Ultrasensitive label-free miRNA-21 detection based on MXene-enhanced plasmonic lateral displacement measurement


 miRNAs are small non-coding RNA molecules which serve as promising biomarkers due to their important roles in the development and progression of various cancer types. The detection of miRNAs is of vital importance to the early-stage diagnostics and prognostics of multiple diseases. However, traditional detection strategies have faced some challenges owing to the intrinsic characteristics of miRNAs including small size, short sequence length, low concentration level and high sequence homology in complex real samples. To overcome these challenges, we proposed a MXene-enhanced plasmonic biosensor for real-time and label-free detection of miRNA. By utilizing MXene nanomaterial which possesses unique characteristics including large surface area and strong carrier confinement abilities, we tuned the absorption of our plasmonic sensing substrate to reach a “zero-reflection” state and induced an extremely sharp phase change at the resonance angle. Combined with the sensing mechanism based on phase-induced lateral displacement measurement, this MXene-enhanced plasmonic biosensor can achieve a much superior sensing performance compared to traditional SPR devices. Based on this biosensing scheme, the ultrasensitive detection of target miRNA with a detection limit down to 10 fM has been successfully demonstrated. More importantly, single-base mismatched miRNA can be easily distinguished from the target miRNA according to the sensing signal. Furthermore, our plasmonic biosensor is capable of detecting miRNA in complex media such as 100 % human serum samples without compromising the detection sensitivity. This MXene-enhanced plasmonic sensing scheme has the ability of detecting miRNAs with extremely low concentration levels in complex surrounding media without the need of introducing extra labels or amplification tags, which holds great potential in various biological applications and clinical diagnostics.


Optimization of the plasmonic sensing substrate
The reflection is tuned by optimizing the thickness of each layer in the Kretschmann configuration.The parameters used to optimize the sensing substrate are shown in Table S1.The thickness of MXene was tuned from 0 nm to 6 nm.As shown in Figure S1, the total reflectivity of the sensing substrate will first decrease and then experience an increase.
Figure S1 Optimization of reflectivity by tuning the thickness of MXene.
Also, when the thickness of Au changes from 35 to 50 nm, the reflection will also be affected.Therefore, we need to tune the parameters to achieve the "almost zero" state.The optimized parameters we used for the plasmonic sensing substrate are 2.5 nm Ti, 40 nm Au, and 2-3 nm MXene.
The sensitivity of SPR sensing based on lateral displacement is in close relationship with the reflectivity.The bulk sensitivity is defined as  = ∆ ∆ (the change in lateral displacement with respect to the changes in refractive index) and can be quantified through the Matlab program.Here, we simulated the signal change when the refractive index of the sensing medium changed.In our optimized setting, minute refractive index changes (10 -6 RIU) can also be detected with a bulk sensitivity of 2.96× 10 6 / .Figure S2 showed the bulk sensitivity of the MXene-on-Au substrate and Au-only substrate.

Figure S2 Lateral displacement changes with respect to the refractive index changes based on
MXene-on-Au and Au-only substrate.

Experimental setup
The GH shift-based SPR plasmonic sensing system is depicted in Fig. S3.A commercial position sensor is employed to detect the lateral displacement associated with the molecule binding process.We

Characterization of MXene nanosheets
The monolayer colloid MXene solutions were purchased from Beike 2D materials Co.Ltd.We have characterized the morphology and thickness of MXene nanosheets through Atomic Force Microscopy (AFM).The thickness of monolayer MXene nanosheets is measured to be 0.89 nm on average.

Detection of miRNA-21 based on conventional SPR device
To further demonstrate the superiority of the proposed sensing method, we have done some sensing experiments based on the conventional SPR method.As shown in Figure S6, the wavelength shift is negligible when detecting miRNA-21 with a concentration level down to 10 -13 mol/L.When the concentration increased to 1 pM, the wavelength shift signal turned out to be detectable.The total wavelength shift was detected to be around 0.26 nm.These results showed that the detection limit of the conventional SPR scheme is around 10 -12 mol/L, which is two orders of magnitude higher than our proposed sensing scheme.The detection experiments on miRNA-21 with various concentration levels have been repeated three times.Figure S8 presented a summary of these measurements, which showed the capability of distinguishing single base mismatched miRNA with a detection range from 10 fM to 10 nM.[2] Srivastava, Akash, et al. "A theoretical approach to improve the performance of SPR biosensor using MXene and black phosphorus."Optik 203 (2020): 163430.
have used a He-Ne laser (Wavelength: 632.8 nm) as the light source.As shown in the figure, the incident light beam is split into p-polarized and s-polarized lights by a beam splitter and pass through an optical chopper.The light beams will reach the sensing substrate through prism coupling.The prism is fixed at the SPR resonance angle for obtaining a large lateral position shift signal change.A microfluidic reaction chamber is designed here to be integrated with the plasmonic substrate, which ensures the convenient transportation of target analytes onto the sensing region.As shown in FigureS4, the reaction chamber is made of PMMA and placed on top of the multi-layered plasmonic substrate with an o-ring in between to seal the liquid inside the PMMA chamber.The differential signal between p-polarized and s-polarized light will be measured by a position sensor to achieve a high signal-to-noise ratio since only p-polarized to the SPR effect.The results are collected and analyzed using LABVIEW and MATLAB programs.

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Figure S3 GH shift-based SPR optical setup.

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Figure S4 Detailed schematic diagram of the integrated SPR sensing module including prism, SPR substrate and the reaction chamber.

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Figure S6 Detection of miRNA-21 based on commercial SPR device.

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Figure S7 Real-time lateral displacement signal change in miRNA detection.

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Figure S8 Summary of the detection results ranging from 10 fM to 10 nM.
Table S1 Parameters used in optimizing the sensing substrate