Methylene Blue

A Trendy Photosensitizer in Medicine and in Solar-Energy Conversion Systems

Filipa Pires, Margarida Coelho, Paulo A. Ribeiro and Maria Raposo

Centre of Physics and Technological Research, CEFITEC, Department of Physics, Faculdade de Ciências e Tecnologias,

FCT, Universidade Nova de Lisboa, Caparica, Portugal

Keywords: Layer-by-layer, LbL, Methylene Blue, DNA, UV-Vis Spectroscopy, Solar Cell, Photodynamic Therapy.

Abstract: The photosensitizer methylene blue (MB) has been investigated as a deoxyribonucleic acid (DNA) intercalant

with the main objective of understanding the chemical/physical processes, which occur when adequate

wavelength light is impinging on DNA intercalated with MB. This understanding is crucial for the creation

of dynamic phototherapy procedures and for the development of new dye sensitized solar cells. During this

work we developed and optimized all the conditions to efficiently produce [MB/DNA] multi-layered films by

self-assembly. Our study revealed that pH strongly influences the growth of our multilayer films. Our UV

studies revealed that the UV radiation causes damage of DNA through opening of aromatic ring and by

breaking the DNA phosphate groups. The FT-IR studies on cast films with [MB/DNA] revealed that the

denaturation ratio decreases as the irradiation increases, meaning that MB is an intercalant of DNA chain.

This study paves the way to develop new dye sensitized solar cells which employ inexpensive materials and

take advantage of the intercalation process in order to adjust the intermolecular arrangement of the

donor/acceptor molecules to improve the device performance.

1 INTRODUCTION

Radiation exposure carries with it the risk of diseases

once it can damage crucial biomolecules such as

deoxyribonucleic acid (DNA), compromise the

imulogical and the nervous systems and contribute to

the development of cancer. (Teoule, 1987).

Several studies reported that ionizing radiation as

UV, X-rays, β and γ particles have enough energy to

excite or ionize the biomolecules. Interestingly, low-

energy species (secondary electrons) also induce

chemical and physical modifications in DNA such as

change of nucleobases, deletion of molecular groups

(phosphate and sugar) and both single and double

DNA strand formation.(Li et al., 2003) In healthy

physiological conditions, living systems have several

enzymatic DNA repair systems, which efficiently

remove this damage from DNA. If this damage is not

repaired in a cell, serious genetic changes such as

mutations occur, thus leading to cancer.

Photodynamic therapy destroys the target

malignant cells using a photosensitizer, a light

sensitive dye, which in the presence of oxygen,

activates and forms reactive oxygen species to induce

the cellular destruction. (Martinez and Chacon-

Garcia, 2005, Mansuri-Torshiza et al., 2001).

In 1992, Decher et al., introduced the layer-by-

layer technique: a revolutionary adsorption technique

consisting in the production of thin films by

immersing the film alternately in solutions of

oppositely charged materials with rinse steps in

between to remove any material unbound to the

surface.(Decher and Schmitt, 1992) LbL technique

requires small amounts of material offering a fine

control over the materials structure and is a cost-

effective, reproducible, robust and user-friendly

technique.

Lbl technique have been used in different areas

which integrate the health, electronics and

environment in order to develop smart nanostructured

devices, drug delivery systems, sensors and solar

cells.

In this paper, MB/DNA films were prepared using

the LbL technique and was assessed the influence of

several factors such as the pH, drying process and

immersion time in the loading of MB molecules. The

results showed that the pH value of MB solution has

a significant effect on the adsorption of the dye into

the film.

This study paves the way to develop new dye

Pires, F., Coelho, M., Ribeiro, P. and Raposo, M.

Methylene Blue - A Trendy Photosensitizer in Medicine and in Solar-Energy Conversion Systems.

DOI: 10.5220/0005843303790383

In Proceedings of the 4th International Conference on Photonics, Optics and Laser Technology (PHOTOPTICS 2016), pages 381-385

ISBN: 978-989-758-174-8

381

sensitized solar cells (Wang et al., 2010) which

employ inexpensive materials and take advantage of

the intercalation process in order to adjust the

intermolecular arrangement of the donor/acceptor

molecules to improve the performance of the device.

2 EXPERIMENTAL DETAILS

The LbL films were prepared from MB (MW= 373.90

g/mol) and DNA sodium salt from calf thymus,

obtained from Aldrich. The MB was dissolved in pure

water supplied by a Milli-Q system from Millipore

(resistivity of 18 MΩcm) to a concentration of 10 mM

and pH 7.The DNA aqueous solutions were 0.5

mg/mL concentration. The MB/DNA LbL films were

adsorbed onto quartz supports that had been

hydrophilized in a Piranha solution for 30 min.

Deposition comprised the following steps: (i)

immersion of the support in MB solution for 5 s; (ii)

washing the support plus MB layer with pure water;

(iii) immersion of the support plus MB layer into the

DNA solution for 60 s; and (iv) washing the substrate

MB/DNA bilayer with pure water. The number of

deposited bilayers is equal to the number of

repetitions of steps (i)–(iv).

Film growth was monitored by measuring the UV

visible using a Shimadzu UV-2101PC

spectrophotometer. The damage caused by UV

exposure was characterized using a FTIR

spectrophotometer Thermo Scientific Nicolet-model

530 (Waltham, MA, USA).

3 RESULTS AND DISCUSSION

3.1 [MB/DNA] Film Growth

The LbL technique is based on physical adsorption

processes resulting, mostly, from electrostatic

interactions but also the existence of van der Waals

forces, hydrogen bonding and hydrophobic forces.

(Oliveira Jr et al., 2001, Oliveira Jr et al., 2002).

Multilayers were deposited on quartz substrates

using the alternate dipping method into dilute

aqueous solutions of MB (pH=4) for 5 sec and DNA

(pH=6.8) for 60 sec. Contrary to what was expected,

the growth of the film is reduced, since the

absorbance values not suffered meaningful changes

as the increasing of the number of bilayers, since in

according to the literature, the amount adsorbed is

proportional to the number of bilayers.

Absorption bands at 260 nm (characteristic of

DNA and MB) and at 600 nm e 670 nm (characteristic

of MB) are clear evidences that some molecules were

adsorbed in the surface of substrates, possible

reflecting the non-ionic physical interactions. The

question we asked ourselves was: can the pH

influence the multilayers formation?

Figure 1: Absorption spectra of [MB/DNA]1 (black line)

and [MB/DNA]5(red line) films with AM at pH=4.

Figure 2: Absorbance spectra of [MB/DNA]5, a pH (MB) 4

(black line) and 7 (red line).

3.2 PH Affects MB Ionization and,

Consequently, the Growth of LbL

Films [MB/DNA]

Multilayers were deposited on quartz substrates using

dilute aqueous solutions of MB with a pH=7 instead

of a pH=4. Absorbance spectra of LbL films

[MB/DNA] with the same number of bilayers, at

different pH, are depicted in the Figure 2, showed that

the pH dramatically influences the growth of the

films. This can easily justified by the increase of the

degree of ionization of the molecules. In fact, at pH

7, the MB molecules are electrically charged which

contribute to the formation of the films due to the

ionic forces.(Impert et al., 2003) The absorption

AOMat 2016 - Special Session on Advanced Optical Materials

382

spectra of the LbL films, with the MB at pH = 7 shows

the characteristic absorption bands, (mentioned

before) of MB and DNA in the visible and ultraviolet.

Given that the assembly conditions were

optimized, the quartz substrates were coated with 20

bilayers of MB/DNA. The absorption band at 618 nm

and 676 nm correspond to the dimeric and monomeric

form of MB, respectively. (Spencer and Sutter,

1979)In Figure 3, we can also see an absorption band

at 285 nm (characteristic of DNA and of MB) and

other at 292 nm which is characteristic of MB and,

according to literature, this assignment correspond to

→∗ transitions. It should be referred also that, for

these LbL films, the absorbance in the 600 to 700

range is lower than the absorbance in the 280 nm and

the absorbance has a nonzero value at 200 nm. As the

spectra of MB solutions the absorbance is higher in

the 600 to 700 nm than the absorbance at 280 nm and

the absborbance decreases to zero for 200 nm, see

figure 3b), one can conclude that both DNA and MB

molecules are incorporated in the films. These results

proved that both MB forms have the ability to link to

the DNA, allowing the formation of LbL films.

Compared to the absorption peak of aqueous MB

(at 664 nm), the visible maximum peak at 623 nm of

[MB/DNA]

film is blue-shifted by 41 nm.

According to Chao et al., phenothiazine dyes such as

MB, suffered a blue-shift absorption band because

they form H-aggregates via π-π stacking. (Gao et al.,

2008)

The absorbance at the peak (at 285 nm, 618 nm

and 676 nm) was used to monitor the buildup of

multilayers, as illustrated in Figure 4. One can see that

the data follow straight lines, indicating that the films

increase linearly with the number of bilayers,

meaning that each deposited bilayer contributes an

equal amount of deposited polymer.

The amount of MB adsorbed per bilayer was

calculated by taking the intensity of the 618 nm and

667 nm peak in the UV-Vis spectra for [MB/DNA]

From the absorbance intensity using the Beer-

Lambert law, we concluded that the MB adsorbed

layer per unit area was 96±5 µg/m

During this work, we assessed the influence of the

drying process as well the imersion time on the

morphology of the LbL films. The drying process

using a nitrogen stream (data not shown) seems to be

an important factor during the preparation of LbL

films since it promotes conformational changes of the

molecules through modifications of their

arrangement in space, which allows a greater

interaction between charges and facilitates the

bonding between the materials.

Figure 3: Absorption spectra of: a)[MB/DNA]20 LbL film.

b) aqueous solutions of MB and DNA.

The kinetic study of DNA imersion time (data not

shown), showed that imersion in DNA solution for 5

sec is enough to obtain a thin [MB/DNA] film with

all characteristic absorption bands of each material.

The imersion for long times can lead to a desorption

phenomena.

Figure 4: Maximum absorbance versus number of bilayers

of MB/DNA self-assembled films adsorbed on quartz

substrates.

200 300 400 500 600 700 800

0.0

0.2

0.4

0.6

MB (7.5M)

DNA (0.0075 mg/mL)

Wavelenght (nm)

Absorbance

0.00

0.05

0.10

0.15

Absorbance

Methylene Blue - A Trendy Photosensitizer in Medicine and in Solar-Energy Conversion Systems

383

3.3 FTIR Characterization of Films

The damage caused by UV radiation on DNA in the

presence of the intercalator MB, are characterized by

fourier transform infrared spectroscopy (FTIR). The

FT-IR results obtained to [MB/DNA]

100

film were

inconclusive since the adsorbed amount of MB and

DNA was not sufficient to obtain a spectra with

adequate resolution.

Cast films were prepared with mixture of DNA

and MB, and were irradiated for different time

intervals.

Several changes in the IR spectra of DNA and

mixture MB/DNA have seen observed after

irradiation, as illustrated in Figure 5a) and b). In order

to better analyse the infrared spectra changes, spectra

baselines were removed and the peaks which did not

change as a result of exposure to UV radiation were

identified. According to Gomes et al., the FT-IR band

detected near 1018 cm

-1

is very stable since it does

not suffer any change when exposed to a radiation

with 140nm. (Gomes et al., 2009)This peak is due to

Figure 5: Infrared absorbance spectra of: a) DNA cast

sample before and after irradiation with 254 nm UV light;

b)a mixture of MB/DNA cast sample before and after

irradiation with 254 nm UV light.

furanose vibrations and was used to normalize the

obtained data, dividing the other peaks areas by the

area of this peak, avoiding the possibility that the

small changes due to the measurement of the infrared

spectra in different regions of the sample are affecting

the observed peak areas decrease or increase.

The vibrations associated with C-O stretching of

nucleic acid sugar (1067 cm

-1

) and PO

- stretching

(1089 cm

-1

) decreased with the irradiation time.

According to Paulo et al.,

13-15

UV radiation causes

damage of DNA through opening of aromatic ring

and by breaking the DNA phosphate groups.

Figure 6: Normalized infrared intensity ratios at 965 cm-1

(CC stretch of backbone), 1067 cm-1 (CO stretch of the

furanose backbone), 1089 cm-1 (Symmetric PO2-

stretching of the backbone ) relative to peak area at 1020

cm-1 of a DNA cast sample irradiated for different periods

of time with 254 nm UV light. The solid lines are

guidelines.

As illustrated in Figure 6, as the irradiation time

increases the ratio of the peaks 1089 cm

-1

and 1067

-1

decrease, meaning that radiation preferentially

affects the PO

groups than sugars.

One of the goals of this work was understand if

the UV radiation leads to a DNA denaturation in the

MB/DNA film. The infrared intensity ratio at 1690

-1

(C2=O2 strength of thymine single stranded or

double stranded and C6=O6 stretching of guanines),

was normalized relative to peak at 1652 cm

-1

(C2=O2

strength of cytosine single stranded or double

stranded and C4=O4 strength of thymine single

stranded or double stranded) of a DNA cast sample

irradiated for different periods of time with 254 nm

UV light. As the irradiation time increases the ratio

1690/1652 cm

-1

decreases, meaning that the presence

of MB induces an intercalation process which forces

the opening of the DNA chain.

AOMat 2016 - Special Session on Advanced Optical Materials

384

4 CONCLUSIONS

This work showed an efficient protocol to produce

[MB/DNA] multi-layered films by self-assembly.

The pH value should be 7, since at this pH the MB

molecules are electrically charged.

An MB adsorbed layer per unit area of 96±5

µg/m

was achived if the film is dried with nitrogen

at the end of each bilayer and immersed for 60 sec in

the DNA solution.

The UV studies revealed that the UV radiation

causes damage of DNA through opening of aromatic

ring and by breaking the DNA phosphate groups. Our

results also showed that UV radiation preferentially

affects the PO

groups than sugars.

The FT-IR studies on cast films with [MB/DNA]

revealed that the denaturation ratio decreases as the

irradiation increases, meaning that MB is an

intercalant of DNA chain.

ACKNOWLEDGEMENTS

This work was supported by the Portuguese research

Grant UID/FIS/00068/2013 through FCT-MEC, the

"Plurianual" financial contribution of "Fundação para

a Ciência e Tecnologia" (Portugal). Filipa Pires

acknowledges the fellowship from RABBIT Doctoral

Programme (Portugal).

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