-1 cancer cells from the complex sample with a good recovery values. The nanostructured FA-PB can also be employed for other applications such as drug delivery, imaging or sensor applications.

Conflicts of interest

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There are no conflicts of interest to declare.

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