In Situ Probing the Structure Change and Interaction of Interfacial Water and Hydroxyl Intermediates on Ni(OH)2 Surface over Water Splitting.

There is growing acknowledgment that the properties of the electrochemical interfaces play an increasingly pivotal role in improving the performance of the hydrogen evolution reaction (HER). Here, we present, for the first time, direct dynamic spectral evidence illustrating the impact of the interaction between interfacial water molecules and adsorbed hydroxyl species (OHad) on the HER properties of Ni(OH)2 using Au/core-Ni(OH)2/shell nanoparticle-enhanced Raman spectroscopy. Notably, our findings highlight that the interaction between OHad and interfacial water molecules promotes the formation of weakly hydrogen-bonded water, fostering an environment conducive to improving the HER performance. Furthermore, the participation of OHad in the reaction is substantiated by the observed deprotonation step of Au@2 nm Ni(OH)2 during the HER process. This phenomenon is corroborated by the phase transition of Ni(OH)2 to NiO, as verified through Raman and X-ray photoelectron spectroscopy. The significant redshift in the OH-stretching frequency of water molecules during the phase transition confirms that surface OHad disrupts the hydrogen-bond network of interfacial water molecules. Through manipulation of the shell thickness of Au@Ni(OH)2, we additionally validate the interaction between OHad and interfacial water molecules. In summary, our insights emphasize the potential of electrochemical interfacial engineering as a potent approach to enhance electrocatalytic performance.

Systematic Optimization of Universal Real-Time Hypersensitive Fast Detection Method for HBsAg in Serum Based on SERS.

Hepatitis B virus (HBV) is a major cause of liver cirrhosis and hepatocellular carcinoma, with HBV surface antigen (HBsAg) being a crucial marker in the clinical detection of HBV. Due to the significant harm and ease of transmission associated with HBV, HBsAg testing has become an essential part of preoperative assessments, particularly for emergency surgeries where healthcare professionals face exposure risks. Therefore, a timely and accurate detection method for HBsAg is urgently needed. In this study, a surface-enhanced Raman scattering (SERS) sensor with a sandwich structure was developed for HBsAg detection. Leveraging the ultrasensitive and rapid detection capabilities of SERS, this sensor enables quick detection results, significantly reducing waiting times. By systematically optimizing critical factors in the detection process, such as the composition and concentration of the incubation solution as well as the modification conditions and amount of probe particles, the sensitivity of the SERS immune assay system was improved. Ultimately, the sensor achieved a sensitivity of 0.00576 IU/mL within 12 min, surpassing the clinical requirement of 0.05 IU/mL by an order of magnitude. In clinical serum assay validation, the issue of false positives was effectively addressed by adding a blocker. The final sensor demonstrated 100% specificity and sensitivity at the threshold of 0.05 IU/mL. Therefore, this study not only designed an ultrasensitive SERS sensor for detecting HBsAg in actual clinical serum samples but also provided theoretical support for similar systems, filling the knowledge gap in existing literature.

Enhancing Light Out-coupling in Perovskite Light-Emitting Diodes through Plasmonic Nanostructures.

Perovskite light-emitting diodes (PeLEDs) have emerged as promising candidates for lighting and display technologies owing to their high photoluminescence quantum efficiency and high carrier mobility. However, the performance of planar PeLEDs is limited by the out-coupling efficiency, predominantly governed by photonic losses at device interfaces. Most notably, the plasmonic loss at the metal electrode interfaces can account for up to 60% of the total loss. Here, we investigate the use of plasmonic nanostructures to improve the light out-coupling in PeLEDs. By integrating these nanostructures with PeLEDs, we have demonstrated an effectively reduced plasmonic loss and enhanced light out-coupling. As a result, the nanostructured PeLEDs exhibit an average 1.5-fold increase in external quantum efficiency and an ∼20-fold improvement in device lifetime. This finding offers a generic approach for enhancing light out-coupling, promising great potential to go beyond existing performance limitations.

SERS-Based Hydrogen Bonding Induction Strategy for Gaseous Acetic Acid Capture and Detection.

Surface-enhanced Raman scattering (SERS) can overcome the existing technological limitations, such as complex processes and harsh conditions in gaseous small-molecule detection, and advance the development of real-time gas sensing at room temperature. In this study, a SERS-based hydrogen bonding induction strategy for capturing and sensing gaseous acetic acid is proposed for the detection demands of gaseous acetic acid. This addresses the challenges of low adsorption of gaseous small molecules on SERS substrates and small Raman scattering cross sections and enables the first SERS-based detection of gaseous acetic acid by a portable Raman spectrometer. To provide abundant hydrogen bond donors and acceptors, 4-mercaptobenzoic acid (4-MBA) was used as a ligand molecule modified on the SERS substrate. Furthermore, a sensing chip with a low relative standard deviation (RSD) of 4.15% was constructed, ensuring highly sensitive and reliable detection. The hydrogen bond-induced acetic acid trapping was confirmed by experimental spectroscopy and density functional theory (DFT). In addition, to achieve superior accuracy compared to conventional methods, an innovative analytical method based on direct response hydrogen bond formation (IO-H/Iref) was proposed, enabling the detection of gaseous acetic acid at concentrations as low as 60 ppb. The strategy demonstrated a superior anti-interference capability in simulated breath and wine detection systems. Moreover, the high reusability of the chip highlights the significant potential for real-time sensing of gaseous acetic acid.

Construction of nanoparticle-on-mirror nanocavities and their applications in plasmon-enhanced spectroscopy.

Plasmonic nanocavities exhibit exceptional capabilities in visualizing the internal structure of a single molecule at sub-nanometer resolution. Among these, an easily manufacturable nanoparticle-on-mirror (NPoM) nanocavity is a successful and powerful platform for demonstrating various optical phenomena. Exciting advances in surface-enhanced spectroscopy using NPoM nanocavities have been developed and explored, including enhanced Raman, fluorescence, phosphorescence, upconversion, etc. This perspective emphasizes the construction of NPoM nanocavities and their applications in achieving higher enhancement capabilities or spatial resolution in dark-field scattering spectroscopy and plasmon-enhanced spectroscopy. We describe a systematic framework that elucidates how to meet the requirements for studying light-matter interactions through the creation of well-designed NPoM nanocavities. Additionally, it provides an outlook on the challenges, future development directions, and practical applications in the field of plasmon-enhanced spectroscopy.

Reliable quantitative detection of uric acid in urine by surface-enhanced Raman spectroscopy with endogenous internal standard.

Abnormal levels of uric acid (UA) in urine serve as warning signs for gout and metabolic cardiovascular diseases, necessitating the monitoring of UA levels for early prevention. However, the current analytical methods employed suffer from limitations in terms of inadequate suitability for home-based applications and the requirement of non-invasive procedures. In this approach, creatinine, a metabolite with a constant excretion rate, was incorporated as an endogenous internal standard (e-IS) for calibration, presenting a rapid, pretreatment-free, and accurate strategy for quantitative determination of UA concentrations. By utilizing urine creatinine as an internal reference value to calibrate the signal fluctuation of surface-enhanced Raman spectroscopy (SERS) of UA, the quantitative accuracy can be significantly improved without the need for an external internal standard. Due to the influence of the medium, UA, which carries a negative charge, is selectively adsorbed by Au@Ag nanoparticles functionalized with hexadecyltrimethylammonium chloride (CTAC) in this system. Furthermore, a highly convenient detection method was developed, which eliminates the need for pre-processing and minimizes matrix interference by simple dilution. The method was applied to the urine detection of different volunteers, and the results were highly consistent with those obtained using the UA colorimetric kit (UACK). The detection time of SERS was only 30 s, which is 50 times faster than UACK. This quantitative strategy of using urinary creatinine as an internal standard to correct the SERS intensity of uric acid is also expected to be extended to the quantitative detection needs of other biomarkers in urine.

Tailoring Fluorescence-Phosphorescence Emission with a Single Nanocavity.

Realizing the dual emission of fluorescence-phosphorescence in a single system is an extremely important topic in the fields of biological imaging, sensing, and information encryption. However, the phosphorescence process is usually in an inherently "dark state" at room temperature due to the involvement of spin-forbidden transition and the rapid non-radiative decay rate of the triplet state. In this work, we achieved luminescent harvesting of the dark phosphorescence processes by coupling singlet-triplet molecular emitters with a rationally designed plasmonic cavity. The achieved Purcell enhancement effect of over 1000-fold allows for overcoming the triplet forbidden transitions, enabling radiation enhancement with selectable emission wavelengths. Spectral results and theoretical simulations indicate that the fluorescence-phosphorescence peak position can be intelligently tailored in a broad range of wavelengths, from visible to near-infrared. Our study sheds new light on plasmonic tailoring of molecular emission behavior, which is crucial for advancing research on plasmon-tailored fluorescence-phosphorescence spectroscopy in optoelectronics and biomedicine.

Ultrasensitive detection of SARS-CoV-2 S protein with aptamers biosensor based on surface-enhanced Raman scattering.

A rapid and accurate diagnostic modality is essential to prevent the spread of SARS-CoV-2. In this study, we proposed a SARS-CoV-2 detection sensor based on surface-enhanced Raman scattering (SERS) to achieve rapid and ultrasensitive detection. The sensor utilized spike protein deoxyribonucleic acid aptamers with strong affinity as the recognition entity to achieve high specificity. The spherical cocktail aptamers-gold nanoparticles (SCAP) SERS substrate was used as the base and Au nanoparticles modified with the Raman reporter molecule that resonates with the excitation light and spike protein aptamers were used as the SERS nanoprobe. The SCAP substrate and SERS nanoprobes were used to target and capture the SARS-CoV-2 S protein to form a sandwich structure on the Au film substrate, which can generate ultra-strong "hot spots" to achieve ultrasensitive detection. Analysis of SARS-CoV-2 S protein was performed by monitoring changes in SERS peak intensity on a SCAP SERS substrate-based detection platform. This assay detects S protein with a LOD of less than 0.7 fg mL-1 and pseudovirus as low as 0.8 TU mL-1 in about 12 min. The results of the simulated oropharyngeal swab system in this study indicated the possibility of it being used for clinical detection, providing a potential option for rapid and accurate diagnosis and more effective control of SARS-CoV-2 transmission.

In situ electrochemical Raman spectroscopy and ab initio molecular dynamics study of interfacial water on a single-crystal surface.

The dynamics and chemistry of interfacial water are essential components of electrocatalysis because the decomposition and formation of water molecules could dictate the protonation and deprotonation processes on the catalyst surface. However, it is notoriously difficult to probe interfacial water owing to its location between two condensed phases, as well as the presence of external bias potentials and electrochemically induced reaction intermediates. An atomically flat single-crystal surface could offer an attractive platform to resolve the internal structure of interfacial water if advanced characterization tools are developed. To this end, here we report a protocol based on the combination of in situ Raman spectroscopy and ab initio molecular dynamics (AIMD) simulations to unravel the directional molecular features of interfacial water. We present the procedures to prepare single-crystal electrodes, construct a Raman enhancement mode with shell-isolated nanoparticle, remove impurities, eliminate the perturbation from bulk water and dislodge the hydrogen bubbles during in situ electrochemical Raman experiments. The combination of the spectroscopic measurements with AIMD simulation results provides a roadmap to decipher the potential-dependent molecular orientation of water at the interface. We have prepared a detailed guideline for the application of combined in situ Raman and AIMD techniques; this procedure may take a few minutes to several days to generate results and is applicable to a variety of disciplines ranging from surface science to energy storage to biology.

Rapid Point-of-Care Assay by SERS Detection of SARS-CoV-2 Virus and Its Variants.

Addressing the spread of coronavirus disease 2019 (COVID-19) has highlighted the need for rapid, accurate, and low-cost diagnostic methods that detect specific antigens for SARS-CoV-2 infection. Tests for COVID-19 are based on reverse transcription PCR (RT-PCR), which requires laboratory services and is time-consuming. Here, by targeting the SARS-CoV-2 spike protein, we present a point-of-care SERS detection platform that specifically detects SARS-CoV-2 antigen in one step by captureing substrates and detection probes based on aptamer-specific recognition. Using the pseudovirus, without any pretreatment, the SARS-CoV-2 virus and its variants were detected by a handheld Raman spectrometer within 5 min. The limit of detection (LoD) for the pseudovirus was 124 TU µL-1 (18 fM spike protein), with a linear range of 250-10,000 TU µL-1. Moreover, this assay can specifically recognize the SARS-CoV-2 antigen without cross reacting with specific antigens of other coronaviruses or influenza A. Therefore, the platform has great potential for application in rapid point-of-care diagnostic assays for SARS-CoV-2.

Quantitatively Revealing the Anomalous Enhancement in Shell-Isolated Nanoparticle-Enhanced Raman Spectroscopy Using Single-Nanoparticle Spectroscopy.

Surface-enhanced Raman spectroscopy (SERS) is an ultrasensitive spectroscopic technique that has been extensively applied in the studies of catalysis, electrochemistry, material science, etc.; however, it is substrate and material limited. The development of shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) effectively offsets this limitation that attracts enormous attention due to its potential to be applied to any surface. As the core of the SHINERS technique, the inert shell prevents the exposure of the active metal surface, however, also significantly enlarges the metallic gap where the light is trapped. Consequently, the shell is widely considered a side issue to debilitate the coupling efficiency and hinder the sensitivity of SHINERS without systematic studies. Herein, we investigate the shell and structural effect of SHINERS by performing the quantitative optical and structural characterization of single nanostructures. By a statistic of over two hundred nanostructures, we observe that the field enhancement loss due to the shell could be overcome by optimizing the coupling geometry of the shell-isolated nanoparticles (SHINs). An example of SHIN dimers shows even higher field enhancement than their bare Au nanoparticle counterparts as confirmed and explained by FDTD simulations. We demonstrate the signal enhancement of SHINERS saturates with the increasing number of hot spots but could be further optimized by altering the aggregation geometries of the nanoparticles. The sensitivity improvement of the SHINERS technique will boost its broader applications in material science.

Ultrafast and field-based detection of methamphetamine in hair with Au nanocake-enhanced Raman spectroscopy.

The disaster and devastation from abuse of Methamphetamine (MAMP) have a serious impact on people's mental and physical health. Developing a rapid and accurate method to screen drug suspects and thus control MAMP abuse is essential to social security. Hair analysis for MAMP detection is considered to be one of the most potential methods for monitoring drug abuse due to its convenient sample collection, easy for storage and long traceability period. However, the current accurate detection of MAMP in hair primarily utilizes hyphenated mass spectrometry (MS) techniques, but it is not suitable for field-based detection due to the bulky instrument. Hence, developing alternative portable detection techniques for rapid on-site detection of MAMP in hair is an urgent problem to be solved. Here, the high-performance Au nanocakes (Au NCs) were constructed as surface-enhanced Raman spectroscopy (SERS) substrates to detect MAMP in hair, realizing 5 min ultrafast and ultrasensitive detection utilizing a portable Raman spectrometer. Experiments and finite-difference time-domain (FDTD) simulations show that Au NCs have stronger enhancement than Au nanospheres (Au NPs), and 0.5 ppb (3.35 × 10-9 M) MAMP standard is stably detected by Au NCs as an enhanced substrate. A strategy of liquid-liquid microextraction was exploited to eliminate the interference of complex matrices in hair. This method exhibited excellent reproducibility and temporal stability across different drug addicts (relative standard deviation was 5.14% within 160 s). Our approach shows great promise in public safety, providing a rapid and accurate method to detect in hair by SERS.

Shell-Isolated Nanoparticle-Enhanced Electrochemiluminescence.

Enhanced electrochemiluminescence (ECL) aims to promote higher sensitivity and obtain better detection limit. The core-shell nanostructures, owing to unique surface plasmon resonance (SPR) enabling distance-dependent strong localized electromagnetic field, have attracted rising attention in enhanced ECL research and application. However, the present structures usually with porous shell involve electrocatalytic activity from the metal core and adsorption effect from the shell, which interfere with practical SPR enhancement contribution to ECL signal. Herein, to exclude the interference and unveil exact SPR-enhanced effect, shell-isolated nanoparticles (SHINs) whose shell gets thicker and becomes pinhole-free are developed by modifying pH value and particles concentration. Furthermore, allowing for the distribution of hotspots and stronger enhancement, excitation intensity and ECL reaction layer thickness are mainly investigated, and several types of SHINs-enhanced ECL platforms are prepared to fabricate distinct hotspot distribution via electrostatic attraction (submonolayer) and a layer-by-layer deposition method (monolayer). Consequently, the strongest enhancement up to ≈250-fold is achieved by monolayer SHINs with 10 nm shell, and the platform is applied in a "turn-off" mode sensing for dopamine. The platform provides new guidelines to shell preparation, interface engineering and hotspots fabrication for superior ECL enhancement and analytical application with high performance.

Succession changes of fermentation parameters, nutrient components and bacterial community of sorghum stalk silage.

To better understand the ensiling characteristics of sorghum stalk, the dynamic changes of fermentation parameters, nutrient components and bacterial community of sorghum stalk silage were analyzed by intermittently sampling on day 0, 1, 3, 7, 14, 28, and 56 of ensiling duration. The results showed that high-moisture sorghum stalk was well preserved during ensiling fermentation, with the DM loss of 4.10% and the little difference between the nutrients of sorghum stalk before and after ensiling. The pH value of silage declined to its lowest value of 4.32 by Day 7 of ensiling, and other fermentation parameters kept steady since Day 28 of ensiling. The amplicon sequencing analysis revealed that the alpha diversity parameters of silage bacterial community including Shannon index, observed features, Pielou evenness and Faith PD gradually declined (P < 0.01) with ensiling duration. Principal coordinate analysis (PCoA) revealed that bacterial profiles of raw material would experience a succession becoming a quite different community during ensiling fermentation. Taxonomic classification revealed a total of 10 and 173 bacterial taxa at the phylum and genus level, respectively, as being detected with relative abundances higher than 0.01% and in at least half samples. LEfSe analysis revealed that 26 bacterial taxa were affected by sampling timepoint (P < 0.05 and LDA score > 4). When focusing on the dynamic trend of silage bacterial taxa, lactic acid bacteria successfully dominated in the bacterial community on Day 1 of ensiling, and the bacterial community almost came to a plateau by Day 28 of ensiling, with Lactobacillus and Leuconostoc as the dominant genera. In a word, the succession of fermentation parameters, nutrient components and bacterial community indicate a successful dominance establishment of LAB and a fast advent of fermentation plateau, suggesting that high-moisture sorghum stalk can be ensiled directly, but the pH of mature silage is a little high.

Self-Calibration 3D Hybrid SERS Substrate and Its Application in Quantitative Analysis.

Surface-enhanced Raman spectroscopy (SERS) has been widely applied in many fields as a sensitive vibrational fingerprint technique. However, SERS faces challenges in quantitative analysis due to the heterogeneity of hot spots. An internal standard (IS) strategy has been employed for correcting the variation of hot spots. However, the method suffers from limitations due to the competitive adsorption between the IS and the target analyte. In this work, we combined the IS strategy with the 3D hybrid nanostructures to develop a bifunctional SERS substrate. The substrate had two functional units. The bottom self-assembly layer consisted of Au@IS@SiO2 nanoparticles, which provided a stable reference signal and functioned as the calibration unit. The top one consisted of appropriate-sized Au octahedrons for the detection of target analytes, which was the detection unit. Within the 3D hybrid nanostructure, the calibration unit improved the SERS performance of the detection unit, which was demonstrated by the 6-fold increase of SERS intensity when compared with the 2D substrate. Meanwhile, the reproducibility of the detection was greatly improved by correcting the hot spot changes through the calibration unit. Two biomedical molecules of cotinine and creatinine in ultrapure water and artificial urine, respectively, were sensitively determined by the 3D hybrid substrate. We expect that the developed bifunctional 3D substrate will open up new ways to advance the applications of SERS.

Atomic overlayer of permeable microporous cuprous oxide on palladium promotes hydrogenation catalysis.

The interfacial sites of metal-support interface have been considered to be limited to the atomic region of metal/support perimeter, despite their high importance in catalysis. By using single-crystal surface and nanocrystal as model catalysts, we now demonstrate that the overgrowth of atomic-thick Cu2O on metal readily creates a two-dimensional (2D) microporous interface with Pd to enhance the hydrogenation catalysis. With the hydrogenation confined within the 2D Cu2O/Pd interface, the catalyst exhibits outstanding activity and selectivity in the semi-hydrogenation of alkynes. Alloying Cu(0) with Pd under the overlayer is the major contributor to the enhanced activity due to the electronic modulation to weaken the H adsorption. Moreover, the boundary or defective sites on the Cu2O overlayer can be passivated by terminal alkynes, reinforcing the chemical stability of Cu2O and thus the catalytic stability toward hydrogenation. The deep understanding allows us to extend the interfacial sites far beyond the metal/support perimeter and provide new vectors for catalyst optimization through 2D interface interaction.

Label-free SERS strategy for rapid detection of capsaicin for identification of waste oils.

Identification of waste oils is challenging in the field of food safety due to the lack of common markers and straightforward analytical methods. Herein, we developed a novel label-free surface-enhanced Raman spectroscopy (SERS) strategy to identify waste oils using Ag nanoparticles solution (Ag NPs sol.) as a SERS substrate to significantly enhance the Raman signal of capsaicin marker molecule usually contained in the waste oils. The enhanced signal was directly detected by a portable Raman spectrometer with the limit of detection (LOD) of 2.9 µg L-1 within 10 min. Concentration-dependent SERS investigation showed the linear relationship between the SERS signal intensity of the characteristic peaks and the concentrations of capsaicin in the range of 10-2500 µg L-1 and the correlation coefficient was 0.9895. Our findings show the sensitivity, accessibility, and reliability of this method for the rapid identification of waste oils and furthermore for the practical applications in the field of food safety.

Direct and Simultaneous Identification of Multiple Mitochondrial Reactive Oxygen Species in Living Cells Using a SERS Borrowing Strategy.

Identification of different mitochondrial reactive oxygen species (ROS) simultaneously in living cells is vital for understanding the critical roles of different ROS in biological processes. To date, it remains a great challenge to develop ROS probes for direct and simultaneous identification of multiple ROS with high specificity. Herein, we report a SERS-borrowing-strategy-based nanoprobe (Au@Pt core-shell nanoparticles) for simultaneous and direct identification of different ROS by their distinct Raman fingerprints. Isotope substitution experiments and DFT calculations confirmed the ability of Au@Pt nanoprobe to capture and identify different mitochondrial ROS (i.e. ⋅OOH, H2 O2 , and ⋅OH). When functionalized with triphenylphosphine (TPP), the Au@Pt-TPP nanoprobe located to mitochondria and detected multiple ROS simultaneously in living cells under oxidative stimulation. Our method offers a new tool for the study of the functions of various ROS in biological processes.

Unmasking the Critical Role of the Ordering Degree of Bimetallic Nanocatalysts on Oxygen Reduction Reaction by In Situ Raman Spectroscopy.

Precise control and accurate understanding of the ordering degree of bimetallic nanocatalysts (BNs) are challenging yet crucial to acquire advanced materials for the oxygen reduction reaction (ORR). AuCu BNs with various ordering degrees were synthesized to evaluate the influence of ordering degree on the ORR at a molecular level using in situ Raman spectroscopy. The activity of AuCu BNs was improved by over 2 times after a disorder-to-order transition, making the performance of highly ordered AuCu BNs exceed that of benchmark Pt/C. Direct Raman spectroscopic evidence of key intermediate (*OH) demonstrates that the active site is the combination site of Au and Cu. Moreover, two distinct *OH species are observed on the ordered and disordered structure, and the ordered site is more beneficial for ORR due to its lower affinity to *OH. This work deepens the understanding on the important role of ordering degree on BNs and enables the design of improved catalysts.

Rapid and Simple Analysis of the Human Pepsin Secondary Structure Using a Portable Raman Spectrometer.

Human pepsin is a digestive protease that plays an important role in the human digestive system. The secondary structure of human pepsin determines its bioactivity. Therefore, an in-depth understanding of human pepsin secondary structure changes is particularly important for the further improvement of the efficiency of human pepsin biological function. However, the complexity and diversity of the human pepsin secondary structure make its analysis difficult. Herein, a convenient method has been developed to quickly detect the secondary structure of human pepsin using a portable Raman spectrometer. According to the change of surface-enhanced Raman spectroscopy (SERS) signal intensity and activity of human pepsin at different pH values, we analyze the change of the human pepsin secondary structure. The results show that the content of the ß-sheet gradually increased with the increase in the pH in the active range, which is in good agreement with circular dichroism (CD) measurements. The change of the secondary structure improves the sensitivity of human pepsin SERS detection. Meanwhile, human pepsin is a commonly used disease marker for the noninvasive diagnosis of gastroesophageal reflux disease (GERD); the detection limit of human pepsin we obtained is 2 µg/mL by the abovementioned method. The real clinical detection scenario is also simulated by spiking pepsin solution in saliva, and the standard recovery rate is 80.7-92.3%. These results show the great prospect of our method in studying the protein secondary structure and furthermore promote the application of SERS in clinical diagnosis.