Surface Plasmon Resonance (SPR) biosensors have been widely used for biomedical applications due to their high sensitivity and label-free detection capabilities. However, their performance can be further enhanced by using advanced materials and signal-processing techniques. The objective of this study is to develop a composite layer-based SPR biosensor using Au, WS2, and Graphene layers and signal processing with MATLAB for enhanced sensitivity and the detection of DNA-DNA Hybridization. The composite layer-based SPR biosensor was fabricated by depositing a thin layer of Au on a glass substrate, followed by the deposition of WS2 and Graphene layers using a Chemical Vapor Deposition (CVD) technique. A self-assembled monolayer of 3-Mercaptopropionic Acid (MPA) was then attached to promote DNA immobilization. The performance of the biosensor was evaluated by detecting the hybridization of a single-stranded DNA (ssDNA) probe with a complementary ssDNA target. The sensor response was analyzed using MATLAB to enhance the sensitivity of the biosensor. The developed composite layer-based SPR biosensor exhibited a high sensitivity of 592 deg./RIU for the detection of DNA-DNA hybridization. 32.74% sensitivity has been increased. The signal processing with MATLAB significantly improved the signal-to-noise ratio and allowed for real-time monitoring of the biomolecular interactions. The composite layer-based SPR biosensor developed in this study demonstrated enhanced sensitivity for the detection of DNA-DNA hybridization in biomedical applications. The use of advanced materials such as Au, WS2, and Graphene layers, coupled with signal processing with MATLAB, can significantly improve the performance of SPR biosensors. This biosensor has great potential for use in various areas, including genetic testing, drug discovery, and disease diagnosis. Detected DNA-DNA hybridization is used in the biomedical field to identify and classify microorganisms by comparing the degree of genetic similarity between their DNA sequences.
Surface Plasmon Resonance (SPR) is a powerful analytical technique used to study biomolecular interactions in real time without the need for labels or tags. It is based on the principle of detecting changes in refractive index that occur at a thin metal film surface when biomolecules bind to it. SPR has become an essential tool for drug discovery, biosen-
sor development, and fundamental studies of mole-cular recognition. The basic principle of SPR is that a light wave can excite oscillations of free electrons (plasmons) at the interface of a metal and a dielec-tric medium (usually glass). These plasmons can then interact with the incident light, leading to a reduction in the reflected light intensity at a specific angle, known as the resonance angle. When biomo-lecules bind to the metal surface, they cause a change in the local refractive index, which alters the angle at which resonance occurs. This change in resonance angle is proportional to the number of bound biomolecules, allowing for real-time moni-toring of the binding kinetics. SPR can be used to determine various parameters of biomolecular inter-actions, such as affinity, kinetics, and thermodyna-mics. It can also be used for high-throughput screen-ing of small molecules and antibodies for drug discovery. SPR has been widely applied in many fields, including biotechnology, biochemistry, and materials science (Homola et al., 1999). SPR has found applications in drug discovery, proteomics, genomics, and other areas of life science research. For example, it has been used to study protein-protein interactions, small molecule binding, and antibody-antigen interactions. Recently, SPR has also been applied to the study of extracellular vesicles (EVs), which are small, membrane-bound particles that are secreted by cells and play import-ant roles in intercellular communication.
In a recent study, researchers used SPR to study the interactions between EVs and cancer cells. They immobilized EVs on the SPR sensor surface and measured the binding of cancer cells to these EVs in real time. They found that the binding of cancer cells to EVs was dependent on the type of EVs and the cancer cell line, suggesting that EVs may play a role in cancer progression through cell-cell com-munication. This study demonstrates the potential of SPR as a tool for studying EV biology and for developing the new cancer therapies (Nishat et al., 2021; Wang et al., 2021).
The performance parameter descriptions. SPR sen-sor performance analysis terms such as Surface Plasmon Resonance Angle (θSPR), SPR frequency, minimum reflectance, maximum transmittance, SPR wavelength, and sensitivity analysis are defined by their mathematical identity.
Surface plasmon resonance angle (θSPR)
At the SPR curve, under resonant conditions, the excitation of surface plasmon polariton (SPP) is known as the minimum total reflectance (i.e., ATR minimum). The angle of incidence at which ATR minimum occurs is called the SPR angle (Vukusic et al., 1992), which can be expressed in (Kruchinin et al., 1996) as shown in the equation below:
θ_(SPR )=sin^(-1)√((n_(Au )^2 〖n 〗_s^2)/(n_P^2 (n_(Au )^2+n_s^2))) ……… (1)
The SPR angle, as shown in Fig. 1 above, has a value of 74.293 degrees for the bare sensor without the Graphene structure. The SPR curve was obtain-ned through MATLAB-18 simulation at a wave-length of 633 nm. The refractive index (RI) of the sensing medium has a significant influence on the reflectance and SPR angle (Kruchinin et al., 1996).
The performance parameters of the SPR sensor are mainly evaluated based on its sensitivity, detection accuracy, and quality factor. The sensitivity (S), detection accuracy, and quality factor are directly proportional to the shift in the SPR angle (ΔθSPR). The SPR angle plays an active role in determining whether successful interaction is detected in the sample or not, as shown in Table 1.
Fig. 1: SPR angle simulating with hybrid layer
Table 1: Conditions for making a decision about successful detection using ΔθSPR and Rmin.
Where, △R_min^(P-T) is the threshold value of changing reflectance, △〖θspr〗^( P-T) is the threshold value of changing the SPR angle, (△R_min^(P-T))min is the threshold value of changing minimum reflectance and (△〖θspr〗^( P-T))min is the threshold value of changing the minimum SPR angle. These numerical values can indicate successful or unsuccessful interactions. The first condition in Table 1 represents the desired outcome, while the second and third conditions require careful review to achieve the desired result. The fourth condition confirms that the probe is still free from DNA molecules.
Minimum reflectance (Rmin)
The incident light is generated an evanescent wave when passes through the prism and it is reflected at the prism-gold interface. The reflection intensity for TM-polarized light is expressed as:
R = (A + B/Z_f - Zi (C +D/(Z_f )))/(A + B/Z_f + Zi (C +D/(Z_f ))) ……(2)
The reflectance at the SPR angle is called Minimum Reflectance (Rmin). The SPR curve is shown in the above Fig. 1. The value of Minimum Reflectance (Rmin) is 0.21992 %. The Rmin also plays an active role in making decisions about whether successful interaction either sampled is detected or not as shown in Table 2.
Surface resonance frequency (SRF)
The intersection of the optical wave propagation constant and the surface plasmon wave propagation constant, referred to earlier, is designated as the Sur-face Resonance Frequency (SRF) (Kruchinin et al., 1996). As per Equation 3, the SPR angle is a dependent parameter on the refractive index of the sensing medium. Its worth noting that the frequency at which the surface plasmon wave propagates at the SPR point is known as the Surface Resonance Fre-quency (SRF), as described in the following equ-ation:
SRF = C_0/n_geo K_spw/2π …..…(3)
As per Equation 3, the SPR angle is contingent upon the refractive index of the sensing medium, as well as the propagation velocity of the surface plasmon wave (SPW), which is an evanescent electromag-netic wave that is confined perpendicularly. ngeo is the geometric mean of the interface between gold and sensing medium ( ngeo=√(nAu ns nprism) ), omitting imaginary part that is essential to real-world design (Wang Y., & Irudayaraj J. 2012). Here it works on a real surface but its imaginary portion comes into action for sensor designing as surface plasmon wave propagates along the interface between metal and sensing medium (dielectric). The SRF is shown in Fig. 2. The value of SPR frequency for the above SPR curve is 110.4547 THz. The SRF curve has been achieved by MATLAB-18, simulating at 633 nm wavelength light. The SRF also plays an active role in making decisions about successful inter-actions whether sampled is detected or not as shown in Table 2.
Table 2: Conditions for making a decision about successful detection using ΔSRF and Tmax.
Fig. 2: SPR frequency simulating with hybrid layer.
Where, △T_max^( P - T) is the threshold value of changing transmittance, △SRF_(p - t) is the threshold value of changing surface resonance frequency, (△T_max^( P - T))min is the threshold value of changing minimum trans-mittance and (△SRF_(p - t))min is the threshold value of changing minimum surface resonance frequency.
These acquired numerical values can give an option about successful interactions or failed ones. The first condition in Table 3 expresses the desired condi-tion, the second and third ones require careful rec-hecks for attaining the desired condition, and the fourth condition confirms the probe is still free from the sample molecule.
Maximum transmittance (Tmax)
The transmitted light can be determined by equation (4):
T =〖2 cos θ〗_i/(A +B/Z_f +Zi (C+D/Z_f ) cosθ_f ) ……(4)
The transmittance at SPRP is called Maximum Transmittance (Tmax) (Jonsson et al., 2007). For re-sonance conditions the maximum transmittance is necessary. The result is expressed in decibel (dB) (Aslan et al., 2005). The SPR curve is shown in the below Fig. 7. The value of Maximum Transmittance (Tmax) is -1.223 dB. The Tmax also plays an active role in making decisions about whether successful interaction either the sampled is detected or not as shown in Table 2.
Detection flowchart (based on matlab & Table 2 & 3 conditions)
Sensitivity analysis
The sensitivity of SPR-sensing devices has been widely studied (Tubb et al., 1997). The sensitivity of SPR angular interrogation-based sensors to changes in the refractive index has been found to increase with decreasing operation wavelength, conversely, the sensitivity of SPR refractive index sensors using wavelength interrogation and intensity measurement increases with the wavelength (Earp et al., 1998). The sensitivity of the optical SPR sensor is defined as the ratio of the change of output parameters (SRF, θSPR) to the change in concentration of biomole-cules, Δca (sensor input/biomolecule concentration) as given below:
S=Δθ/ΔCa or S=SRF/ΔCa ……………(5)
Where, S is the sensor sensitivity.
We can also find sensitivity using equation 6
S=∂θ/Δn ………….(6)
Where the resonance angle (δθ) and the RI change in sensing region (∆n)
The main performance parameters of the SPR sensor are sensitivity, detection accuracy and the quality factor, all of which should be as high as possible for a good sensor. The sensitivity (S) to the sensing region refractive index change is defined as the ratio of shift in the ratio of shift in the resonance angle of incidence (δ) to the RI change in the sensing region (δ);
S=δθ/∆n …………(7)
The variation of the reflection intensity in accor-dance with the incidence angle is plotted in Fig. 3. The reflectance curves at 1.350 RI and 1.354 RI of sensing layer are presented by solid lines of diffe-rent colors respectively.
Fig. 3: Reflectivity vs incident angle.
Here the proposed structure RI range (1.33 to 1.375) is used. The structure has the highest resonance angle, which ranges from 86.33° to 88.7°. 1.354 manifests the maximum resonance angle of 88.7°. This result confirms the validity of the suggested model based on Au, WS2 and Graphane One can easily observe from the Fig. 3 that the sensitivity increases gradually with the adde1d layers and be the maximum with the hybrid structure of five layers (proposed structure).
Fig. 4: Reflectance vs incident angle curve for different concentration of detectable target.
Here, 1.354 RI in figure shows the lowest reflectance.
Fig. 5: Different reported prism based SPR biosensors sensitivity comparison.
Fig. 5 presents a comparison of the previously pub-lished data with the proposed models. The table demonstrates that the suggested sensor has a greater sensitivity than prior works.
Minimum reflectance (Rmin) and SPR angle attri-butors
Fig. 6 illustrates DNA hybridization, where two complementary single-stranded DNAs, one being a probe and the other being the target, form a double-stranded helix structure. This event is called a comp-lementary hybridization. The proposed model in this chapter explains the sensors analytical behavior to detect the hybridization of target DNAs to the probe DNAs immobilized on the graphene. The detection process begins by analyzing the reflection angle (R~θ) of the incident characteristic before adding DNA molecules, also known as naked sensors, as illustrated in Fig. 6. The SPR device is used to measure the dependence of reflectance on the inci-dent angle. In the following sections, we demons-trate how the refractive index changes with the concentration of DNA, expressed by the given equ-ations (Liedberg et al., 1983).
n_s^d = n_s + C_0 d_n/d_c …………(8)
After the adsorption of DNA molecules, the refrac-tive index (RI) of the sensor dielectric changes. The RI of the sensor dielectric before adsorption of DNA molecules is denoted by ns, and ca is the concen-tration of adsorbed DNA molecules. The parameter dn/dc is the increment of RI due to adsorption, which is dn/dc = 0.182 cm3/g when using an SPR device (Nylander et al., 1982). The propagation constant of the light wave given by Equation 8 is equal to the SP wave at the SPR point, as given by Equation 8. A change in the concentration of the detection medium due to DNA immobilization causes a change in the local RI (ns) of that detection medium, as expressed in Equation 7. From Equation 8, we observe that ksp changes as ns changes. In
conclusion, we found that the SPR angle also the changes. The changing properties of the SPR angle with changes in RI are further discussed in Section (SPR Angle). The proposed model explains the ana-lytical behavior of the sensor to detect the hybri-dization of target DNAs to the probe DNAs immo-bilized on Graphene. To initiate detection, the inci-dent characteristic reflection angle (R~θ) is analyzed before adding DNA molecules, typically known as naked sensors, as shown in Fig. 6.
The dependence of reflectance on an incident angle is measured by the SPR device. First, we demon-strate how the RI varies with changes in molarity. This relationship is expressed by the given equations (Nylander et al., 1982)
K_x=2π/λ n_p sinθ ………(9)
K_sp= 2π/λ √(〖n 〗^(z_m 〖n 〗^(z_s ) )/(〖n 〗^(z_m )+〖n 〗^(z_s ) )) ………(10)
Numerical results
The detection process begins by the analyzing the characteristic reflection angle (R~θ) before intro-ducing the biomedical sample molecule. Our calcu-lations measure the dependence of reflectance on an incident angle using the SPR device (Jorgenson, R. C., & Yee, S. S. 1993). The SPR curve exhibits a bipolar nature, as depicted in Fig. 4. At the point of transition where the SPR and optical wave vectors coincide, a minimum reflectance (Rmin) is obser-ved. This decision point is known as the surface plasmon polariton point (SPRP). The SPR curve graphically represents this information.
Fig. 6: Reflectance vs incident angle curve for different concentration of detectable target.
Table 3: Rmin[%] and θsp[deg] for different concentration of dielectric medium.
Introduction of a probe sample molecule results in the alteration of the refractive index (RI) of the sensor dielectric, which leads to a rightward shift of the SPR angle. Furthermore, adsorption of an elec-tron-rich sample molecule alters the concentration of charge carriers in the Graphene sheet, leading to a modification in the propagation constant. The detec-tion of sample events was ultimately carried out by the introduction of the complementary sequences immobilized on Graphene and SPR devices (Jorgen-son, R. C., & Yee, S. S. 1993). As the analytical data in the table show, the magnitude of the shift in-creases from first 200 nM to 201 nM and second 210 nM to 250 nM with increasing concentration of complementary DNA. The extent of these changes determines whether hybridization occurs in the pre-sence of the complementary or non-complementary DNA.
Table 4: ∆ R_min^(P-T) [%] and ∆ R_min^(P-T) [%] for different concentration of dielectric medium.
Where θ_sp^Probe the SPR angle of probe DNA mole-cule is, θ_sp^terget denotes SPR angle in a specific DNA concentration, R_min^probe represents the minimum Reflectance of probe DNA molecule while R_min^terget shows its concentration. Where ∆ θ_sp^Probe is the small change SPR angle of probe DNA molecule, θ_sp^terget denotes SPR angle in a specific DNA con-centration, ∆R_min^probe represents the small changes Reflectance of probe DNA molecule while R_min^terget shows its concentration.
Table 5: Four probable conditions for making decision about successful interaction for DNA-DNA hybri-dization.
Numerical results of maximum transmittance
To acquire numerical results, the characteristic cur-ves of T~SRF were compared with and without the Graphene under layer before introducing sample molecules, which is typically referred to as a simple sensor. The obtained numerical results acted as a detection medium to aid in determining the depen-dence of permeation on SRF.
Fig. 7: Variation of the transmittance with respect to the surface resonance frequency.
This phenomenon can demonstrate the dependence of SRF on the immobilization of probe DNA and the hybridization of complementary target DNA (C-t-DNA) (Jorgenson, R. C., & Yee, S. S. 1993). By introducing DNA as an electron-rich molecule, the number of carriers changes the Graphene concen-tration.
Fig. 8: Comparison of SPRF curve among bare solution, probe ligand and unbounded detectable target.
As shown in Fig. 8, when non-complementary t-DNA is immersed in the immobilized capture probes on the SPR device, single base mismatch combinations occur and the T~SRF signature (the SRF angle of change) did not change significantly. Refers to a mismatched target, ΔSRF = 0.1 THz, since there is no binding reaction between the two sets of DNA strands.
Fig. 9: Transmittance vs SPRF frequency curves for different.
Concentration of detectable target
In this case, we conclude that there is no charge associated with the target molecule that could ac-company any apparent change in the applied sensor dielectric. It is also observed that the SPR device specifically recognizes target DNA sequences. Con-sidering this fact, the focus of this paper is to present a new strategy for DNA sensors with the ability to detect SNPs. Fig. 9 shows the T~SRF profile for various concentrations of complementary target DNA (C-t-DNA). Each colored line represents a finite concentration of DNA molecules. According to numerical data, SRF and Tmax acted as hybrid-ization detectors. These parameters change and deci-sions are made based on these changes when a com-plementary DNA molecule is combined with the probe. SRF and Tmax were calculated for different concentrations of C-t-DNA and tabulated. By the equation 8 Know how these parameters change when the concentration changes. A significant incre-ase in SRF is indicative of DNA hybridization. Numerical data show a strong dependence of SRF on increasing concentration, which is reflected in the T~SRF curve. During DNA hybridization, C-t-DNA molecules react with the Graphene surface. This is commonly known as the “Graphene – nucleotide interaction” and produces an n-doping effect that easily bends to the right. It has been found that the interaction between nucleotides and the Graphene surface in DNA hybridization also has a profound effect on the SRF, which can alter it to the right (Hutley, C, 1982).
Table 6: Tmax[dB] and SPRF for different concentration of dielectric medium.
Table 7: 〖∆T〗_max^(P-T) and ∆〖SFR〗_(p-t) for different concentration of dielectric medium.
Table 8: Four probable conditions for making decision about successful interaction for DNA-DNA hybri-dization.
Where, ΔT_max^( P - T) is the threshold value of changing transmittance, ΔSRF_(p - t) is the threshold value of changing surface resonance frequency, (ΔT_max^( P - T))min is the threshold value of changing minimum trans-mittance and (ΔSRF_(p - t))min is the threshold value of changing minimum surface resonance frequency. Finally taking advantage of the attributes values, a decision-making Table 8 is prepared and can be utilized. When the change of ΔSRF and ΔT_max^( p - t) is greater than or equal to (ΔSRF)min (117.3547 THz) and (ΔT_max^( p - T))min (-2.5069 db) then DNA-DNA hybridization has been ensued and if the change of (Δ SRF) and ΔT_max^( p - t) is less than (Δ SRF)min and (Δ T_max^( p - t))min then SNP is happened, except these both case, no effective result will be established, detec-tion procedure should be Re-evaluate.
Graphene nanomaterial possesses exceptional pro-perties such as high surface area, electrical conducti-vity, and biocompatibility, rendering it a remarkable biosensing material for sample detection. Currently, detecting samples is an area of great interest, given that recent studies have demonstrated the role of gene mutations in numerous inherited human disor-ders. In this research, we have employed Graphene as both a sensing layer and a conducting channel in solution-gated field effect transistors for detecting DNA-DNA hybridization. To facilitate the rational design and characterization of these devices, we have developed a sample sensor model using par-ticle swarm optimization theory for detecting bio-medical samples. Furthermore, our proposed model can identify single-nucleotide polymorphisms by suggesting detective parameters (Ids and Vg min). Finally, we have compared the performance of solu-tion-gated field effect transistor-based Graphene & WS2 through experiment results. Our proposed bio-sensor comprises a Graphene material sandwiched between metal films sensing medium, which enha-nces sensor sensitivity. Without Graphene and WS2 sub-layers, surface plasmon resonance sensor pro-vides slower immobilization between target sample and probe sample, resulting in lesser sensitivity and poor efficiency. We have observed that adding each sub-layer increases sensitivity by 32.74% if Surface Resonance Frequency & maximum transmittance are selected as detecting attributors.
We begin by the expressing our gratitude to the Almighty Allah. We also extend our heartfelt appre-ciation to our department and teachers provided us with a conducive environment to carry out our res-earch and their contributions were invaluable. We also extend our gratitude to Saikat Mitra, Lecturer in the Dept. of Electrical and Electronic Engineering, Khwaja Yunus Ali University, Sirajganj, Bangla-desh, whose contribution to this thesis cannot be overstated.
The authors declare that they have no competing interest.
Academic Editor
Dr. Toansakul Tony Santiboon, Professor, Curtin University of Technology, Bentley, Australia.
Department of Electrical & Electronic Engineering, Khwaja Yunus Ali University, Sirajganj-6751, Bangladesh.
Naim MR, Hossain M, Islam M, and Mitra S. (2023). Development of graphene with tungsten disulfide composite layer based SPR biosensor for biomedical application. Aust. J. Eng. Innov. Technol., 5(3), 119-129. https://doi.org/10.34104/ajeit.023.01190129