Plasmonic biosensor for the study of blood diseases by analysis of hemoglobin concentration

The plasmonic materials and phenomena are widely studies and applied in multiple fields for an extended time. One among the foremost promising applications is within the engineering of biosensor devices, Diseases of the red blood cells are among the diseases of the blood. Anemia as an example is an abnormal reduction within the concentration of hemoglobin within the blood. When the amount of red blood cells decreases, so does the concentration of hemoglobin. The tissues and organs not receive sufficient oxygen to work normally. This numerical study will give a contribution or approach on the behaviour of blood with surface plasmons resonance SPR by analysing the performance of SPR with any changes in hemoglobin concentrations (refractive index) for the application of technique in the detection of blood diseases.


Introduction
Surface plasmon resonance (SPR) sensors offer great advantages over conventional analytical techniques, as they allow the direct, unlabelled detection of many biological tissues such as hemoglobin and chemicals in real-time. Its advantages include high specificity, sensitivity, small size, and profitability. Many advanced concepts and many multidisciplinary approaches, including micro-electronics, micro, and nanotechnology, molecular biology, biotechnology, and chemistry, have been applied in the implementation of new optical biosensors [7]. Depending on the method of signal transduction, biosensors can classified as optical, electrochemical, thermometric, piezoelectric, or magnetic. The most commonly reported class of biosensors is optical biosensors [2]. various optical techniques such as grating coupled interferometry (GCI), resonant waveguide gratings (RWGs), quartz crystal microbalance QCM, colorimetry, Raman scattering, fiber gratings, microstructure 1 3 234 Page 2 of 14 waveguides surface enhanced infrared absorption spectroscopy, and surface-enhanced fluorescence can be used to detect pathology [17].
The optical field interacts with a biorecognition element to perform optical detection. Label-free optical biosensing and label-based optical biosensing are the two broad categories of optical biosensing [29]. The detected signal in a label-free mode they generated directly by the interaction of the tested material with the transducer. Label-based sensing, on the other hand, uses a label to generate an optical signal, which is then processed using a colorimetric, fluorescent, or luminescent method [29].As biorecognition elements, the biosensor can use a variety of biological materials such as enzymes, antibodies, antigens, receptors, nucleic acids, whole cells, and tissues [17]. Optical resonators, surface plasmon resonance (SPR) and localized surface plasmon resonance (LSPR), evanescent wave fluorescence, and optical waveguide interferometry to detect the interaction of the biorecognition element with the analyte use the evanescent field near the biosensor surface [20]. Table 1 provides the most common optical sensor techniques for detecting various diseases and microorganisms.
Analytical advancements have occurred in parallel by coupling various techniques (fiber optics, Raman, fluorescence, luminescence) to SPR in order to improve sensitivity. Future advancements will embrace instrument miniaturization to hand-held procedures compatible with smart phone imaging and analysis simplification.
Sensing techniques based on plasmon resonance (SPR) are a promising new method for detecting pathogens quickly and at a high sensitivity; as a result, this method has progressed to replace other traditional diagnostic methods [2]. This technology can detect diseases and pathogens including viruses and bacteria in real time [20].
The limit of detection (LOD) is a performance factor that measures the change in analyte or biomolecule concentration in the sensing section. It is defined as the ratio of the change in refractive index (Δn) to the shift in resonance angle (Δθres). The table 2 shows the smallest practical value obtained for the limit of detection in terms of size and concentration for each technique listed in the Table 1.
When using an SPR biosensor under the Kretschmann configuration (Fig. 1), a high refractive index prism, one surface of which is covered with a thin layer of metal, is usually applied to an excitation SPR, in which the intensity of the incident light that satisfies the plasmonic condition changes in response to the change in refraction index [36,30]. A thin metallic gold film used in SPR sensors because of its stable optical and chemical properties and because it maintains surface plasmon polaritons propagation (SPP) in the visible wavelength range. The principle of the SPR sensor is an optical phenomenon in which a beam of p-polarized light satisfies certain resonance conditions and generates oscillations in charge density known as surface plasmon waves, SPW, which propagate along with the metal-dielectric interface. The plasmon resonance condition is articulated as [31]: When the above ccondition fulfilled, resonance appears in the form of a sharp decrease in the optical output signal at the θ SPR resonance value of the incident angle. Every change in refractive index around the metal-insulator interface causes a shift in the value of the resonance angle [31].   [33] high temporal resolution, high sensitivity, biocompatible imaging agents, and non-invasive properties [21] Difficulties in obtaining the binding molecule's valency [33] Slightly slow detection; time consuming; application restricted (small molecule) [ High reproducibility Rapidity [28] Low selectivity (improving) Bulky size [17] Narrow concentration range [28] Optical waveguide interferometry Yes Study of cellular responses and processes Virus detection [39] Low cost, compact structure, good robustness, high sensitivity Fragile structure, poor repeatability, temperature cross interference and transmissive structure [5] Bioluminescent Optical fibre

No
Response of cells to genotoxic agents [14] The multi-detection of genotoxins by live cell array provided sensitivity in the parts per billion range. [4] Flexibility in fiber probe configuration (D shaped, U shaped and tapered) High sensitivity and specificity of such light-emitting enzymatic reactions [38] Loss of light as it travels through the fiber limitation of the bandwidth of the signals [38] Surface-enhanced Raman spectroscopy Yes Detection of cancer proteins Protein biomarker in environment High sensitivity, specificity, multiplexing capability and photostability [40] Can be fabricated through controllable techniques [22] Low detection limit of biomolecules [37] The complexity of exosome populations secreted from diverse sources, such as those isolated from human blood, has severely limited this approach's viability as a diagnostic tool. [10] In addition to various biological parameters, SPR based sensors were tested for pesticides, membrane proteins, immunoassays, and human blood. SPR sensors can also be potential candidates for the effective detection of other biological properties, such as hemoglobin concentration, an important parameter in several diseases such as anemia (Fig. 2). The logic behind this argument is that blood samples with different hemoglobin concentrations should have different distribution ratios due to the corresponding variations in their chemical and biological composition [30].

Theory and method
• SPR system design. Figure 1 shows the three layers of SPR sensor configuration, where the first layer is the BK7 glass prism with refractive index n 1 . The second layer is gold followed by a sensing medium. An incident light beam propagating through the prism undergoes total internal reflection at the prism-gold layer interface generating an evanescent wave. This generated evanescent wave penetrates through the gold layer and propagates in the x-direction (Fig. 3). The magnitude of propagating wave vector in the x-direction can be expressed as [11].
Firstly, we talk about the different components of our configuration sensor with their optical and other properties.

BK7 glass
The technique based on the Kretschmann configuration attenuated total reflection ATR; the coupling device considered as BK7 glass prism with refractive index n 1 . The Sellmeier dispersion relations of the BK7 prism is given as [6]: SPR [24] Optical waveguide interferometry [18] Bioluminescent optical fibre [16] Surface-enhanced Raman spectroscopy [1,19] Limit of detection

Metal layer
In Fig. 1, as shown the base of the prism have been coated with a thin gold layer with n 2 refractive index and 50 nm of thickness. According to the free-electron Drude model the wavelength depending on the complex dielectric function ε m of any metal can be written as [12]: where, ε r , ε i the real part and imaginary part permittivity's of metal, which is depending also on the incident wavelength. The thickness of the metal layer is dependent on which material glass is used. To efficiently couple the incoming light due to the much larger difference between the refractive index of glass and metal [12].

Sensing medium
The refractive index of the sensing medium (blood) in the visible range can be described by [30]: It's varies linearly with the hemoglobin concentration (C H ) and can be written in the form of Barer's expression given as [26]: Where ( ) is specific refractive increment dependent on the wavelength. The imaginary part (k) of the refractive index varying with hemoglobin concentration can be calculated at any wavelength by using the corresponding data for molar extinction coefficient measured by Prahl [25].
The sizes of the hemoglobin molecules are much smaller than the wavelength so that scattering can be neglected then we use this relation [8].
where, µa is the absorption coefficient in cm -1 , e is the molar extinction coefficient and M H is the molar mass of hemoglobin.

SPR and blood pathologies
The diagnosis of blood pathologies such as anemia based on the detection of hemoglobin levels in blood samples. Hemoglobin is a protein found in red blood cells that is responsible for transporting oxygen. When hemoglobin is lost, damaged, or compromised, decreased blood oxygen levels cause dizziness, fatigue, shortness of breath, and an irregular heartbeat. Low hemoglobin is diagnosed as anemia caused by more than seventeen different diseases, mainly hemoglobinopathy, iron deficiency, and malaria [35].
Optical density (OD) or absorption, a non-uniform quantity is typically used to describe the transmission and absorption of light by biological media. OD has a logarithmic relationship with transmission (T) = I 0 /I and expresses light absorption as a function of hemoglobin concentration, as shown in [9,35].
where, I 0 is the light intensity of incident light, I is the light intensity of transmitted light, ζ is the extinction coefficient of hemoglobin, C H is the concentration of hemoglobin, and L is the length of light path through solution. The propagation vector of surface plasmon involves the refractive index of gold n 2 , and the hemoglobin n 3 , because the wave propagation at the metal-surrounding medium interface occurs partly in both the material. Therefore, the surface plasmon propagation vector K sp can be defined as [3].
The SPR excited by adjusting the incident angle θ, so that the propagation vector of the evanescent field, Kx, matches with the Ksp. Therefore, when this condition satisfies Kx = Ksp, reflected light intensity drops sharply [29]. The reflectivity curve at this resonance condition referred in the SPR curve and the angle corresponding to reflectivity minimum referred to as SPR angle. The reflectivity of the p-polarized incident light has been calculated using the 3-layer model for the most common Kretschmann configuration [34].
Two aspects determine the performance of the SPR sensor. The first is the sensitivity aspect, which means that the SPR resonance angle shift must be as large as possible for a given change in the refractive index of the sensitive layer. The second is the aspect of accuracy, which assumes that the full width at half maximum (FWHM) according to the SPR curve should be as small as possible so that the error in determining the resonance angle is minimal. Therefore it can be assumed that the detection accuracy of the SPR-based sensor is inversely proportional to the FWHM of the SPR curve [12,13].
FWHM can be determined by calculating the full width at the maximum half reflection drop (Δθ 0.5 ). The angular sensitivity S of the SPR sensor is defined as the ratio of the change in the resonance angle δθ to the change in the refractive index of the sensitive medium δn according to [30]: The simulations and design of SPR biosensors are very important aspects to base on before its conception. And is usually performed by estimating the reflectivity and equivalent electromagnetic field at each layer by a well-known N-layer model, which is mostly, based on the 2 × 2 Abeles matrix approach [32].

Resultes and discussion
This paper analyses the reaction of SPR with hemoglobin by numerical simulation using the Matlab mathematical model mentioned above, the assumed active metal is gold.

Evolution of optical density with pathology
First, optical density is calculated by Eq. (10) for different concentrations in order to determine the absorbance light of hemoglobin in terms of wavelength in the visible range. Figure 4 shows the absorbance of hemoglobin in the range of wavelength from 250 to 700 nm, the spectrum depicts that hemoglobin has a distinct variation of absorption. We are interested in the visible field; hemoglobin presents the highest point absorption at 435 nm and another at 555 nm. They not equal to zero in all points but it gives a low value compared to these two points. We also distinguish by the Fig. 5 that the absorbance increases when the concentration increase, is then possible to conclude that there is a direct relationship between optical density and blood pathology.

SPR reflectivity
Initially, composite structure shown in Fig. 1 was simulated for a different combination of detection mediums. For this purpose, air, water, and hemoglobin with refractive indices of 1,0002, 1,332 and 1,358 respectively for hemoglobin at 100 g/L (Hb) and 633 nm wavelength, The corresponding SPR curve in Fig. 6 showing the variation of the reflected light intensity with the angle of incidence for 3 sensitive medium. The shape of the SPR curve strongly influenced by the type of the third medium, each indicating the same behavior. The angle of resonance of the SPR curve progresses by increasing the refractive index of the sensing medium.

Optimization of wavelength and hemoglobin thickness
In order to optimize the effect of thickness and wavelength on the hemoglobin SPR sensor at C Hb =100 g/L, reflectance has been calculated and plotted. Figure 7 shows the variation of reflectance as a function of angle. Results show that the resonance angles for different wavelengths of reflectance curves are varying. From this figure, it is clear that the FWHM of the SPR dip decreases upon increasing the value of wavelength, the depth of the dip decreases upon increasing the value of wavelength. The form of the SPR curve is almost the same for all wavelengths Except for 435 nm because gold absorption begins at ~ 2.4 eV correlated to a wavelength 520 nm [15]. The optimum wavelength will considered us the one that has the minimum dip. Although that 435 nm presents high absorbance of hemoglobin but it has clear that 633 nm wavelength is the calculated optimal wavelength for the resonance angle condition. The Fig. 7 shows the best behavior when approaching to the infrared, which indicates the curve at 700 nm. It suggests that it is better to work at higher wavelengths such as 850 nm rather than at smaller wavelengths such as 400-500 nm to ensure sufficient coupling of incident light with surface plasmons [31].
Subsequently, from Fig. 8 the thickness of the sensing medium varies from 30 to 300 nm whereas keeping the thickness of Au film fixed at 50 nm and 100 g/L concentration of Hb in the wavelength 633 nm. The thickness of Hb for obtaining the best SPR reflectance curve is found to be around 105 nm, With increasing thickness of Hb (> 200 nm), the SPR reflectance curve degrades due to the high energy absorbed by Hb. Figure 9 shows the SPR curves of fixed wavelength at 633 nm for the detection of hemoglobin with concentrations (refractive indices) ranging from 0 to 250 g/L. this increasing variation induces an increase in the refractive indices.

SPR refractive index sensor (hemoglobin sensing)
It is observable from Fig. 9 that the reflectance curves are approximately the same, with a difference in resonance angle and minimum reflectance, the minimum reflection intensity varying from 0.00627 to 0.01884 UI, and in the angle of resonance from 64.6° to 72.4° respectively. Moreover, the full width at half maximum (FWHM) increases between 4.6° to 6°and the shift in resonance angle δθ value is 7.5°, which represented in the Table 3. Because of the high presence of hemoglobin molecules the absorption increase, this occurs due to important energy loss caused by an increase of the dielectric constant imaginary part. Figure 9 illustrates the behavior of SPR with hemoglobin for all the changes in concentrations corresponding to a variation of 0.0652 RI, the SPR exhibits high sensitivity at a high concentration equivalent to 137.4°/RIU and 106.87°/RIU than for low concentration. Therefore, a very small adjustment in the refractive index can clearly appear in real-time and affect the sensitivity of SPR.
As can be seen from the study, the FWHM is a little big and even the total shift of the resonance angle δθ is not large enough to judge the high performance of SPR which limits this study in the visible range and which influences the sensitivity of SPR. It's recommended to work in the infrared range (region of pathology) or used other materials with gold such as graphene to improve the performance of SPR.

Conclusion
SPR sensor suggested to help understand blood interaction with light in the visible range by studying hemoglobin concentration in the blood to accelerate diagnosing and treating human disease and in order to reduce testing on the patients,also to find the best sensor sensitivity to improve the accuracy of sensitivity measured results, FWMH has been analyzed, minimum reflection with regard to gold thickness After improving the wavelength that should be large, the thickness of hemoglobin is less than 200 nm so as not to lose energy. Note that the sensitivity of the proposed blood disease detection system is sufficient, but it is important to minimize FWHM and reflect the good performance of sensors, and the results obtained are comparable with some techniques used by biologists. This technique have great advantages like some of the best sensors in indexing affinity or catalytic receivers. They have greater sensitivity and versatility, which enables faster, real-time measurements, and can be adapted for multichannel and multiparameter detection as matched to some other technologies like scattring, fluoresence and bioluminesence, because it is a non-destructive technique, relatively low cost and easy utilization.