Mesoporous Ag–TiO₂ based nanocage like structure as sensitive and recyclable low-cost SERS substrate for biosensing applications

Highlights

• This manuscript reports a facile method of fabrication of susceptible, low-cost, and unique nanocage-like structure of Ag–TiO₂ SERS film for the first time.

• The fabrication technique is simple, low-cost, and acceptable for mass production.

• The fabricated SERS substrate is optimised, and a 3D FDTD simulation investigates the enhancement mechanism.

• The reproducibility and reusability of the optimised SERS substrate were tested considering the R6G molecule as a probe using a portable Raman spectrometer.

• The recyclability of developed SERS substrate was explored by placing the SERS substrate under UV illumination for 130 min.

• The low-cost reusable SERS substrate is sensitive to detecting various urea concentrations up to 1 mM level covering the critical range of blood urea level.

Abstract

We fabricated a porous Ag–TiO₂ film with a nanocage (NC) like structure as a sensitive, recyclable, and low-cost SERS substrate using facile methods. The developed nanocage structure of TiO₂ film was first coated on a glass plate by doctor-blade method, and then thin film of silver with an optimum thickness of 10 nm was deposited onto it using thermal evaporation method. The unique mesoporous cage-like structure of TiO₂ film leads to the exceptional and non-uniform growth of Ag film. The developed Ag nanostructure is also porous, caps spherical in shape, and the average separation between two consecutive nanostructures is 10 nm. These closely decorated nanostructures of Ag produce hotspots by localising the incident electric field within the minimal volume. The unique nanocage structure of TiO₂ film provides a larger effective surface area of adsorption to the analytes. Thus, the synergistic effects of Ag–TiO₂ NC structure exhibit significant enhancement of 10⁸ considering rhodamine-6g (R6G) molecule as a probe using a portable handheld Raman spectrometer. The amount of electromagnetic enhancement was further investigated by 3D FDTD simulation that matches well with the experiments. The recyclability of the developed SERS substrate was explored by placing the SERS substrate under UV illumination for 130 min. The enhanced photocatalytic activity of Ag–TiO₂ NC SERS film degrades the dye molecules under UV radiation multiple times successively. Further, the Ag–TiO₂ SERS substrate is highly sensitive to detect various concentrations of urea up to 1 mM covering the vital range of blood urea level. Thus, the optimised, low-cost, reusable SERS substrate can be used as level free biosensors for routine analysis of biomolecules in clinical applications.

Introduction

Surface-enhanced Raman spectroscopy (SERS), discovered in 1974 by Fleishmann et al., has become a valuable technology in detecting and identifying numerous biological and chemical species in sensing applications [[1], [2], [3]]. Since Raman spectroscopy provides crucial information on molecular vibrational and rotational energy levels, SERS technique is utilized to amplify the usually weak Raman signal to 10⁸–10¹⁰ times using various nanostructured surfaces or nanoparticles of solution form [4,5]. The enhancement arises due to the localized surface plasmon resonance (LSPR) effect. LSPR occurs due to the longitudinal coherent collective oscillation of nanoparticles under an oscillatory electromagnetic field. If the oscillation frequency matches the incident laser frequency (resonance condition), the incident electric field will be trapped within the minimal volume creating hotspots [6]. Thus, the amplified local electric field (electromagnetic enhancement) causes enhancement in the scattered Raman signal of the analyte placed on a hotspot [6]. The nanostructures of noble metals like silver (Ag) or gold (Au) are exclusively used for their valuable plasmonic properties in the visible region of light for SERS-based technology [7]. Another enhancement mechanism, chemical enhancement, occurs due to a charge-transfer (C-T) resonance between the fermi level of metallic nanostructure and the orbital of an adsorbed analyte [8]. The amount of enhancement is tremendously dependent on the shape and size of nanostructures, interface, and type of molecules [6].

It is well established that a patterned SERS substrate provides better efficiency in enhancement compared to an irregular SERS substrate [9]. However, the techniques for patterning nanostructures like e-beam lithography, nanosphere lithography are costly, time-consuming, and not affordable for mass production [9,10]. Nanoparticles dispersed in solution are easy to produce, but it is difficult to fabricate a periodic nano-structured SERS substrate using solution-based nanoparticles [11]. One cost-effective method for fabricating patterned SERS substrate is the glancing angle deposition (GLAD) technique [12]. But in the case of the GLAD process, there is a considerable wastage of costly materials like Au or Ag to fabricate columnar-like structures. Other low-cost techniques include the deposition of nanoparticles using a natural pattern surface like cicada wings, butterfly wings etc. [[13], [14], [15]]. Apart from the natural patterned surface, low-cost porous materials like carbon arrays, alumina membrane, aluminum nanorods combined with Ag or Au exhibit efficient Raman signal enhancement [[16], [17], [18], [19], [20]]. But either the fabrication method is complex, or the produced SERS substrates are not recyclable. Hence, it is challenging to fabricate a reproducible, recyclable, and sensitive SERS substrate using facile methods. Zhao et al. have reported MoS₂ based heterojunction substrate that exhibits enhanced SERS and photocatalytic performance [21]. Research articles on tunable SERS using pyroelectric nanogenerator, hydrophobic multiscale cavities of MoS₂ grown on pyramidal silicon wafer composed of in-situ reduced Au nanoparticle, Ag nanoparticles in multiple graphene layers have shown enhanced SERS and self-cleaning ability [[22], [23], [24]]. Other photocatalytic materials like ZnO and TiO₂ combined with Ag or Au nanostructures have been explored to meet all the existing challenges in practical SERS based applications [25,26]. But TiO₂ is superior compared to ZnO as it is non-toxic and highly stable [27]. Further, TiO₂ is also highly scattering material in the visible range. Zhao et al. have fabricated Ag decorated TiO₂ nanowires by hydrothermal methods that exhibit enhanced Raman signal due to the chemical enhancement process [28]. Zhang et al. have fabricated a recyclable porous Ag/TiO₂ nanocomposite using photochemical deposition of Ag on spin coated TiO₂ films [29]. Lancu et al. have detected environmental pollutants using Ag–TiO₂ nanocomposites synthesized by sol-gel and chemical synthesis methods [30]. Zhou et al. have fabricated Ag loaded Ti³⁺ doped TiO₂ photonic crystal as SERS substrate using a specially designed anodization process [31]. Li et al. have fabricated highly structured Au semi-shells on TiO₂ spheres using nanosphere self-assembly and atomic layer deposition method [32]. Thus, the existing low-cost methods suffer low reproducibility and sensitivity. At the same time, the patterned sensitive substrates are not practical for the high cost. All published literatures contain the fabrication of TiO₂ nanowires, nanorods, and nanospheres in SERS based applications.

In this study, we have developed a facile method of fabrication of a highly controllable low-cost mesoporous Ag–TiO₂ based nanocage (NC) like structured film as a SERS substrate. The NC structure is unique and advantageous as it provides 3D platform for the decoration of Ag nanostructure, and it also offers a larger effective surface area of adsorption to the analyte [33,34]. A scalable method like doctor-blade has been utilized to fabricate the porous TiO₂ film over glass slides. Then 10 nm thin film of Ag was grown over TiO₂ film by thermal evaporation. The method of deposition is highly controllable and low cost. Most importantly, it is reproducible and ideal for mass production in practical purposes. The optimised Ag–TiO₂ based SERS substrate exhibits an enhancement factor of 10⁸ considering rhodamine 6G (R6G) molecules as a probe. The amount of enhancement was investigated by a three-dimension finite difference time domain (3D FDTD) simulation for the developed nanostructure. The recyclability of the fabricated Ag–TiO₂ SERS film was tested by placing it under UV light illumination. It completely degraded the dye molecules and was recycled five times successively.

Further, the fabricated SERS substrate was used for the detection of urea for various concentrations. Urea is a waste product produced after protein metabolism in the human body that the kidney removes to maintain its balance in blood serum [35]. But the amount of blood urea can be imbalanced due to different health issues and causes like malfunctioning of kidneys and other diseases. Hence, it is helpful to develop a fast, low-cost, and sensitive SERS technique for routine detection of urea in human blood to investigate the functionality of the kidney and other clinical conditions [36]. For the diagnosis of urea concentration in blood, the fabricated SERS substrate has been calibrated for the crucial range of blood urea (0.5 M to 1 mM) level. The results are remarkably reproducible, which further confirms the high quality of SERS substrate for clinical purposes. The experimental methods, fabrication and simulation details and results are briefly discussed in subsequent sections.