Detection of Envelope Glycoprotein Domain III (ED III) Based on Surface-Enhanced Raman Scattering for Dengue Diagnosis

Surface-enhanced Raman Scattering (SERS) based ED III protein detection has been conducted as a dengue virus diagnosis. ED III is one of the ectodomains of the envelope located along the surface of the dengue virus and acts as a receptor for the virus with its host cells. The SERS substrate used in this study is On-Spec-lite SERS by NECTEC and fabricated as a grating surface coated with gold. The imaging results from SEM show that the average radius of gold nanoparticles on OnSpec-lite SERS is 60 nm. On the grating-Au surface, the grating period is 588 nm with 50 nm thickness of gold thin film. OnSpec-lite SERS has a more significant increase in Raman intensity than the grating-Au surface when detecting ED III using Raman spectroscopy. The amino acids in the ED III Raman bands are Glycine, Alanine, Histidine, and Proline. The highest intensity Raman band that appears is 850 cm-1. Based on the research done in dengue virus detection, ED III has a typical Raman peak of 850 cm-1 which is the Raman band of Glycine and Alanine with CCN symmetry stretching vibration mode (𝑣(CCN)). This distinctive characteristic of ED III can be used as a diagnostic to detect dengue virus.


Introduction
Dengue hemorrhagic fever is an acute febrile disease spread in tropical climates and is caused by one of the four dengue virus stereotypes, such as DENV1, DENV2, DENV3, and DENV4 [1].The dengue virus is transmitted through the medium of the Aedes aegypti mosquito, which belongs to the Arthropod-Borne Virus, Flavivirus genus, Flaviviridae family.It is known as the main vector for carrying the dengue virus [2].Clinical symptoms caused by the dengue virus include high fever, bleeding manifestation, signs of circulatory failure, and shock due to plasma leakage, which causes death [3].The envelope protein (E) is a protein found on the surface of the dengue virus that interacts with host cells.This protein weight about 3 kDa and has 3 envelope domains (ED), they are ED I, ED II, ED III.[4].ED I is the center of the protein envelope.ED II has a role in the fusion process.ED III, the outermost part of the envelope, has a role in inducing antibody formation and has high immunogenicity properties.Generally, ED III has a role in the development of the dengue virus vaccine [5] It can be used as a candidate for developing dengue infection diagnosis technology [6].
Clinical diagnosis of dengue virus for patients can be made generally by laboratory tests such as checking platelet and hematocrit levels [7].This conventional method has disadvantages such as requiring specific instruments, consuming a lot of preparation medium that can cause biological waste, long analysis time, high cost, and requiring trained technicians to perform sample preparation and process data.The optical method is one of the options because its use only requires a little sample preparation and fast analysis time [8].
Raman spectroscopy was invented to study the structural properties of solid, liquid, and gaseous molecules from scattering spectra.Raman spectroscopy's detection offers a more detailed biochemical fingerprint and is useful for identifying unknown molecules [9].Low Raman signal can be enhanced by Surface-Enhanced Raman Scattering (SERS) technique.SERS is a technique based on the sensitivity of nanostructures or thin surfaces of metals such as gold and silver, thereby increasing the reading of Raman scattering through absorbed molecules [10].The measurements of Raman spectra were carried out on the ED III antigen of Dengue virus.In this research, we fabricated a gold-coated grating Surface as a SERS substrate made from a Digital Versatile Disk (DVD).The DVD can be used as a low-cost SERS substrate that has the characteristics of having a uniform nanostructure surface and is coated by a thin metal layer [11].The DVD has a uniform grating pattern on its surface and can enhance the nearfield effect as the propagating plasmon.This effect also can enhance the Raman signal.The enhancement of the Raman signal can be augmented by the grating-Au surface, which will be compared in performance with a commercially produced OnSpec substrate by the National Electronics and Computer Technology Center (NECTEC).

Fabrication of SERS Substrate
The SERS substrate was fabricated using a DVD made from polycarbonate.The DVD was then cut into small parts and cleaned using ethanol.The dried DVD was then put into an ultrasonic cleaner for 10 minutes to clean the substrate surface using ultrasonic waves.The clean substrate was then prepared for sputtering by Ion beam sputtering.Figure 1 shows the steps of grating-Au substrate fabrication using Ion Beam Sputtering Hitachi MC1000."The sputtering process used Hitachi MC1000 Ion Sputtering with gold in bulk as the target metal.The substrate that has been prepared is then inserted into a vacuum tube with a 6 cm diameter tube base.Argon gas then flows into the tube.The tube and target metal are then given a potential difference and 10 mA of current which causes free gas electrons around the target metal to move away and collide with Argon-neutral atoms, which turn into Argon ions.The high-speed Argon ions will collide with the negatively charged target metal, causing the gold to detach from the target metal surface and accumulate on the substrate.The result of the sputtering was a gold thin film that coated the DVD substrate.This process is carried out for 10 minutes to achieve a thickness of up to 50 nm.The grating-Au and OnSpeclite SERS substrates are examined through a Scanning Electron Microscope (SEM; Hitachi, SU3500) to see the characteristics of both SERS substrates.
A piece of DVD Remove the DVD cover and cut it into pieces with size 1x1 cm 2 .
The gold sputtering will take 10 minutes to reach the 50 nm of thickness. Grating

ED III Detection using Raman Spectroscopy
Grating-Au surface and silicon were cleaned gently using ethanol to remove the residual on the surface.OnSpec-lite SERS does not require the cleaning using ethanol due to its geometry so the analyte step in between its structure.Approximately 10 μL of ED III protein was dripped on the grating-Au, silicon, and OnSpec-lite SERS surface substrates using micropipettes.The detection of ED III using silicon substrates was performed to determine the Raman spectra of ED III before the enhancement of Raman spectra occurred.The sample was then allowed to stand for 10 minutes in a stained cup to adhere to the substrate.The substrate was then placed on a holder and inserted vertically in the Raman spectroscopy (Horiba, MacroRam) sample chamber, followed by closing the sample chamber.The laser with a wavelength of 785 nm was set with 50% (225 mW) power and an acquisition time of 5 seconds.Measurements were taken at three points on the sample, which were then averaged to see the Raman peaks that emerged from the characterization of the ED III sample.

SEM Characterization of OnSpec-lite and grating-Au surface SERS substrate
The surface structure of the SERS substrate coated with gold metal was examined through SEM analysis.Figure 2a displays the top view of the DVD substrate that was created and analyzed using SEM.This analysis revealed that the substrate structure consists of gratings coated with a gold thin film with a 22.63 nm depth and 297 nm and a grating period of 588 nm.The gold thin film was coated along the track pitch of the DVD.Compared to the OnSpec-lite SERS whose surface roughness is derived from a micrometer-scale by laser-engraved on an aluminium substrate, the grating-Au surface substrate has a nanometer-scale and more regular grating-like surface.The OnSpec-lite SERS developed by NECTEC was fabricated by laser-engraving surface on aluminum at the nanoscale and then deposition of Au nanoparticles.This process produces gold nanoparticles with an average radius of 60 nm on a surface with dimensions of 5 ⨉ 5 mm 2 [12].Figure 2b shows the SEM image of a laser engraving on alumunium surface coated with gold nanoparticles.

Analysis of Raman spectra of ED III protein
Before measuring the ED III protein, the SERS substrate was first characterized to obtain the maximum power that the SERS substrate can receive.This measure of power usage is obtained from the base substrate characterization.The result shows that the grating-Au surface and silicon do not give any Raman band by our procedure.Based on this characterization, it is found that the maximum power that can be used on the fabricated SERS substrate is 50%.This is because when the analytes were measured at 20% and 50% laser power, the Raman signals from the analytes appeared with different intensities.But when the laser power is increased by more than 50%, the substrate used is burnt so that the Raman signal from the analyte does not appear.From this characterization, the laser power used is the same on both substrates at 50% (250 mW) of laser power.Figure 3 shows the Raman spectrum of ED III from different substrates such as silicon, grating-Au, and OnSpec-lite SERS substrate.The use of silicon substrate to show ED III spectra before the Raman signal enhancement.The peak of the silicon substrate appears at 520 cm -1 Raman band. Figure 3 shows that the maximum intensity of the ED III spectrum with silicon substrate is very low.The silicon substrate does not appear to have any enhancement in the Raman band.An increase in intensity occurs on OnSpec-lite SERS and grating-Au surface SERS substrates.OnSpec-lite SERS has a higher intensity enhancement when compared to the grating-Au SERS substrate.OnSpec-lite SERS substrate fabricated by NECTEC can increase the intensity to 3.90 times, while the grating-Au SERS substrate can increase the intensity to 1.72 times.The amplitude of the Raman signal enhancement is the ratio between the Raman signal using the SERS substrate (grating-Au surface and OnSpec-lite) and the Raman signal without using the SERS substrate (silicon).
The increase in intensity on the OnSpec-lite SERS is almost twice the increase on the grating-Au surface.The higher signal enhancement on the OnSpec-lite SERS occurs due to the rougher substrate surface that can be seen directly by the eye.Substrates coated with gold will increase the occurrence of plasmonic effects so that the molecular vibrations of the analyte interacting with the Raman wavelength on the metal surface will increase the Raman signal.This rough surface is caused by scratching the aluminum surface using a laser marking machine with a micrometer scale and coating gold nanoparticles using a magnetron sputtering system.When compared, the grating-Au substrate has a flat surface when viewed directly.The grating-Au surface substrate will look rainbow-colored when subjected to visible light.This phenomenon occurs due to the diffraction of light in the grating structure on the nanometer-sized DVD surface [13].The higher field enhancement on the OnSpec-lite SERS substrate than the grating-Au surface substrate is also due to the plasmonic effect on the denser spacing of the gold nanoparticles.The periodic surface of the SERS substrate coated with gold can enhance the Raman signal.This is because a substrate that has a periodic surface and is coated by gold will create many hotspots when given an incident wavelength.When biomolecules interact with the SERS substrate, electron oscillations on the surface of the substrate can produce a very strong electromagnetic field so that there is an increase in the Raman signal from biomolecules on the surface of the substrate.
In Figure 1, the OnSpec-lite SERS with macro-scale engraving is coated by gold nanoparticles with an average radius of 60 nm that form clusters that reach outward from the surface plane.These gold nanoparticle clusters will create many hotspots when the incident field hits the gold nanoparticle clusters so that the increased field created will also increase the Raman signal.Grating-Au surface substrates with nanoscale slits are coated by gold thin film with 50 nm of thickness along the grating.The thickness of the gold thin film is from the datasheet of the Ion Sputtering Beam.The gold thin film on the grating-Au surface substrate is only formed along the substrate plane so that the hotspots created when the incident field will create a field enhancement that is not greater than the OnSpec-lite SERS substrate.
The highest Raman band appears at 850 cm -1 , which can be considered as symmetric stretch vibrations (v(CNC) symmetric stretch) of amino acids Glycine and Alanine, which have simple R groups [14].850-900 cm -1 is the Raman band with the strongest intensity in the amino acid glycine, alanine, and histidine which has a symmetric stretch vibrational mode [15].From the amino acid series, Glycine, Histidine, and Alanine are the amino acids that are abundant in the amino acid groups in ED III, whose Raman bands appear the highest.When viewed from the Infrared (IR) band assignment, glycine appears at 890 cm -1 [16], alanine appears at 850 cm -1 [17], but the spectrum is lower than the Raman band, and histidine appears around 860 cm -1 [18].Glycine and alanine are non-polar amino acids, so they have active Raman and inactive IR characteristics.The 917 cm -1 Raman band is the vibrational mode for the C-C stretch found in the amino acid proline [19].The 1050 cm -1 Raman band is the vibrational mode for the C-O (v(CO)) stretch in the carboxylic acid group of the amino acid group [20].The 1462 cm -1 Raman band is the vibrational deformation mode of CH3 on the R side chain of the amino acid alanine [15].The Raman bands of Threonine, Lysine, and Leucine do not appear in Figure 3 due to their soluble nature in liquid form, so the Raman bands that appear are so weak that they are difficult to detect [21].[23], and Khan et al. [24] have in common the appearance of Raman bands at 850-900 cm -1 with v(CNC) vibration mode.In this Raman band, there is an increase in intensity between samples of healthy people and those infected with the dengue virus.This Raman band indicates the amino acid glycine, the most amino acid constituent of the ED III dengue virus.ED III contained on the entire surface of the dengue virus allows direct interaction with the Raman wavelength so that the scattering causes the Glycine and Alanine Raman bands to appear in samples infected with the dengue virus.Based on the explanation before, the detection of ED III can be used as a parameter to detect dengue viruses that infect humans.

Conclusion
The data and analysis concluded that the OnSpec-lite SERS substrate fabricated by NECTEC can increase the intensity to 3.90 times while the grating-Au SERS substrate can increase the intensity to 1.72 times.Each substrate affects the increase of the electric field, which affects the increase of the Raman signal.Amino acid groups (Glycine, Alanine, Histidine, and Proline.) were detected in ED III using Raman spectroscopy on the surface of each SERS substrates.The detected amino acid is the most abundant amino acid among the 160 amino acid sequences of ED III.

Figure 1 .
Figure 1.The substrate fabrication of grating-Au surface.The sputtering process used Hitachi MC1000 Ion Sputtering with gold in bulk as the target metal.The substrate that has been prepared is then inserted into a vacuum tube with a 6 cm diameter tube base.Argon gas then flows into the tube.The tube and target metal are then given a potential difference and 10 mA of current which causes free gas electrons around the target metal to move away and collide with Argon-neutral atoms, which turn into Argon ions.The high-speed Argon ions will collide with the negatively charged target metal, causing the gold to detach from the target metal surface and accumulate on the substrate.The result of the sputtering was a gold thin film that coated the DVD substrate.This process is carried out for 10 minutes to achieve a thickness of up to 50 nm.The grating-Au and OnSpeclite SERS substrates are examined through a Scanning Electron Microscope (SEM; Hitachi, SU3500) to see the characteristics of both SERS substrates.

Figure 2 .
Figure 2. SEM imaging results of (a) grating-Au surface SERS substrate with 22.63 nm of depth and 588 nm of periodicity, and (b) OnSpec-lite SERS with laser-engraved on aluminum surface.
has 160 amino acid groups whose types vary with each amino acid that makes it up.Glycine (G), Alanine (A), Histidine (H), Threonine (T), and Lysine (K) are the most amino acid in ED III protein.Most of the amino acid sequences in ED III have very few amino acids that have benzene rings, such as Tryptophan, Tyrosine, and Phenylalanine.

Figure 3 .
Figure 3. Raman spectra of ED III protein in three different substrates such as silicon, grating-Au, and OnSpec-lite SERS surface

Table 1 .
Raman band assignment of ED III proteinThe association of ED III protein with the dengue virus can be seen from the Raman band that appears in a person infected with the dengue virus sample.Research fromKhan etal.(Khan et al., 2016), Bilal et al.