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Abstract
An investigation was made to detect the DNA samples of BALB/c rats, exploiting the technique of broadband plasmonic response in the visible spectral regime. In experiments, a non-coherent light beam was physically designed and practically implemented to study the spectral effect due to serial dilution of BALB/c rat’s dried DNA. In particular, three different diluted DNA samples (with ratios of 1:10, 1:20, and 1:40) dried on the surface of a nanolayer gold thin film were considered to retrieve the plasmonic conditions under which the reflectance becomes minimum. The results indicate the most diluted DNA sample exhibits prominent plasmonic conditions, and the resonance wavelengths undergo redshifts with increasing incidence angle (of the p-polarized light). Also, the sensitivity of the configuration is enhanced in the presence of a DNA sample (as compared to the case of non-existence of measurand), which is further increased for larger incidence angles.
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1. Introduction
Deoxyribonucleic acid (DNA) plasmonic analysis is crucial in understanding the optical behavior of the known largest individual polymerized molecular chain. DNA exists in almost every cell nucleus of human body as the hereditary material and contains all necessary chemical codes for gene transcription [1–4]. The DNA sequence of various species is different while the DNA sequence similarity of one group of a certain species is almost similar [5–8]. For instance, in human nature, the DNA of a cell contains of about 3 billion bases (i.e., the information in the form of chemical blocks known as A, C, G and T, respectively called as the adenine, cytosine, guanine, and thymine), and more than 99% of those bases are the same in all [9]. Human genome is composed of 46 molecules of DNA or two pairs of chromosomes. Therein the basic repeating structure contains eight positively charged spherical histones (nucleosomes) and about a meter length of DNA is packed in micrometer size in a unit cell [10,11]. Each base pair (bp) in a DNA strand is about 0.34 nm long which, along with other billions of bps, can be extracted from the nuclei of the cells of blood, semen, skin cells, tissue, organs, muscle, brain cells, bone, teeth, hair, saliva, mucus, fingernails, etc.
The morphology of DNA after extraction is quite large for broadband plasmonic spectrometry, which generally deals with the one- (1D), two- (2D), and three- (3D) dimensional nanoparticles [12–16]. It must be noted that, in broadband plasmonics, the emitted light is incoherent in nature which limits the spectral quality as compared to what one would expect using a single wavelength laser light source. Thus, the extracted DNA, which contains pile of broken (or unbroken) DNA strands, has the dimension several hundred times greater than the scale of visible wavelength range. In this situation, one possible way to reduce the size of the pile appropriately to the dimension of plasmonic sensing range is to apply the process of dilution. But, estimating the limit of dilution for a certain DNA pile for it to be appropriate to achieve plasmonic phenomenon is challenging. However, serial dilution unnecessarily lessens the target DNA template number in the extracted sample. Other exemplary research by Motazedifard and Madani [17] used dilution of rabbit- and human-DNA, and methylene-blue (MB) as a disinfectant on transparent multilayered thin film. They applied non-linear transmittometry optical method, known as spontaneous parametric-down conversion (SPDC), to detect concentrations of about 0.01 ng/µl and 0.1 ng/µl.
Herein, we investigate the plasmonic response of DNA of a living creature such as BALB/c rat. For this, we employ the process of serial dilution that enhances the interaction of light with nanoscale concentration of DNA. Within the context, plasmonic phenomenon greatly depends on the size, shape, the material type in use, the refractive index of nanoscale measurand and the thickness of substrate [18–22]. Every extraction of DNA molecules from any living source has different physical morphologies in micron scale and imposes a random complex structure to the broken DNA strands. In plasmonics, however, the repetition of strong (plasmonic) response requires the replication of the same periodic structure in nanoscale [23–27]. Such explanations determine that DNA can never create a periodic structure on a thin homogeneous layer of gold. In this work, we attempt to investigate the spectral response of BALB/c rat’s dried DNA implementing plasmonic studies, which incorporate the deposition of DNA over a nanolayer thin film of gold.
2. Method and experiment
Chemically, a DNA molecule is polar in nature. A thin surface of gold contains free electrons, and this high electron density property would provide strong binding between the positive polarity of DNA and the negative polarity of gold layer [28–30]. Figure 1 depicts the 2D schematic of DNA, wherein the pile of DNA at high concentration (Fig. 1(a)) has lesser chance of getting a better binding to the thin layer of gold compared to the low DNA concentration (Fig. 1(b)). Clearly, Fig. 1(a) exhibits high scattering of positive charges and weak binding with gold film at high DNA strands concertation, whereas Fig. 1(b) depicts the opposite of it, i.e., low scattering of positive charges and weak binding with gold nanolayer at low DNA strands concertation. In broadband plasmonic phenomenon, the frequency of operation typically varies from ∼375 to ∼750 THz. Thus, low DNA concentration essentially improves the compatibility of plasmonic sensing in such a frequency range.
Fig. 1. Demonstrative schematic of the localized (a) weak, and (b) strong charge bindings when the number of pile (of DNA strands) in a certain unit of the surface are high (a) and low (b), respectively.
In the following subsections, we discuss the progress regarding the extraction, dilution, drying BALB/c rat DNA, and plasmonic spectrometry of the dried DNA deposited over gold nanolayer thin films for exploring broadband plasmonic signature.
2.1 DNA extraction steps from BALB/c rat blood sample
In our work, we chose the target living species as BALB/c rat because of many genomic DNA similarities of this mammal to human. We extracted the DNA directly from the blood sample of BALB/c rat and added immediately 2 ml of ethylenediaminetetraacetic acid (EDTA) as the anticoagulant and prevention factor to degrade DNA as well as inactivate agent for nucleases and metal ion-requiring enzymes [31]. We started the process of extraction by taking 2 ml blood from BALB/c rat and adding 700 µl TRIzol (i.e., acid-guanidinium-phenol based reagent specially designed for the separation of DNA from cells and proteins at high pH) to the taken blood sample [32]. This solution was then mixed using vortex mixer for 30s. The next step was to stabilize pH during cell lysis as the DNA is sensitive to pH variation. Hence, we added tris or hydroxymethyl aminomethane as a pH buffer to the solution and kept in the shaker for 10 minutes.
The next step was separating nucleic acids from other cellular substances. Thus, we added 200 µl cold phenol-chloroform as isoamyl alcohol to the mixture and centrifuged at a speed of 12000 rpm at a temperature range of 4–10°C for 15 minutes. After the liquid phase separation according to the density of substances in the blood samples, the denser liquid phase sediments at the bottom of the microtube container. The 300 µl lighter liquid phase could be removed from surface and replaced with cold isopropanol alcohol for the purpose of removing the H2O hydration shell of phosphate and enhancing the process of DNA extraction [33]. The resultant aqueous solution remained at 20°C for 30 minutes as the process of chemical interactions is time consuming. Further extraction of unwanted blood cellular substances was made by centrifuging at a speed of 10000 rpm for 10 minutes. After separation of the upper liquid phase, 500 µl of 75% ethanol was added to the solution for improving DNA extraction. The final centrifuge at a speed of 8000 rpm for 5 minutes precipitates the DNA strands and the sediment appears at the bottom of microtube. Finally, the extracted DNA was dissolved in 30 µl DPEC (diethylpyrocarbonate) and sterile filtered water, and heated by dry heat blocks at 57°C for 15 minutes, to preserve the solubility of DNA in DEPC water. The spectrometric analysis was made by NanoDrop 8000 UV-Vis spectrophotometer device, which determined the concentration of the extracted DNA to be 883 ng/µl; this was sufficient for dilution progress – the part we discuss in the following subsection.
2.2 BALB/c rat DNA dilution
The discussion in Section 2 determines that, as a preliminary step, the targeted DNA needs to be diluted before depositing over the gold thin film. The process of serial dilution in practice is a way to reduce concentration of a target solute in liquid phase of a certain solution without changing the volume (of that solution). We decrease the DNA concentration for better polarity binding of the bipolar DNA molecules with gold nanolayer as it helps subtracting the biological template applied to the extracted DNA from BALB/c rat to resize the concentration scale from less than 100 ng/µl to less than 1 ng/µl.
The extracted DNA of BALB/c rat is soluble in distilled water. In our experiment, to obtain a low concentration of less than 1 ng/µl, we applied the serial dilution on 1 µl of solution containing 20.825 ng of the BALB/c rat DNA. In practice, we changed the coefficient ratios (i.e., 0.1, 0.05 and 0.0025 ng/µl) in every step for reaching the scale of less than 1 ng/µl in lower number of dilution steps (Fig. 2). In our study, we mainly considered the DNA concentrations of less than 10 ng/µl. Thus, for the following steps, we applied 2.825, 1.04125, and 0.520625 ng/µl for deposition of the DNA on the gold nanolayer thin film.
Fig. 2. Serial dilution of the BALB/c rat DNA with distilled water as the target solution starting from 1 µl to 0.025 µl of the solute.
The extracted DNA of BALB/c rat is soluble in distilled water. In our experiment, to obtain a low concentration of less than 1 ng/µl, we applied the serial dilution on 1 µl of solution containing 20.825 ng of the BALB/c rat DNA. In practice, we changed the coefficient ratios (i.e., 0.1, 0.05 and 0.0025 ng/µl) in every step for reaching the scale of less than 1 ng/µl in lower number of dilution steps (Fig. 2). In our study, we mainly considered the DNA concentrations of less than 10 ng/µl. Thus, for the following steps, we applied 2.825, 1.04125, and 0.520625 ng/µl for deposition of the DNA on the gold nanolayer thin film.
2.3 Drying and deposition of the BALB/c rat DNA
The permittivity of wet DNA varies with the DNA layer thickness and the distribution of DNA molecules on the gold nanolayer. It must be considered that the wet and dry DNAs of the same thickness have different dielectric permittivity values. Optically, the refractive index of DNA changes with thickness, and in direct drying deposition technique, the placement of DNA strands pile in nanoscale takes place randomly. Thus, the thickness at a local point would be different from the neighboring points. This fact is more evident in Fig. 1. Generally, we can decrease the thickness of DNA sample over the gold thin film after drying using the dilution method. Since the DNA molecules assume lattice structures after drying [32], in our experiment, we considered a few measures, namely we (i) made sure that the DNA is independent of environmental factors (such as temperature and humidity) during optical analyses, and (ii) ensured that the permittivity would not change due to lattice structure, and therefore, the DNA thickness is not a variable parameter. For this, we dried the DNA at a constant temperature of 60°C for a period of 24 hours. Besides, what remains on gold thin films after 24 hours should not possess water molecules and we ensured only dried DNA exists over the gold nanolayer surface. For efficient broadband plasmonic spectrometry in the visible spectral range, we deposited 40 nm thick homogeneous gold nanolayer over glass substrate, which we achieved using the sputtering technique.
In our experiment, we used micropipette. Figure 3(a)–(c) exhibits the BALB/c rat DNA substance of concentrations 88.3 ng/40 µl, 44.15 ng/40 µl and 22.075 ng/40 µl, respectively, dripped over the gold nanolayer. We put Fig. 3(d) alongside as the reference condition to compare plasmonic effect when the gold nanolayer did not have a DNA deposition over it. After depositing the DNA samples of different concentrations, we treated all thin films by dry air under a constant temperature of 60°C in an incubator for 24 hours. The fading of stains of dried DNA appeared on the gold thin film surface (as Fig. 3 depicts), which essentially determines the outcome of serial dilution, i.e., reduced DNA concentrations.
Fig. 3. Gold nanolayers with deposited dried BALB/c rat DNA of concentrations (a) 88.3 ng/40 µl, (b) 44.15 ng/40 µl, (c) 22.075 ng/40 µl, (d) air (i.e., non-existing DNA layer). All photographs (in Fig. 3(a)–d) were captured by high definition 48 mega pixel camera.
2.4 Opto-electro-mechanical broadband plasmonic spectroscopy setup
Figure 4 depicts the comprehensive schematic (in the form of block diagram) of the broadband plasmonic spectroscopy setup. Herein, the used optical source emits light in broadband visible regime, and therefore, it is incoherent in nature and cannot provide efficient linear spectrum in all wavelength range. The emitted light needs to be polarized before reaching the thin film surface, which we achieved electronically using the software and a gearbox interface to rotate the Glan-Taylor prism inside the light tube [34,35]. The incoherent light becomes spatially coherent after propagating through the pin hole [36].
The polarized beam, after passing through the glass cylindrical half-circle prism, interacts with the gold thin film surface at different angles. At certain incidence angles (of light), the phenomenon of surface plasmon polariton (SPP) wave vector matching would exist at the interface of gold thin film and dried DNA sample, thereby leading to the condition of surface plasmon resonance (SPR) [37–39]. The observation of SPR in the operating spectral domain is feasible through the polarized reflected light containing the spectral information of the light intensity in different visible wavelength bands at different angles, which we varied in a range of 10°–50°. The designed software controls the incidence angle and spectrometer (STS Vis Ocean) at a step of 0.5° angular move with the help of stepper motors with which the attached components are the mechanical shaft, coupler to the light tube and spectrometer rotary plane (Fig. 4). We performed all spectral measurements in real time and the progress of capturing the signature of broadband plasmonic characteristics for the target sample, which is BALB/c rat’s dried DNA in this experiment. We analyzed the big data as provided by the spectrometer to the software (as Fig. 4 shows). In the following section, we analyze and discuss the spectral results as obtained from this optical measurement setup, which essentially provides the reflectance characteristics of the DNA samples deposited over gold nanolayer surface.
3. Results and discussion
In our experiment, as stated above, we used a 40 nm thick gold film deposited over glass substrate. Over the gold nanolayer, we deposited the BALB/c rat DNA of different dilutions, as Fig. 3 depicts. We considered the p-polarized incidence radiation and, as stated before, we varied the angle of incidence at a step of 0.5° starting from 10°; we kept the upper limit of incidence angle to be 50°. Figure 5 illustrates the typically chosen plots of reflection intensity against wavelength corresponding to different fixed values of the angle of incidence while considering different DNA concentrations, namely 1/10, 1/20, and 1/40 ng/µl. In particular, Fig. 5(a)–h, respectively, correspond to the used angular values as 20.1°, 30.84°, 39.32°, 41.58°, 46.1°, 47.22°, 47.79°, and 48.36°. In all these figures, we also incorporated the case of ‘no DNA deposition over the gold film’ as the reference sample – the situation which we refer to as “Air”, because of the free-space being the ambience of gold film.
Fig. 5. Intensity reflection spectra of the p-polarized light from DNA thin film samples (the measurands) at different incidence angles, namely (a) 20.1$^\circ $, (b) 30.84$^\circ $, (c) 39.32$^\circ $, (d) 41.58$^\circ $, (e) 46.1$^\circ $, (f) 47.22$^\circ $, (g) 47.79$^\circ $, and (h) 48.36$^\circ $. The figure also incorporates the spectral behavior as achieved in the absence of measurand.
While conducting the experiment, we observed the initial presence of plasmonic effect at an incidence angle of 20.1°, as Fig. 5(a) illustrates. The noticeable point in this figure is the intensity spectrum corresponding to the most diluted DNA sample, which exhibits maximum reflection at 20.1° angle. Also, the less diluted DNA samples do not exhibit plasmonic resonance to the extent that the case of most diluted DNA sample shows. Investigators reported that, for homogenous gold layer of 40 nm thickness, the plasmonic resonance happens at higher incidence angles [40].
In Fig. 5, we observe strong noisy behavior of the spectra in a wavelength domain of ∼300–500 nm. This is due to applying the normalization condition to the experimentally measured data. This noise is imposed empirically owing to the scale limitation feature of the used equation
with X being a variable.Figure 5(a)–(h) clearly shows that, with the increase in incidence angle, the resonance wavelengths undergo redshifts, which is less when the angular values are relatively small. For larger incidence angles, the redshifts in transmission minima become more prominent. Also, for large angles, the DNA samples of less dilution do not exhibit plasmonic resonance at all, which becomes obvious from Fig. 5(c)–h. Based on the obtained red shifts in plasmonic resonance, it is worth explaining in short the factors that lead to achieving such spectral behavior. SPPs exist at the interface of two homogeneous layers of gold/DNA, which cannot be excited directly by a light beam as the propagation constant $\beta (\lambda )$ of broadband incidence light is greater than $k(\lambda )$ – the wavevector of light on the DNA-gold side of the interface. As the projection of the momentum along the interface between film and prism has the direction, the phase matching of SPP portions between the gold and DNA sides takes place when the propagation constant of light becomes equivalent to the wavevectors of leaky waves through Eq. (1), as follows [18]
Here, ${\varepsilon _{Au}}$ and ${\varepsilon _{DNA}}$ are the permittivity of gold and DNA, respectively. The condition of SPP excitation relies on the incidence angle $\theta $. The refractive index spectrum of DNA in the visible region is nearly constant whereas that of gold undergoes significant change with wavelength, as Fig. 6 depicts. The dependence of SPP excitation to the incidence angle $\theta $ and wavelength, and the refractive index of gold and BALB/c Rat DNA are the imperative factors to determine the observed red shifts in Fig. 5.
The shift in resonance wavelength with operating condition essentially determines the sensitivity S of the device, which can be mentioned as $S = \Delta \lambda /\Delta \theta $. Figure 7 illustrates the sensitivity plots of the configuration in the absence as well as presence of (the most diluted) DNA thin film. We clearly see the sensing configuration acquires larger sensitivity when the DNA sample is in use, though it varies with changing incidence angle. Typically, for relatively small incidence angles, the device yields $S = 7.2$ nm/degree and $S = 3.2$ nm/degree, respectively, in the cases of presence and absence (Air) of DNA sample. For large incidence angles, however, the same situations provide the respective sensitivity values as $S = 22.4$ nm/degree and $S = 4.8$ nm/degree. The results clearly indicate the plasmonic sensor configuration can be efficiently used to detect the significantly diluted DNA samples in use.
Fig. 7. Plots of resonance wavelengths against incidence angle corresponding to the situations of presence and absence (the case of “Air”) of DNA samples.
4. Conclusion
The afore mentioned experimental investigation pivoted to the extraction of diluted BALB/c rat DNA and its possible sensing characteristics implementing the technique of plasmonic spectrometry reveals that the plasmonic signature can be observed provided the DNA is diluted to the maximum possible extreme (i.e., of the nanoscale concentration). Discussions are made of the ways to extract the dried DNA samples and their subsequent deposition over gold nanolayer thin film. To conduct the experiment, proper rotary mechanical stages were constructed along with the software design to capture the spectrum at small steps of every 0.5° angle. It is found that the plasmonic resonance wavelengths exhibit redshifts with increasing angle of incidence (of the incoming radiation); the redshifts are more for larger angles. This way, the configuration yields enhanced sensitivity for larger angles of incidence. Apart from this, the obtained results also indicate the configuration to achieve higher sensitivity is when the DNA measurand appears (over the gold nanolayer) as compared to the situation of the absence of measurand.
Funding
Zhejiang University (1113000*194232301/002); Xiamen University (XMUMRF/2023-C12/IECE/0046).
Acknowledgement
This research was partially supported by the Xiamen University (Malaysia) Research Fund (Grant No.: XMUMRF/2023-C12/IECE/0046) and that by the Zhejiang University (China; Grant No.: 1113000*194232301/002). Also, the authors are thankful to the two anonymous reviewers for constructive criticisms which elevated the status of the manuscript.
Disclosures
The authors declare no conflict of interest.
Data availability
The produced data for this article including photos and numerical data are available upon reasonable request.
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