# DNA Hybridization Detection Based on Resonance Frequency Readout in Graphene on Au SPR Biosensor

Authors: Md. Biplob Hossain; Md. Masud Rana
Journal: Journal of Sensors
 (2016)

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## Abstract

This paper demonstrates a numerical modeling of surface plasmon resonance (SPR) biosensor for detecting DNA hybridization by recording the resonance frequency characteristics (RFC). The proposed sensor is designed based on graphene material as biomolecular recognition elements (BRE) and the sharp SPR curve of gold (Au). Numerical analysis shows that the variation of RFC for mismatched DNA strands is quiet negligible whereas that for complementary DNA strands is considerably countable. Here, graphene is used to perform faster immobilization between target DNA and probe DNA. The usage of graphene also changes the RFC that ensure hybridization of DNA event by utilizing its optochemical property. In addition, proposed sensor successfully distinguishes between hybridization and single-nucleotide polymorphisms (SNP) by observing the variation level of RFC and maximum transmittance. Therefore, the proposed frequency readout based SPR sensor could potentially open a new window of detection for biomolecular interactions. We also highlight the advantage of using graphene sublayer by performing the sensitivity analysis. Sandwiching of each graphene sublayer enhances 95% sensitivity comparing with conventional SPR sensor.


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## Body

## 1. Introduction

DNA hybridization is a biochemical process to find out the DNA nucleotide bonding in proper orientation (between adenine (A) and thymine (T) or between guanine (G) and cytosine (C)) or in mutated sequence shown in Figure4, in [1]. DNA mutation is a piece of information that indicates hepatitis B [2], cancer, and other complex congenital diseases [1]. Nowadays, biosensors have been intensely researched owing to their importance for many industry applications such as medical diagnostics, enzyme detection, food safety, and environmental monitoring [3, 4]. Today, a number of biosensor schemes have been developed; among them fiber-optic surface plasmon resonance (SPR) biosensor bears the advantages of compactness, light weight, high sensitivity, the ease of multiplexing and remote sensing, and so forth [5]. Fiber-optic surface plasmon resonance (SPR) biosensor is an evanescent guided electromagnetic wave mode [6] that propagates along a metal-dielectric interface (e.g., gold and biomolecule) by utilizing the surface plasmon waves (SPW) [5]. The variation of the biomolecules concentration will produce the local change of the surrounding refractive index (SRI) near the sensor surface that results in changing the propagation constant of the SPW and thus the resonance frequency [5]. The SPR technique has been successfully applied in various fields, such as chemical and biochemical sensing, film characterization, and beam polarization selection [7]. In biochemical sensing applications, SPR is used for DNA-DNA hybridization [1, 5–8], protein-protein [9, 10], protein-DNA [11, 12], enzyme-substrate or inhibitor [11, 13], receptor-drug [14, 15], lipid membrane-protein [16, 17], protein-polysaccharide [18], cell or virus-protein [19–21], and so forth.In this paper, a numerical modeling of graphene based SPR biosensor is proposed for DNA hybridization detection which results in faster immobilization by monitoring the resonance frequency characteristics (RFC). Since graphene has outstanding properties such as high surface area [22], electrical conductivity [23], and biocompatibility [1, 23], so graphene monolayer is sandwiched between metal films and sensing medium as biomolecular recognition elements (BRE) for enhancing sensor sensitivity 95% better than the conventional sensitivity. Carrier concentration is varied due to the immobilization of DNA molecule on the sensor surface that changes the refractive index (RI) near the graphene and gold (Au). The RI change will in turn lead to change in the SPR angle that translates a change in propagation constant of SPW. Moreover, graphene layer on gold will also modify the propagation constant of SPW, thereby varying the sensitivity due to RI change.


## 2. Numerical Modeling and Sensor Methodology

We introduce a numerical model using Kretschmann prism arrangement [8, 24] described in Figure 1(a) that shows the schematic configuration of the proposed graphene-on-gold surface plasmon resonance biosensor, where the first layer is a SF10 glass prism (RI n
p
=
1.723), second layer is gold film (thickness and RI are d
Au
=
50 nm and n
Au
=
0.1726
+
i
3.4218, resp. [8]), third layer is graphene (thickness and RI are d
G
=
0.34 nm and n
G
=
3
+
i
1.149106, resp. [8]), and the final layer is water (RI n
s
=
1.33 [24]). Then, a TM polarized He-Ne (wavelength, λ
=
633 nm) light wave is used, which passes through the prism and some portion is reflected at the prism-gold interface.Figure 1
(a) Schematic of the 4-layerd model for surface plasmon resonance (SPR) biosensor: prism, Au (50 nm), graphene (0.34
L nm, where L is the number of graphene sublayers), and sensing medium (water). (b) TheT~SRF curves for the conventional biosensor (L
=
0) and graphene coated biosensor (for L
=
1,2
,
…
,
10) before the adsorption of DNA molecules.
(a)
(b)While impinging light energy to prism-gold interface, an evanescent wave is generated which is known as surface plasmon wave (SPW) that propagates with the different propagation constant from optical wave which is defined in [8, 25]. The propagation constant of SPW can be adjusted to be equal to the propagation constant of optical wave that is also defined in [8]. The point at which optical wave propagation constant equals SPW propagation constant is called surface plasmon resonance point (SPRP) [8]. Equation (1) suggests that SPR angle is a dependent parameter on RI of sensing medium. At SPRP, the frequency at which SPW propagates is called surface resonance frequency (SRF) and the angle of incidence is called SPR angle that can be given in [25] shown as follows: (1)
θ
SPR
=
s
i
n
-
1
n
Au
2
n
s
2
n
p
2
n
Au
2
+
n
s
2
.When submersing single strand target DNA (t-DNA) into probe DNA (p-DNA) on the sensor surface, then the RI of sensing medium is mathematically modified as [26, 27](2)
n
s
d
=
n
s
+
c
a
d
n
d
c
.Here, n
s
d is the RI of the sensing dielectric after adsorption of DNA molecules, n
s is RI of the sensing dielectric before adsorption of DNA molecules, c
a is the concentration of adsorbed DNA molecules, and d
n
/
d
c is the RI increment due to the adsorbate (DNA). The RI increment parameter is d
n
/
d
c
=
0.153 cm3/gm in the case of water [26, 27]. If SPR angle changes, the propagation constant of SPW also changes which has been explained in the literature [1, 8, 28, 29] as given in the following:(3)
k
SPW
=
2
π
λ
n
p
s
i
n
θ
SPRand finally if propagation constant of SPW changes it makes the surface resonance frequency (SRF) change which can be explained by the following equation:(4)
SRF
=
c
0
n
geo
k
SPW
2
π
,where c
0
/
n
geo is the propagation velocity of SPW that is a perpendicularly confined evanescent electromagnetic wave [6, 8] and n
geo is the geometric mean of interface between gold and sensing medium (n
geo
=
n
Au
n
s
n
G
3, omitting imaginary part that is essential to real world design [25]) linked by graphene because SPW propagates at the interface between the metal and the dielectric (i.e., sensing medium) [6, 8]. If the incident angle of optical wave is tuned, SPR condition is achieved in which reflectance (R) of reflected wave is minimum and transmittance (T) is maximum and then SPW penetrate at SPF along the x-direction. We define a plot “transmittance versus surface resonance frequency (T~SRF),” as surface resonance frequency response (SRFR) curve. To generate a SPFR curve, we used Fresnel equations [30] for four-layered heterooptical system to determine transmitted light intensity [25].


## 3. Numerical Results

At first numerical analysis is presented by comparing theT~SRF characteristics curve in between with graphene sublayer and without graphene sublayer before adding DNA molecule which is normally known as bare sensor, as depicted in Figure 1(b). The SPR frequency without graphene is 91.70 THz and with single layer of graphene is 92.65 THz. The SPR frequency is increased (ΔSRF) to 0.95 THz in the right side of theT~SRF curve due to sandwiching each graphene film whose phenomenon accounts for enhanced sensitivity [5, 8] described in the sensitivity analysis section. The change of detecting attributors (ΔSRF and  T
max
⁡) due to adding graphene film is provided in Table 1. The data of Table 1 has been extracted from Figure 1(b) where results without graphene layer are used as reference in order to calculate other ΔSRF and T
max
⁡.Table 1
T
max
⁡ and SRF with graphene film and without graphene film.
Graphene layer
T
max
⁡ [dB]
SRF [THz]
ΔSRF [THz]
No layer (L
=
0 nm)
0.2267
91.70
0.00
One layer (L
=
1
∗
0.34 nm)
0.5494
92.65
0.95
Two layers (L
=
2
∗
0.34 nm)
0.9400
93.80
1.90
Three layers (L
=
3
∗
0.34 nm)
1.5870
94.95
2.85
Four layers (L
=
4
∗
0.34 nm)
2.2630
95.10
3.80
Ten layers (L
=
10
∗
0.34 nm)
6.2180
101.20
9.50In our SPR device, water is used as sensing medium that helps to measure the dependency of transmittance on SRF. Further work is continued by assuming that our sensor is susceptible of differentiating between complementary and single-based mismatched DNA with regard to the analysis of mutations and polymorphisms [1]. Since the change of concentration is due to the immobilization of DNA within the sensing medium, the local RI (n
s) of the sensing medium is also changed described by (2). From (1) we know that SPR angle changes if n
s change which finally translates k
SPW change observed from (3). Then, at the transition point, where the SPW wavevector and optic wavevector are equal to each other, the maximum transmittance (T
max
⁡) is detected.This point is called SPRP which is mentioned in Section2. It is observed from Figure 2(b), by immobilization of the probe DNA (p-DNA), either complementary or mismatched, on the graphene surface, the SPRP is considerably right shifted by 1.15 THz. This phenomenon can demonstrate the dependency of SRF on the immobilization of the probe DNA and hybridization of the complementary target DNA (C-t-DNA) [1]. Introducing DNA as electron-rich molecules, the number of carriers would change the graphene concentration which has let to varying the propagation constant. Finally, the hybridization event has been performed by adding complementary DNA sequences [1, 31].Figure 2
(a) Schematic of initial stage detection concept. (b) Surface resonance frequency response curves before and after the adsorption of biomolecules, blue line for bare sensor and red line for sensor with 1000 nM probe DNA.
(a)
(b)As illustrated in Figure3, single-based mismatched combination occurred with the submersion of the noncomplementary t-DNA to the immobilized capture probe on SPR device; then no significant change ofT~SRF characteristics resulted (change of SRF angle reference presenting mismatched target is ΔSRF = 0.1 THz) due to its nonbonding reaction between two pairs of DNA strands since they cannot be hybrid. In this case we conclude that there are no associated charges with the target molecule that can incorporate an obvious change to the applied sensing dielectrics. It is also observed that the SPR device specifically recognizes the target DNA sequences. In light of this fact, the focus of this paper is to present a new strategy for DNA sensor with the capability of detection of SNP.Figure 3
(a) Schematic configuration of mismatched event in SPR sensor. (b) Comparison of SPRF curve before adding DNA molecules (bare sensor, blue line) and after adding p-DNA (red line) and after adding mismatched t-DNA (black line).
(a)
(b)Figure4(b) describes theT~SRF characteristics for different concentrated complementary target DNA (C-t-DNA), where each color line depicts finite concentration of the DNA molecules. According to the numerical data, SRF and T
max
⁡ have acted as hybridization detectors. These parameters will vary and decision will be made based on these variations when complementary DNA molecules are combined with probe.Figure 4
Immersing complementary DNA solution. (a) Schematic after immersing the device in complementary DNA solution and (b)T~SRF characteristics response for different concentrated C-t-DNA.
(a)
(b)SRF andT
max
⁡ are calculated for different concentrated C-t-DNA and tabulated in Table 2, in order to know how these parameters would change with changing the concentrations. A considerable increase of SRF that determines DNA hybridization is pointedly seen. The numerical data apprises the strong dependency of the SRF on the concentration increment that reflects in theT~SRF characteristics curve. With DNA hybridization, the C-t-DNA molecules are reacted with the graphene surface, commonly known as “graphene-nucleotide interaction” and generated n-doping effect which is liable to turn right of SPRP. It is stated that the interaction between nucleotide and graphene surface upon DNA hybridization has an enormous influence on SRF too, which can change it rightwards [32]. For clarifying DNA hybridization, Δ
SR
F
p
-
t and Δ
T
m
a
x
p
-
t are represented as the detecting attributors. The following equations are used to determine these attributors:(5a)
Δ
SR
F
p
-
t
m
i
n
=
SR
F
p
-
SR
F
t
=
1000
nM
=
0.72
THz
,
(5b)
Δ
T
m
a
x
p
-
t
m
i
n
=
T
m
a
x
p
-
T
m
a
x
t
=
1000
nM
=
0.003
dB
.In (5a) and (5b) SR
F
p, SR
F
t, T
m
a
x
p, and T
m
a
x
t represent the SRF of p-DNA, SRF of specific concentrated C-t-DNA, maximum transmittance of p-DNA, and specific concentrated C-t-DNA, respectively. Finally taking advantage of the attributor’s values, a decision making table (Table 3) is prepared and can be utilized. When the change of ΔSRF and Δ
T
m
a
x
p
-
t is greater than or equal to (
Δ
S
R
F
m
i
n (0.72 THz) and Δ
T
m
a
x
p
-
t
m
i
n (0.003 dB), then DNA-DNA hybridization is ensued and if the change of ΔSRF and Δ
T
m
a
x
p
-
t is less than (
Δ
S
R
F
m
i
n and Δ
T
m
a
x
p
-
t
m
i
n, then SNP happens; except both of these cases, no effective result will be established, and detection procedure should be restarted.Table 2
Δ
T
max
⁡
p
-
t and Δ
SRF
p
-
t for different concentration of DNA molecules.
Concentration (c
a) [nM]
Δ
T
m
a
x
p
-
t [dB] = T
m
a
x
p
-
T
m
a
x
t
Δ
SRF
p
-
t [THz] = SRF
p
-
SRF
t
1000 (target)
0.0030
0.72
1001 (target)
0.0043
0.87
1010 (target)
0.0052
1.05
1100 (target)
0.0065
1.25Table 3
Decision making table based upon different conditions which happened to detective parameters.
Conditions
Decision
Δ
T
m
a
x
p
-
t
≥
Δ
T
m
a
x
p
-
t
m
i
n
&&
Δ
SRF
p
-
t
≥
Δ
SRF
p
-
t
m
i
n
Hybridization happened
Δ
T
m
a
x
p
-
t
≥
Δ
T
m
a
x
p
-
t
m
i
n
&&
Δ
SRF
p
-
t
≤
Δ
SRF
p
-
t
m
i
n
Try again
Δ
T
m
a
x
p
-
t
≤
Δ
T
m
a
x
p
-
t
m
i
n
&&
Δ
SRF
p
-
t
≥
Δ
SRF
p
-
t
m
i
n
Try again
Δ
T
m
a
x
p
-
t
<
Δ
T
m
a
x
p
-
t
m
i
n
&&
Δ
SRF
p
-
t
<
Δ
SRF
p
-
t
m
i
n
SNP happened


## 4. Sensitivity Analysis

The sensitivity of the proposed optical sensor is defined as the ratio of the SRF change,Δ
SR
F
T (sensor output), to the change in concentration of biomolecules, Δ
c
a (sensor input), as given in the following: (6)
S
G
=
Δ
SR
F
T
Δ
c
a
=
Δ
SR
F
0
+
Δ
SR
F
L
Δ
c
a
=
Δ
SR
F
0
+
L
Δ
SR
F
L
=
1
Δ
c
a
=
Δ
SR
F
0
+
0.95
L
Δ
SR
F
0
Δ
c
a
=
1
+
0.95
L
Δ
SR
F
0
Δ
c
a
or
S
G
=
1
+
0.95
L
S
0
,where Δ
SR
F
T is the SRF change of sensor summing with and without graphene sublayers, S
0 is the sensitivity of the devices without graphene film, and S
G is the sensitivity for different number of graphene sublayers (L
=
1,2
,
3
,
…). The study on graphene sandwiched SPR sensor ensures 1 + 0.95L times higher sensitivity. If single graphene sublayer is considered then sensitivity has increased to 95% for DNA detection which is a great achievement compared to other results that had been 34% in [8]. The sensitivity has been improved due to the changes of detecting attributors. In this study, we consider surface resonance frequency and maximum transmittance as the detecting attributors, whereas, in [8], surface resonance angle and minimum reflectance are considered for detecting attributors.


## 5. Conclusion

In this paper, a numerical modeling of SPR biosensor is proposed for detecting DNA-DNA hybridization by monitoring the resonance frequency characteristics (RFC). The proposed biosensor has been designed based on graphene material which is sandwiched between metal film and sensing medium for enhancing sensor sensitivity. Without graphene sublayers SPR sensor provides slower immobilization between target DNA and probe DNA as well as lesser sensitivity which are the causes of poor efficiency. It is observed that 95% sensitivity has been increased for adding each sublayer if surface resonance frequency and maximum transmittance are chosen as the detecting attributors.



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*Source: 6070742-2015-12-21.xml*