A Novel Biosensing Approach: Improving SnS2 FET Sensitivity with a Tailored Supporter Molecule and Custom Substrate

Abstract The exclusive features of two‐dimensional (2D) semiconductors, such as high surface‐to‐volume ratios, tunable electronic properties, and biocompatibility, provide promising opportunities for developing highly sensitive biosensors. However, developing practical biosensors that can promptly detect low concentrations of target analytes remains a challenging task. Here, a field‐effect‐transistor comprising n‐type transition metal dichalcogenide tin disulfide (SnS2) is developed over the hexagonal boron nitride (h‐BN) for the detection of streptavidin protein (Strep.) as a target analyte. A self‐designed receptor based on the pyrene‐lysine conjugated with biotin (PLCB) is utilized to maintain the sensitivity of the SnS2/h‐BN FET because of the π–π stacking. The detection capabilities of SnS2/h‐BN FET are investigated using both Raman spectroscopy and electrical characterizations. The real‐time electrical measurements exhibit that the SnS2/h‐BN FET is capable of detecting streptavidin at a remarkably low concentration of 0.5 pm, within 13.2 s. Additionally, the selectivity of the device is investigated by measuring its response against a Cow‐like serum egg white protein (BSA), having a comparative molecular weight to that of the streptavidin. These results indicate a high sensitivity and rapid response of SnS2/h‐BN biosensor against the selective proteins, which can have significant implications in several fields including point‐of‐care diagnostics, drug discovery, and environmental monitoring.


Detailed reaction mechanism for the synthesis of PLCB
The pyrene-lysine-biotin construct, a molecule used to support PLB, was created using Fmoc chemistry.The H-Rink amide resin (substitution value = 0.54 mmol/g) was purchased from PCAS BioMatrix company.A benchtop manually operated solid-phase peptide synthesizer (SPPS) was used for the synthesis process.Before starting synthesis, a fixed quantity of resin was swelled using DMF for half an hour.For deprotection using 20% piperidine in N, Ndimethylformamide (DMF), a standard set of conditions was used, i.e., heating at 90 °C followed by 75 °C for 15 s and 50 s respectively.The resin was washed with DMF and dichloromethane (DCM) between the deprotection and coupling steps.The synthesis began by coupling Fmoc-Lys (Biotin)-OH moiety in assistance with activated amino acid (DIPEA).The coupling reaction was performed by adding 0.5 M diisopropyl carbodiimide (DIC) and 1 M Oxyma in the SPPS synthesizer.The reaction was performed at the same temperature conditions mentioned above but increased reaction time by 55%.After deprotection, 0.2 M 1pyrene butyric acid solution was used for coupling keeping the same concentration.The final product was dried and cleaved from resin support using trifluoroacetic acid (95%), tri-isopropyl silane (2.5%), and the rest deionized water.The product was filtered under a nitrogen atmosphere, precipitated using ether (diethyl ether), and lyophilized to prepare a solution of desired concentration for all experimental investigations.The concentration of our PLCB was estimated by measuring the absorbance of its solution at 335 nm.An optimized concentration of 1 nM was used for substrate functionalization to avoid stacking.For further details on the reaction mechanism, please refer to Figure S1a.The synthesized PLCB was characterized via UV-spectra for the presence of pyrene and successful biotinylation, as shown in Figure S1b.
The appearance of a clear peak at ~ 202 nm confirms biotinylation, whereas, a clear and sharp peak at 335 nm confirms the conjugation of pyrene.At each stage of synthesis, the Kaiser test was used to confirm coupling and deprotection as shown in Figure S1c.

PLCB and streaptavidin nteraction.
The target protein (streptavidin) has a strong interaction capacity (dissociation constant in the order of ~ 10 −14 mol L −1 ) for the biotin (coupled with the PLCB construct, facing upwards).The interaction is marked by a formidable non-covalent bond (illustrated in Figure S2), whereby charges on the PLCB construct were shared, actively participating in the binding process with the target molecule (streptavidin).There is a total of 10 amino acids binding with the biotin including eight polar groups binding with hydrogen bonds and two non-polar groups binding with the van der Waals forces [1] .This combination of forces is not applicable to any other moiety present in the system, hence, making possible the precise and prompt capturing of streptavidin by the PLCB construct.Insight represents a clear view of 10 binding sites per streptavidin molecule.The construct (PLCB) is engineered in such a way that upon functionalizing, the Pyrene moiety plays its role and aligns with the hexagonal surface of SnS2.While, after functionalization, the biotin (facing upwards) is fully available to interact specifically with the target protein (streptavidin) withwhich it has strong binding ability.

Raman Analysis of a few layers thick h-BN and SnS2
The Raman spectra of the pristine h-BN exhibit an intense peak at ~1373 cm −1 which represents the E2g peak, i.e., the high-frequency vibration mode of h-BN [2] , as shown in Figure S3a.In the comparison of this peak with the bulk mode (E2g located at 1366 cm -1 ), a 6 cm -1 upshift can be observed which is inconsistent with the results reported earlier.Moreover, the FWHM of the A1g peak (main resonance) is related to the h-BN crystal dimensions which are about 10.17 cm −1 to suggest a few layers of h-BN with excellent crystallinity as compared to the results reported earlier [3] .
The Raman spectra of pristine SnS2 represent a clear and sharp peak around ~315.4 cm -1 (A1g) and ~200 cm -1 (Eg) as shown in Figure S3b, confirming the pristine material nature.The appearance of two sharp peaks represents two active phonon modes of phase (2H) SnS2 [4] .
Moreover, the presence of a single peak in the 190-225 cm -1 range clearly indicates the pure crystalline SnS2 at its ground state with 2H polytype.Applying the Gaussian fit, the full width at half maximum (FWHM) of A1g (main resonance peak) is calculated, and it is 9.85, representing the crystalline nature of the material.Further, a sharp second resonance peak at 200 cm -1 represents the small number of layers of SnS2 material as a broad peak at this position is attributed to the overlapping of a large number of layers [5] .Due to the pristine nature of SnS2 material, the intensity ratio (A1g / Eg) is very high and equivalent to 18.Moreover, the detailed Raman analysis of the SnS2 surface before and after functionalization with PLCB and streptavidin is also illustrated in Figure S3 c-d.The intensity ratio and FWHM are plotted over the SiO2 substrate for pristine SnS2, after functionalization with PLCB, and after streptavidin detection.(b-c) The output curves acquired from the SnS2 FET over the SiO2 substrate at various gate voltages changing from -60 to 60 V.

Stacking of PLCB over SnS2 surface.
For the device functionalization, a solution (2.5 µL, 1 nM) containing our engineered pyrenebased supporter construct (PLCB) is poured over the channel containing SnS2/h-BN.The hexagonal surface of the SnS2 flakes provides an effective platform for the hexagonal pyrene rings (present in our PLCB construct) to assemble over it via - bonding without lying over as shown in Figure S5.Pyrene is a polycyclic aromatic hydrocarbon consisting of four fused benzene rings which contribute a total of 16 π-electrons to its aromatic system.The aromaticity of pyrene arises from the presence of a continuous ring of π-electrons.Principally, pyrene can interact with other materials through various interactions, such as van der Waals forces, π-π stacking, and hydrogen bonding, etc.These interactions can occur between the aromatic rings of pyrene and other aromatic or electron-deficient systems in other materials (SnS2), where the π-electron clouds of the aromatic rings align and interact through various attractive forces.

Real-Time Detection of Streptavidin via SnS2 FET over h-BN and SiO2 substrates.
At a fixed biasing voltage (Vds = 1 V), the comparison of the SnS2 FET over h-BN and SiO2 substrates is plotted together, as shown in Fig. S6a.The real-time detection of the streptavidin over the h-BN substrate is showing more promising results as it detected the minimum concentration up to 0.5 pM while the SnS2 FET over the SiO2 substrate can detect the lowest concentration of streptavidin up to 1 pM.Furthermore, the response time of the SnS2 device over the h-B substrate is significantly lower than the SiO2 substrate against the 1 pM streptavidin concentration as shown in Fig. S6b.Additionally, the selectivity test for the SnS2 FET against streptavidin and BSA protein is also investigated at both substrates as shown in  The SnS2 FET exhibited faster and more sensitive detection of streptavidin over the h-BN substrate as compared to the simple SiO2 substrate.This performance improvement may be due to several factors.Firstly, the h-BN substrate possesses a higher surface area and greater hydrophobicity than the SiO2 substrate, which may facilitate the binding of streptavidin to the SnS2 FET and enhance the detection sensitivity [6] .Secondly, the surface charge of the substrate could also play a role in the detection efficiency, with the surface charge of h-BN potentially promoting greater binding of streptavidin and therefore higher detection sensitivity.Thirdly, the properties of the SnS2 FET itself may differ depending on the substrate, such as higher density or improved crystallinity when deposited on h-BN, contributing to the enhanced detection efficiency observed [7] .

Selectivity Test.
Hence, we extended the selectivity testing by making various real simulated solutions of BSA (nearly similar M. Wt. of 66.4 kDa) and Lysozyme (the egg protein, having a different M. Wt. of 14.6 kDa) than our target protein (streptavidin).Each solution has the same analyte concentration of 0.5 pM.The results showed the selectivity of our device in capturing the target analyte (streptavidin) only as shown in Figure S7.

Figure
Figure S1.(a) The synthesis and characterization of our engineered PLCB construct.(a) The

Figure S2 :
Figure S2: The binding of streptavidin by the biotin coupled onto the PLCB construct.The biotin is confined in the active site of streptavidin by ten different binding moieties involving eight hydrogen bonds (among polar groups) and two van der Waals interactions (among nonpolar groups).The distances are estimated using the Pymol software and mentioned in Å.

Figure S3 .
Figure S3.(a)The Raman spectroscopy analysis of a few layers thick hexagonal boron nitride (h-BN) sample, shows characteristic Raman peaks at 1366 cm -1 (E2g mode), confirming the SnS2 over the Si/SiO2 substrate.The electrical characterizations of the SnS2 channel materials were also investigated over the SiO2 substrate as a comparative analysis.The red circle area is the SnS2 FET device over the SiO2 substrate without h-BN as illustrated in Figure S4a.The transfer curves extracted at various biasing voltages are illustrated in Figure S4b, showing an ascending current as the biasing voltage is increased from 0.5-2.0V.Even the SnS2 FET exhibited a slightly high current over the SiO2 substrate as compared to the h-Bn substrate as a result of the single-layer dielectric of SiO2.Furthermore, the linear Ids versus Vds curves at various gate voltages are also plotted for the SnS2 FET over the SiO2 substrate, as shown in Figure S4c.The straight lines of Ids-Vds are presenting good Ohmic electrodes due to the negligible barrier height as shown in the energy band diagram of the main manuscript (Figure 5a-c).

Figure S4 :
Figure S4: (a) The schematic diagram is illustrating the SnS2 FET over the h-BN and SiO2

Figure S5 :
Figure S5: The SnS2 (channel top layer) FET is functionalized with the PLCB construct.The Pyrene present in PCLB is stacked over the hexagonal surface of the SnS2 sheet via - bonding (represented by Black lines).The transferring of electrons (those involved in loan pairs but not involved in bonding) from Pyrene-based PLCB to SnS2 is presented in RED lines.

Figure S7 :
Figure S7: The device selectivity.The current response of the device is recorded for 0.5 pM