The paper presents our research on the optical properties of thin-film deoxyribonucleic acid (DNA) complexes based on cetyltrimethylammonium (DNA-CTMA) deposited onto an optical elastomer substrate, which may be suitable for the construction of biophotonic devices. The study involved the measurement of Raman spectra, absorption spectra from the visible to the near-infrared region and the values of the refractive indices by m-line spectroscopy at five wavelengths (473, 632.8, 964, 1311, and 1552 nm). The samples were proved to have waveguiding properties from visible to infrared spectrum and high contrast of refractive index - the value is 0,0457. Photoluminescence measurement was done under excitation at three wavelengths (355, 405 and 450 nm) and showed one broadband with maxima at 437, 520 and 530 nm depending on the excitation wavelengths. The study has demonstrated that this combination of polymers, because of its unique properties, has great potential for the implementation of all-polymer structures in the applications of high-density photonics and biocompatible optical devices.
© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
Deoxyribonucleic acid (DNA) is a basic building block of living organisms and has been the fundamental basis in life-science and biotechnology research. Because of its unique properties, since its discovery [1–3], it has become an actively studied material with many applications in interdisciplinary scientific research. Particularly, the DNA-based polymers in solid-state thin layers possess many excellent mechanical, electrical and optical properties such as tuneable electrical resistivity , low propagation losses and high transmission from visible to infrared spectrum . DNA layers have also exhibited excellent thermal stability (up to 230°C), which is much higher than the most polymer materials used in optics and biophotonics . Such attractive properties have already found to utilization of the DNA polymer in realization of a long list of structures used in many applications as, e.g., organic field-effect transistors , organic light-emitting diodes , electro-optic waveguide modulator , dye-doped nanofiberes , single-mode-channel waveguides  saturable absorber for laser mode locking  or biocompatible optical fiber temperature sensor . Also, surface plasmon resonance imaging biosensor based on waveguide coupled surface plasmon resonance, which can be used for wide applications in the field of high-throughput measurements including proteins or DNA microarray [13,14] have been presented as well.
However, even though so many structures based on the DNA (see above) it seems that the potential of the DNA polymers has not been yet expired and research of new DNA-based photonics structures has been still actual, mainly what concerns those where optical planar waveguides are fundamental key elements.
One of the most important factors in the realization of waveguiding structures are refractive indices of the materials, mainly their contrast, which plays a fundamental role in determining of the optical waveguides; and higher index contrast that permits to realize high-density photonics structures components using complex waveguide interconnections within a small area . The refractive index contrast plays a very important role not only for determining dimensions of the single mode waveguides, coupling losses efficiency, minimum bending radius, polarisation effects, fabrication process, materials availability and etc.
Here we are going to focus on the construction of optical planar waveguides represented by the DNA cetyltrimethylammonium complexes (DNA-CTMA) with a polymer substrate. The substrate will be optical elastomer polydimethylsiloxane (PDMS) because of its low absorption from visible and near-infrared region, high thermal and chemical stability, biocompatibility and, what is the most important, a low value of its refractive index (1.4028 at 1321 nm), which would allow for achieving of high refractive index contrast between the core and cladding. Another important advantage of the PDMS is, that it can be prepared in a form of thin free-standing layer allowing for a realization of a flexible bendy structure .
The aim of this study is to predominantly report on waveguiding properties of the DNA-CTMA thin layers deposited onto PDMS elastomer flexible substrate. The combination of these materials allows achieving a higher refractive index contrast between deposited DNA-CTMA layer and PDMS substrate. The waveguiding structures with higher refractive index contrast can by applied for complex high-density integrated optical circuits. Moreover, the DNA-CTMA and PDMS polymers are excellent biocompatible materials, therefore combinations of these materials also allow fabrication of biocompatible photonic devices.
For the precise design of the photonic devices the values of refractive indices as well as waveguiding properties are important to know, therefore, we measured these properties by m-line spectroscopy from visible to infrared spectral ranges. We hope that this knowledge will in the near future allow to realize new structures with better understanding of their function.
2. Sample fabrication
In order to utilise the conventional spin-coating process based on organic solutions, various surfactants were dissolved in the standard DNA aqueous solution to obtain DNA-lipid complex precipitates [12,17,18]. These precipitates were vacuum-dried and subsequently dissolved in organic solvents, which were then used in the conventional spin-coating process to make thin solid films and devices. Cetyltrimethylammonium chloride (CTMA-Cl) has been one of the best-known cationic lipids to form DNA–lipid complexes. In an attempt to make the DNA-CTMA precipitation process more reproducible, Soxhlet-dialysis has been used to rinse CTMA remnants off . However, this rinsing cannot alter the fundamental precipitation process itself, which would significantly affect the material properties of the DNA-CTMA thin solid films.
We have developed a unique refinement process to chemically enhance the bonding of CTMA on the DNA backbone . Pristine DNA-CTMA precipitates were formed in two processes: A) drops of CTMA aqueous solution were injected into the DNA aqueous solution, B) drops of DNA aqueous solution were injected into CTMA aqueous solution. These processes provided A1 and B1 DNA-CTMA samples in Fig. 1. A1 and B1 were then supersaturated in ethanol, where drops of water were poured to re-precipitate DNA-CTMA. This study worked with the B2 DNA-CTMA sample, which exhibited the highest level of recrystallisation. An appropriate amount of ethanol was also used to control the OH contents. Using the DNA-CTMA precipitates, two basic samples were made, with one of them containing 100 ppm and the other one containing 500 ppm of DNA-CTMA in Hexafluoroisopropanol (HFIP) solvent. The process preparation of the DNA-CTMA solution was similar to that in the already published paper . The prepared solutions were deposited by spin coating (the spin-coating process started at 500 rpm/5sec and continued at 2000 rpm/30sec) onto the PDMS elastomer Sylgard 184 (Dow Corning) substrate. The substrates were made by mixing A (base agent) and B (curing agent) in the 10:1 ratio. After the thorough mixing of the components, the solutions were evacuated for up to 60 minutes in a desiccator to eliminate all the bubbles. Curing in the oven at 80 °C was then applied for 4 hrs. The thickness of the layers was determined by the method of talystep. According to these measurements, the thickness of the DNA-CTMA 100 ppm layer is around 2.05 µm and for the DNA-CTMA 500 ppm it is 4.95 µm.
3. Sample characterization
3.1 Raman spectroscopy
Raman spectroscopy as a method used here for checking the composition of the deposited layer was performed on a DXR Raman microscope spectrometer of the Thermo Scientific company equipped with a confocal Olympus microscope. A solid-state Nd:YAG laser (wavelength: 532 nm, maximum power: 10 mW) was used as the excitation source together with 900 lines/mm grating. Detection was performed using a multichannel thermoelectrically cooled CCD camera has 50x magnification and a measuring trace of approx. 1 µm2. The measurement conditions were as follows: the power of 8 mW, 100 accumulations of 30 s scans, and the aperture being a 25 µm pinhole.
The Raman spectra presented here are the results of three measurements of each sample, which were then averaged. Since the substrates were rather thick compared with the deposited layers, the reference spectrum of the PDMS substrate level was then subtracted from the obtained spectra of the DNA-CTMA samples.
3.2 UV-VIS-NIR absorption spectroscopy
Absorption spectra of the DNA-CTMA layers deposited onto the PDMS substrates were collected with the UV-VIS-NIR spectrometer (UV-3600 Shimadzu) in the spectral range of 180–1650 nm.
3.3 M-line spectroscopy
Waveguiding properties and effective refractive indices of the DNA-CTMA thin layers deposited onto the PDMS substrate were determined by m-line spectroscopy, also known as dark-mode spectroscopy or prism-coupling technique. The principle of the method has already been presented in [20–22]. The measurement was done using the Metricon model 2010/M prism coupler  equipped with five lasers operating at the wavelengths of 473, 632.8, 964, 1311, and 1552 nm and measurement was done for transverse-electric (TE) polarisation. The optical coupling of light into the measured DNA-CTMA samples was done via a coupling prism with the refractive index 2.1558 at 632.8 nm, which enabled the measurement in the range of refractive indices between 1.2 and 2.02 (which was more than sufficient for the measurement of the DNA samples). The effective refractive indices of the measured layers were then determined by measuring the critical angle of incidence θ and calculated from the Eq. (1) :
3.4 Photoluminescence spectroscopy
The steady-state photoluminescence properties of the prepared samples were measured on the HORIBA Jobin-Yvon Fluorolog-3 Extreme using FluorEssence 3 software. The spectra were collected at room temperature within the range of 250–1000 nm for the visible region (VIS) by a photomultiplier tube (PMT) with thermoelectric cooling and a Ce:InGaAs photocathode (model number R955) in the detection range of 185–900 nm. The samples were excited using a 450 W xenon continuous-wave (CW) lamp at three different wavelengths: 355, 405 and 450 nm. Various wavelengths were selected by means of a double-diffraction-grating monochromator at the entrance and a single-diffraction-grating monochromator at the exit. Photoluminescence spectra were collected in a reflective arrangement with the samples being tilted at an angle of approx. 60°. In the VIS region, the photoluminescence radiation was collected at the front-facing exit. For spectral evaluation, all the measured luminescence spectra were transformed to the base level and after subtraction of the background, they were normalised with the reference PDMS substrate.
4.1 Raman spectroscopy
The Raman spectra were measured in the range of 0–3200 cm-1 and we observed several strong bands at 161, 191, 491, 617, 689, 711, 791, 2853, 2905 and 2965 cm-1 (not shown in the Fig.), which belong to the PDMS substrate. Figure 2 shows the spectrum in the range of 900–1800 cm-1 of the sample made from the solutions containing 500 ppm of DNA-CTMA, where the spectrum at the bottom concern the PDMS substrate, the spectrum in the middle show the as-measured spectrum of the DNA-CTMA sample, and the top spectrum show the subtracted spectrum from the substrate PDMS spectra. The bottom spectrum in Fig. 2 shows two strong bands at 1264 and 1412 cm-1 and an additional three weaker bands at 989, 1264, 1412, 1486 and 1598 cm-1, all belonging to the PDMS substrates. The spectrum in the middle of Fig. 2 shows the as-measured spectra of the samples and the upper spectra show the spectrum subtracted from the substrate PDMS spectra. It means that the results clearly evidenced that the 500 ppm samples really possessed the DNA-CTMA layer. The spectra of the 100 ppm samples had very similar features but a much weaker intensity so that they are not shown. In the spectrum at the top of Fig. 2, there are the bands of particular components of the DNA. The assignment of those peaks was done according to [25,26] and is also depicted on the left side of Fig. 2. Namely, the peaks of cytosine (cyt), guanine (gua), adenine (ade) and thymine (thy) at 1182, 1241, 1255, 1301, 1332, 1369, 1482, 1573 cm-1 are clearly apparent as well as were clearly identified the peaks of deoxyribose phosphate backbone at 965, 1008, 1064, 1094 and 1441 cm-1. It means that DNA can be easily recognised in the samples having its high concentrations, as in this case was the 500 ppm one. The bands attributed to the particular bases are slightly broader or shifted against the data published by  obtained for the water solution of the DNA-CTMA. In our case, Raman spectroscopy clearly identified the presence of DNA-CTMA on the surface of PDMS, however an explanation of the exact position of the band and the vibration modes changes is a complex as well complicated task and would require a detailed study.
4.2 UV-VIS-NIR absorption spectra
The absorption spectra of DNA-CTMA of two different amounts of DNA-CTMA (100 and 500 ppm) layers deposited onto PDMS substrates and collected by the UV-VIS-NIR spectrometer (UV-3600 Shimadzu) in the spectral range of 200–1700 nm. Measurement was done for both samples 100 and 500 ppm. In Fig. 3 the spectra after subtraction of the PDMS substrate are given.
The absorption spectra of DNA-CTMA samples show strong absorption in the UV wavelength range with the maximum at around 260 nm which correspond well with results presented in [11,27]. The DNA-CTMA 100 ppm sample was transparent from 300 nm to 1700 nm (which was the highest wavelength we used). The sample with a concentration of 500 ppm DNA-CTMA had higher total absorption compared to the 100 ppm sample.
4.3 M-line spectroscopy
The actual measurement was preceded by an attempt to make a simple estimation of the waveguiding properties of the deposited DNA layers in order to determine the critical minimum thickness of our deposited layers. The estimation was based on the refractive index values given in  as n = 1.5445 at 632.8 nm and n = 1.4846 at 1550 nm for deoxyribonucleic acid-hexadecyltrimethylammonium chloride (DNA-HCTAC) reported in  and the dispersion Eq. (2), which has been formulated on the principle of transverse resonance [22,30]:
The calculations have shown that in the case of supporting TE0 modes, the minimal thickness for the wavelength of 632.8 nm is 0.163 µm and for the wavelength of 1550 nm, it should be 0.561 µm. In the case of the higher TE1 and TE2 modes, the minimum thickness values should be 0.669 µm or 1.175 µm at 632.8 nm, respectively. For the wavelength of 1550 nm, it should be 2.149 µm (TE1) or 3.737 µm (TE2). The results of mode calculations are also shown in Fig. 4, where Fig. 4(a) concerns the wavelength of 632.8 nm and Fig. 4(b) concerns that of 1550 nm.
Figure 5 shows the results of our m-line measurement, specifically the assessments of the DNA-CTMA effective refractive indices (so-called mode spectra, i.e. the dependence of the reflected intensity of the beam on the angles of incidence θ). The arrows in the Fig. refer to TE0 modes of the DNA-CTMA planar waveguides.
Figures 5(a), 5(b) and 5(c) give the mode spectra at 473, 632.8 and 964 nm, respectively, and show that both 100 ppm and 500 ppm samples always exhibited only one mode. Figures 5(d) and 5(e) (at 1311 and 1552 nm, respectively) do not show any supported mode with the 100 ppm sample and only one mode with the 500 ppm sample. Figure 5(f) depicts the measured curves for the reference sample of the PDMS substrates at all five wavelengths used.
It arises from the results shown in Fig. 5 and Table 2 that the thicker DNA-CTMA layer exhibited waveguiding properties for all the measured wavelengths while the thinner ones (100 ppm sample) transited light only at the shorter wavelengths (473, 632.8 and 964 nm). However, the numbers of the supported modes, as predicted by the calculation, are to be higher. At 632.8 nm, the 2.05 µm thick layer is, according to the simulation, to support four modes (TE0, TE1, TE2 and TE3) and the 4.95 µm thick layer at 1550 nm is expected to support three (TE0, TE1 and TE2) modes. The lower number of the observed guided modes could be a consequence of a poorer optical contact between the coupling prism and a gooseneck (sag) of the rather soft PDMS substrate and deposited the DNA layer.
The aim of our work was to achieve a higher contrast between the refractive indices of the deposited DNA-CTMA layers and that of a substrate, in our case the PDMS. There was a good agreement between our refractive index values and the results published by other authors dealing with a similar subject as:
- c) Effective refractive index values at 632.8 nm of a large variety of DNA-CTMA samples in dependence on the humidity of the environment were presented in . The obtained values for the TE mode were in the range of 1.507–1.480.
Since the used material of the substrate does not affect the effective refractive indices of the deposited DNA-CTMA layers much (as expected), the considered refractive index contrast could obviously be provided by a suitable choice of the substrate bearing in mind that the material of such substrate should allow for deposition of high quality, compact and homogeneous layer of the DNA-CTMA. For that reason, the PDMS seems to be a good choice.
The refractive indices of the PDMS substrate can be obtained from the DNA-CTMA/PDMS samples from Figs. 5(a)–5(e) and for the PDMS substrate from Fig. 5(f) for all the measured wavelengths by inserting the angles of incidence (done with the edge in the m-line spectra in Fig. 5) into Eq. (1) . This way obtained values (depending on the wavelength) of the refractive indices of the PDMS substrates were in very good agreement with the results presented in our earlier paper .
In this work, when the DNA-CTMA optical waveguides were deposited onto the PDMS substrate, we reached the refractive index contrast Δ = 0.0456 (n1 = 1.4709, n2 = 1.4022, λ = 1550 nm). It means, our approach allows for constructing smaller waveguides that would enable significantly increase the density of integration and therefore realize complex photonics structures within a smaller area.
4.4. Photoluminiscence spectra
The photoluminescence spectra of the DNA-CTMA layers deposited on the PDMS substrate are shown in Fig. 7 for the excitation wavelength λex of 355 nm. The luminescence measurement were done for three excitation wavelengths of 355, 405 and 450 nm. It was found that the intensity I(λex) for all three excitation wavelengths decreased in the following order: I (355 nm) > I (405 nm) > I (455 nm) ≈ 100% > 78% > 68%. The highest luminescence intensities were therefore found for the excitation wavelength of 355 nm. Based on the spectra the PDMS substrate had one main luminescence band at around 437 nm, which is in accordance with the literature . The spectra of both samples, containing 100 ppm and 500 ppm of DNA-CTMA, had two wide luminescence bands positioned at around 437 nm (originating from the PDMS substrate ) and 503 nm (belonging to the DNA). Generally, the higher concentration of DNA-CTMA leads to the higher-intensity luminescence.
In our discussion, we are going to focus on the comparison of our results with published experiments. Nevertheless, the specific system DNA-CTMA and its properties have been published rarely, so far. We will also discuss the comparison with DNA-CTMA doped with various dyes.
First, we focus on the usable wavelength range. In our experiments, the absorption spectra of DNA-CTMA samples show that the strong absorption appears in the UV wavelength range, with the absorption edge at 275 nm for both 100 and 500 ppm DNA-CTMA samples. More precisely the maximum was found at 260 nm for both samples. As to the longer wavelengths from 325 to 1700 nm, absorption of the 100 ppm DNA-CTMA sample was comparable to the absorption of the PDMS substrate. As expected, the 500 ppm DNA-CTMA sample has a stronger absorption than that containing 100 ppm of the DNA-CTMA. We assume that absorption at 260 nm was caused by the π −π* absorption transition in the nucleotide bases  and it corresponds well with results presented in [11,27]. The authors quoted the absorption maximum at 261 nm concerning the 0.2 mm thick DNA-CTMA sample and did not find any absorption in the commonly used communication wavelength range (850, 1310 or 1550 nm). From the literature as well as from our experiments it follows that DNA-CTMA waveguide structures could be used in all three telecommunication windows and for all wavelengths above about 300 nm.
M-line spectroscopy measurements showed that the refractive index values of the PDMS substrate were similar to those ones already presented in our paper , similarly as the values of the effective refractive indices of the DNA-CTMA layers, which also were in a good agreement with our former data given in . High density integration photonics structure requires optical waveguides with high relative index difference Δ. In the case of all-polymer structures, there are only a few possibilities to combine materials to have a large contrast of refractive index. The optical waveguides made from epoxy polymer EpoCore and a cladding layer made from EpoClad (refractive indices are as follows: n1(EpoCore) = 1.5743 and n2(EpoClad) = 1.5635 both for 1550 nm ) have the refractive index contrast Δ = 0.0068. Concerning siloxane-based polymer LIGHTLINK XP-6701A and core polymer LIGHTLINK XH-100145 Clad (refractive indices: n1(core) = 1.5024 and n2(clad) = 1.47815 at λ = 1550 nm) have the index contrast Δ = 0.016 . Biomaterial silk fibroin optical waveguides deposited onto silica-on-silicon substrate had the index contrast Δ = 0.0197, where refractive index of the silk fibroin core was n1 = 1. 4733  and refractive index of the silica-on-silicon layer generally is n2 = 1.444, both for 1550 nm. The structures realized from DNA-CTMA on PDMS substrate prepared by our group achieved the refractive index contrast Δ = 0.0456. Therefore, the prepared DNA-CTMA layers on PDMS allows the realization of flexible high-density photonics structures.
Concerning the photoluminescence spectra, a broad luminescence band at around 503 nm for all the excitation wavelengths (namely 355, 405 or 450 nm) was detected; the excitation at 355 nm yielded the strongest luminescence. We have not found any relevant data in the literature concerning the fluorescence measurements of the un-doped DNA-CTMA. However, the reports dealing with the DNA-CTMA doped with riboflavin and curcumin reported – similar to our measurements – one main fluorescence band at around 530 and 550 nm, respectively, for the excitation wavelength of 405 and 450 nm, respectively [37,38].
Last but not least, the stability of the potentially developed devices is a rather important factor for their practical utilization. We have concerned it and measured the important characteristics of the samples – Raman and m-line spectra – within 12 months. The samples were stored on common conditions on air at room temperature and we found that they did not change.
The paper reports on the properties of thin-film deoxyribonucleic acid complexes based on cetyltrimethylammonium deposited on the polydimethylsiloxane substrate. The motivation of the research was to create the background for the construction of a new type of biocompatible photonic structure. The actual aim of the presented work was to find and experimentally verify a proper combination of the functional DNA layer and a suitable substrate having a low value of the refractive index. For that, we prepared DNA-CTMA layers with different DNA concentrations and different thicknesses deposited onto the PDMS substrates. The measurement of the Raman spectra of the samples showed the bands of particular components of the DNA, namely cytosine, guanine, adenine and thymine. The absorption spectra of the 100 ppm DNA-CTMA showed that the absorption from 325 to 1700 nm is practically negligible and therefore the realized structures are suitable for use at the main three telecommunication wavelengths of 850, 1300 and 1550 nm. The effective refractive indices of the prepared DNA layers were evaluated for 5 different wavelengths. The waveguiding properties were proved for all of the measured wavelengths from 473 to 1552 nm (visible to infrared spectrum). Moreover, the deposition of the DNA-CTMA waveguides onto the PDMS substrate allows reaching a high refractive index-contrast of 0.0457, which is a significantly higher value comparing with the structures of epoxy polymers EpoCore/EpoClad (0.0068) and siloxane-based polymer LIGHTLINK (0.016). Our solution will make it possible to realize the DNA-CTMA/PDMS structures for high-density photonics devices or micro-opto-electro-mechanical systems. Moreover, biocompatibility, as well as measurable luminescence at 503 nm of the DNA and PDMS, could be beneficial in the case of utilizing them as biosensors.
Our study has demonstrated that thanks to their unique properties, the DNA-CTMA thin layers deposited onto the PDMS substrate can be used for the implementation of free-standing and flexible polymer structures for the applications in photonics and biocompatible optics.
Centre of Advanced Applied Natural Sciences (Reg. No. CZ.02.1.01/0.0/0.0/16_019/0000778).
This work was supported by the Centre of Advanced Applied Natural Sciences, Reg. No. CZ.02.1.01/0.0/0.0/16_019/0000778, supported by the Operational Program Research, Development and Education, co-financed by the European Structural and Investment Funds and the state budget of the Czech Republic.
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