Optical Slot-Assisted Metasurface for IgG Protein Detection

The sensing of antigens plays a pivotal role in the early diagnosis of tumors by potentially revealing specific biomarkers associated with them. Early detection of tumors can facilitate targeted and less invasive therapeutic strategies. In the realm of Immunoglobulin G (IgG) protein detection, a high concentration may indicate tumor lesions or the presence of symptoms commonly detectable just through invasive diagnostic methods. IgG detection is feasible in blood samples or other biological fluids like saliva or urine. These non-invasive tests offer the advantage of being repeatable over time, reducing the need for invasive or painful procedures. In the pursuit of next-generation medical technologies aimed at flexible and compact sensing platforms, we proposed a refractometric system mainly based on a dielectric metasurface, operating at about 780 nm, featuring a 2D distribution of cells composed of dimers that support the optical slot effect. This effect allows for a large Q-factor, with a small footprint, and strong spatial confinement. Specifically, a Q-factor of 3.19 × 103 with a significant amplitude (> 0.7 a.u.) enables a sensitivity of 25 nm/RIU for IgG protein detection.


Introduction
Liquid biopsy has become a revolutionary diagnosis technique that detects molecules released from easily accessible bodily fluids like blood, urine, sweat, etc. [1].It holds huge potential in detecting and monitoring cancer cells in early stages due to its ability and versatility to provide accurate molecular information as well as its non-invasive nature as compared to traditional organ-specific biopsy.Among various proteins that hold a long history as cancer biomarkers, Immunoglobulin G (IgG) has emerged as a promising biomarker due to its stability, systematic presence in blood as well as proven characteristics of changing its concentration in response to cancerous cells [2].
Detection of proteins like IgG to diagnose cancer in early stages through the advantageous liquid biopsy can be performed through different approaches like electrochemical technique, imaging method as well as photonics [3].Among these techniques, photonics holds a particular advantage due to its peculiar beneficial features like real-time label-free detection, high sensitivity, compact footprint as well as stability.The photonics sensing principle is based on the interaction of a modal field with a sensing medium.To advance the sensitivity, different approaches including surface plasmon resonance, optical fibers, integrated optical waveguides, and metasurfaces have been widely investigated over the years.Among these, metasurface-based sensors recently gained large attention thanks to their enhanced sensitivity, ease in modal excitation as well as compatibility with different detection mechanisms [4].Here, a sensor based on a dielectric slot-assisted metasurface is proposed with IgG as the cancer biomarker which shows a high surface sensitivity around 25 nm/RIU and IgG sensitivity around 1.8 nm/(ng/ml) combined with an excellent Q-factor of 3.19×10 3 .

Proposed system for IgG detection
To propose a system characterized by high sensitivity, specificity, and compactness, we've explored a refractometric technique that relies on detecting changes in amplitude caused by shifts in the refractive index at the sensor surface.This alteration leads to a corresponding shift in the resonance wavelength of the sensor, at the heart of the system, represented by a metasurface.The compact system is made of an LED as the illumination source, the sensor, interleaved between two polarization filters and a photodiode.
A strategic approach involves designing the metasurface resonance to be aligned with one edge of the LED emission spectrum.Consequently, any wavelength shift translates into a discernible variation in transmitted intensity.To further optimize performance, the integration of two polarization filters, strategically positioned before and after the sensor, serves to effectively eliminate unwanted polarization components, thereby significantly boosting the Signal-to-Noise Ratio (SNR).

Design of the sensor
The proposed metasurface consists of a 2D array of engineered cells, spaced at Λ with a duty-cycle (DC) of 70%, deposited on a glass substrate (refractive index n = 1.46).Water (n = 1.33) serves as the ambient medium.Each cell of the array is composed of two dimers, with a thickness of t = 100 nm.These dimers have lengths along the xand y-axes of DC x Λ/2 and DC x Λ, respectively, and are made of amorphous silicon (a-Si:H) with n = 3.6 and losses k ≈ 10 -4 within the operating wavelength range of 700 nm -800 nm [5].The two dimers are separated along the x-axis by a gap g that supports the optical slot effect (Fig. 1).
The design of the metasurface was carried out using a 3D FEM-based solver, assuming excitation occurs along the z-axis with TE-polarized waves along the x-axis.Initially, to design both Λ and DC, a metasurface configuration, where each cell is composed of a single block resulting from the combination of the previously mentioned two blocks, was considered.Therefore, the silicon block has a length along both xand yaxes equal to DC x Λ.A period Λ of 500 nm enables the structure to resonate within the wavelength range of 700 to 800 nm, where water exhibits minimal absorption losses.As expected [6], the metasurface exhibits two resonance peaks, characterized by different distributions of the electric field E in the cell (in-plane and out-of-plane distribution, respectively) and different performance.The resonance with in-plane E field distribution shows a larger Q-factor than the one with out-of-plane distribution, and it results in our interest since a larger Q-factor paves the way to a larger sensitivity.To avoid worsening the SNR in detecting the desired resonance, the resonance wavelength difference has been maximized through the design of DC = 70 %.
To increase the Q-factor while preserving the resonance amplitude, the resonance with a shorter wavelength and in-plane field confinement was tailored using the slot configuration.The slot effect refers to the phenomenon where the presence and specific dimensions of gaps between components in a structure significantly increase the in-plane field confinement, consequently enhancing the Q-factor.The intensity of the effect depends on the gap between the two dimers, as demonstrated in Fig. 2(a).Particularly, it can be observed in Fig. 2(b) that the Q-factor exponentially increases as g increases, at the expense of a decrease in the resonance amplitude.Specifically, to achieve a good compromise between the Q-factor, resonance amplitude (larger than 0.5 for accurate measuring), and manufacturability, a final design of Λ = 500 nm, DC = 70 %, and g = 60 nm has been considered, with a resulting Q-factor of 3.19x10 3 and an amplitude > 0.7 a.u. of the resonance.It shows a Q-factor enhancement of more than 1 order of magnitude with respect to the case g = 0 nm.

Biofunctionalization protocol of the sensor
A proper biofunctionalization of the sensor surface and reliable immobilization of the bioreceptors over the detection area is the most crucial step for achieving high detection performance label-free biosensors.Biofunctionalization protocols involve modifying sensor surfaces to enhance their specificity and sensitivity while preventing the binding of non-targeted molecules to avoid unspecific binding [7].Typically, these protocols involve the surface immobilization of specific ligands, such as antibodies or aptamers [8], capable of selectively binding to IgG protein.In this context, the functionalization protocol includes an initial cleaning process using oxidizing agents or, more strictly, Piranha solution.This solution, comprising a mixture of sulfuric acid and hydrogen peroxide, serves to remove organic contaminants and generate hydroxyl groups (-OH) on the surface [9].The surface is covered by sulfhydryl groups by the salinization with MPTS for 6 hours.Subsequently, anti-IgG antibodies are immobilized on the selected area, and Casein (1% in PBS solution) is added into the area to minimize unspecific binding.Lastly, the IgG proteins are released into a selected surface to bind to the corresponding antibodies [10].
To model the anti-IgG functionalization layer, a 10 nm thick layer (tAB) is commonly considered above the silicon cubes with a refractive index of 1.45, as shown in Fig. 1(d) [10].

Biosensing performance
The surface sensitivity was assessed in evaluating the designed metasurface as a biosensor.The sensor's sensitivity for detecting IgG is crucial in determining how effectively the metasurface can identify and measure the presence of IgG in the sample.Specifically, surface sensitivity is crucial in this context as it measures how effectively the metasurface can detect IgG molecules directly adsorbed or bound to the surface.Following the deposition of the antibody layer, a red shift in the resonance wavelength has been observed, measuring a resonance wavelength shift of ≈ 3 nm (Fig. 3(a)).Consequently, a surface sensitivity of 25 nm/RIU has been calculated, which is higher compared to previously proposed metasurface configurations in literature and comparable to the surface sensitivity of plasmonic configurations, although with larger resonance amplitude.Finally, the sensor's response to varying analyte concentrations has been evaluated.Specifically, a concentration (C) range of IgG from 1 pg/ml to 1 ng/ml results in a refractive index change of the antibody from 10 -4 a.u. to 10 -3 a.u.[10].This effect is due to an increase in the density of the antibody layer with increasing concentration, without affecting its geometric dimensions.The resonance shift (Δλ) to the resonance wavelength position after antibody deposition (≈ 784.28 nm) is shown in Fig. 3(b) by varying IgG concentrations.A resulting IgG sensitivity of ≈ 1.8 nm/(ng/ml) has been calculated.

Conclusions
In summary, a-Si:H -based dielectric metasurface is proposed in slot configuration for the sensing of IgG protein, an important biomarker for cancer.All the parameters of the proposed structure including the grating period, slot gap, and duty cycle are optimized through the well-accurate 3D-FEM method in a wavelength range of 700-800 nm, showing an excellent Q-factor 3.19×10 3 , thanks to the highly confined slot mode.The sensitivity analysis shows a high surface sensitivity of 25 nm/RIU over a 10 nm thick bio-layer, resulting in an exceptional IgG detection sensitivity of 1.8 nm/(ng/ml).The compact geometry and high sensitivity reflect its potential for early-stage cancer detection through liquid biopsy.

Figure 1 .Figure 2 .
Figure 1.(a) Proposed optical slot-assisted a-Si:H metasurface on a glass layer; (b) top-view of a single cell; (c-d) cross-section pre-and post-functionalization with antibody layer, respectively.