Surface-enhanced Raman Scattering Peak Research Articles

Machine learning-assisted SERS sensor for fast and ultrasensitive analysis of multiplex hazardous dyes in natural products

The adulteration of natural products with multiple azo dyes has become a serious public health concern. Thus, on-site trace additive detection is demanded. Herein, we developed a gold-nanorod-based surface-enhanced Raman scattering (SERS) sensor to detect trace amounts of azo dyes, including lemon yellow, sunset yellow, golden orange II, acid red 73, coccine, and azorubine. After optimizing pre-processing steps, the additives were separated and identified through visual observation. The stable and sensitive SERS sensor developed enabled accurate detection of the added colorants. Density Functional Theory confirmed that the characteristic SERS peaks of the six dyes were accurate and credible. The optimized SERS sensor achieved a detection limit of 50 mg of dye per kilogram of raw material. A SERS spectral dataset comprising 960 replicates from all 64 potential dye combinations was generated, forming robust training sets. The K-Nearest Neighbor model exhibited best performance, identifying dye additives in real samples with a 91 % success rate. This model was further validated by screening nine randomly collected safflower batches, identifying three with illegal dye additives, which were subsequently confirmed by HPLC. Summarily, the developed SERS sensor and classification model offer an ultrasensitive, and reliable approach for on-site detection of hazardous dyes in natural products.

Unmasking Bacterial Identities: Exploiting Silver Nanoparticle 'Masks' for Enhanced Raman Scattering in the Rapid Discrimination of Diverse Bacterial Species and Antibiotic-Resistant Strains.

Unraveling bacterial identity through Raman scattering techniques has been persistently challenging due to homogeneously amplified Raman signals across a wide variety of bacterial molecules, predominantly protein- or nucleic acid-mediated. In this study, we present an approach involving the use of silver nanoparticles to completely and uniformly "mask" adsorption on the surface of bacterial molecules through sodium borohydride and sodium chloride. This approach enables the acquisition of enhanced surface-enhanced Raman scattering (SERS) signals from all components on the bacterial surface, facilitating rapid, specific, and label-free bacterial identification. For the first time, we have characterized the identity of a bacterium, including its DNA, metabolites, and cell walls, enabling the accurate differentiation of various bacterial strains, even within the same species. In addition, we embarked on an exploration of the origin and variability patterns of the main characteristic peaks of Gram-positive and Gram-negative bacteria. Significantly, the SERS peak ratio was found to determine the inflection point of accelerated bacterial death upon treatment with antimicrobials. We further applied this platform to identify 15 unique clinical antibiotic-resistant bacterial strains, including five Escherichia coli strains in human urine, a first for Raman technology. This work has profound implications for prompt and accurate identification of bacteria, particularly antibiotic-resistant strains, thereby significantly enhancing clinical diagnostics and antimicrobial treatment strategies.

A novel ratiometric aptasensor based on SERS for accurate quantification of cardiac troponin I

Cardiac troponin I (cTnI) as a biomarker of acute myocardial infarction (AMI) is directly related to myocardial damage. Here, we proposed a ratiometric aptamer-sensor (aptasensor) for quantifying cTnI based on surface-enhanced Raman scattering (SERS), in which the uniform and high-density gold array was decorated with highly specific aptamer Tro4 to form the capture substrate for target cTnI. Firstly, gold array was labeled with 5, 5’-dithio bis-(2-nitrobenzoic acid) (DTNB) as one SERS signal to effectively realize signal self-calibration. In addition, aptamer Tro6 targeting cTnI and 4-mercaptobenzoic acid (4-MBA) as another SERS signal were anchored to porous gold (pAu) to form a pAu signal probe. In the presence of cTnI, the capture substrate and pAu signal probes could build a typical sandwich structure, thus the ratio of SERS peak intensity (I4-MBA/IDTNB) could be used to quantify cTnI. The ratiometric aptasensor showed a good linearity from 0.001 to 100 ng mL−1 and a low detection limit down to 0.27 pg mL−1. Own to dual signal output and the gold array substrate, the aptasensor exhibited good reproducibility, high sensitivity and accuracy. When the aptasensor was applied to the quantification of cTnI in human serum samples, the detection performance was comparable to that of enzyme-linked immunosorbent assay (ELISA), proving that the sensor has a good prospect in the diagnosis of AMI disease.

Internal standard optimization advances sensitivity and robustness of ratiometric detection method.

We design a p-aminothiophenol (pATP) modified Au/ITO chip to determine nitrite ions in lake water by a ratiometric surface-enhanced Raman scattering (SERS) method based on nitrite ions triggering the transformation of pATP to p,p'-dimercaptoazobenzene (DMAB). Intriguingly, by using the SERS peak (at 1008 cm-1) from benzoic ring deforming as an internal standard instead of the traditional peak at 1080 cm-1, the detection sensitivity of the method was improved 10 times.

SERS and MRS signals engineered dual-mode aptasensor for simultaneous distinguishment of aflatoxin subtypes

The accurate monitoring of aflatoxin subtypes is vitally important for food safety. Herein, a dual-mode aptasensor with surface-enhanced Raman scattering (SERS) and magnetic relaxation switching (MRS) signals is developed for the detection of aflatoxin B1, B2 and M1 (i.e. AFB1, AFB2 and AFM1). Au-Ag Janus NPs and Au-mushroom NPs are prepared and show intense and non-interfering SERS peaks without the additional modification of Raman molecules, and are utilized as SERS nanotags for the distinguishment of AFB1 and AFB2. Fe3O4@Au NPs functionalized by AFM1 aptamers are applied as MRS nanoprobes for the monitoring of AFM1. Aptamers engineered SERS nanotags and MRS nanoprobes are assembled, and show strong SERS performances and high transverse relaxation time (T2). AFB1, AFB2 and AFM1 induce the separation of SERS nanotags from the assemblies and the dispersion of Fe3O4@Au NPs, resulting in the decrease of SERS signals at 1278 cm−1 and 1000 cm−1 as well as the reduction of T2 values. The dual-mode but three kinds of detection signals don’t interfere with each other and exhibit a significant linear relationship with the concentration of targets. This platform provides a high throughput monitoring strategy for the simultaneous analysis of different subtypes of mycotoxin.

Chemically renewable SERS sensor for the inspection of H2O2 residue in food stuff

Hydrogen peroxide (H2O2) residue in foodstuffs will bring great harm to human health. We immobilize the composite of the reduced polyaniline (PANIR) modified gold nanoparticles on the surface of ITO (ITO/AuNPs/PANIR) to develop surface-enhanced Raman scattering (SERS) sensor for H2O2.detection. The principle is that PANIR is oxidized by H2O2 to generate a new SERS peak at 1460 cm−1 for realizing quantitative analysis of H2O2. Fe2+-Fenton reaction is introduced to catalytically react with H2O2 to hydroxyl radical, which speeds up the oxidation of PANIR. Before SERS detection, acidic treatment could guarantee the reduced state of PANIR in composite. Limit of detection of ITO/AuNPs/PANIR-based SERS assay for H2O2 is down to 1.78 × 10−12 mol/L and a good linear relationship from 1 × 10−10 to 3.16 × 10−7 mol/L is achieved. Furthermore, the SERS sensor could be regenerated by acidic treatment. As a scenario, the renewable SERS sensor is utilized to monitor H2O2 residues in food and environmental samples.

Simulated enzyme catalytic strategy for surface-enhanced Raman scattering detection of methylmercury by using carbon dots/gold-based nanozyme

Methylmercury (MeHg) as an organometallic is much more toxic than inorganic mercury. Nevertheless, building an organometallic assay remains a great challenge due to the coexisting inorganic metal ions. Herein, we synthesize a gold nanozyme (NP-CDs/AuNPs) via using norepinephrine-based carbon dots (NP-CDs) as reducing agent. As expected, NP-CDs/AuNPs exhibits enhanced peroxidase-like activity in the presence of MeHg to form Au-Hg amalgam (Au@HgNPs). Compared with the NP-CDs/AuNPs, the formed Au@HgNPs could oxidize weak Raman-active reporter o-phenylendiamine (OPD) into the surface-enhanced Raman scattering (SERS) activity 2,3-diaminephenazine (DAP) with a single characteristic SERS peak of C-Hg bond at 477 cm−1. The results of UV–vis–kinetics and SERS-kinetics models demonstrated the better affinity between Au@HgNPs and the substrate than that of NP-CDs/AuNPs, indicating the high sensitivity SERS signal intensity response. The SERS-enhancing mechanism explicitly revealed that the generation of charge transfer complex Au@HgNPs further increases the “hot spot” effect. The work provides guidance in designing the ultrasensitive and specific analysis of organometallic compound via SERS.

Surface-Enhanced Raman Scattering Enantioselective Detection of Gastric Cancer-Related d-Amino Acids in Saliva Based on Enzyme-Mediated Cascade Reaction.

The unusual d-amino acids (d-AAs), as the counter enantiomer of usual l-amino acids (l-AAs), have evoked increasing attention because of their potential relevance with diseases. Accordingly, it is essential to establish sensitive and selective detection methods for d-AAs without the interferences from l-AAs. The surface-enhanced Raman scattering (SERS) technique is efficacious for the detection of molecules but routinely ineffective in enantiomeric differentiation. d-Proline (d-Pro) and d-alanine (d-Ala) are regarded as biomarkers of gastric cancer. Herein, Raman-active boronate modified SERS chips are constructed to develop a d-amino acid oxidase (DAAO)-mediated cascade reaction-based SERS enantioselective assay for d-Pro and d-Ala. The principle is that DAAO selectively catalyzes the deamination of d-Pro and d-Ala, and the produced H2O2 oxidizes boronate to present a new SERS peak at 883 cm-1 for quantitative analysis in a ratiometric way. A linear range from 20 to 400 μmol/L and a limit of detection down to 14.8 μmol/L are reached. In addition, interferences from l-AAs and many other possible species coexisting in biofluids with the detection of d-Pro and d-Ala are ignorable. Enzyme-mediated cascade reaction-based SERS chips are further utilized for saliva sample analysis, and the total levels of d-Pro and d-Ala in salivary samples from gastric cancer patients are much higher than those of healthy persons. This work provides a solution for SERS enantioselective analysis and noninvasive screening chiral biomolecules for disease diagnosis.

Flexible surface-enhanced Raman scattering substrate of cotton swab-Ag combination with smartphone app for chemical warfare agent simulants detection

A simple method was developed for the one-step synthesis of silver nanoparticles (AgNPs), co-stabilized by poly[N-(3-trimethoxysilylpropyl)diethylenetriamine] (PATS) and embedded on a flexible cotton swab (CS). PATS served as the capping agent for AgNPs. The flexible CS-PATS-AgNPs substrate was subjected to surface-enhanced Raman scattering (SERS) for the detection of single and multiple chemical warfare agent simulants (CWAS). SERS peak intensity versus concentration changes were rapidly detected and analyzed using a self-developed smartphone-based app system, which performs quantitative detection of the analyte. The results showed that CWAS (dimethyl methylphosphonate, DMMP; 2-chloroethylethylsulfide, 2-CEES; acetonitrile, ACN can be detected with a high sensitivity, with a limit of detection (LOD) of DMMP, 2-CEES and ACN was 1.0, 46.7 and 57.9 g/L, respectively. The flexible SERS substrate, CS-PATS-AgNPs, in conjunction with a Raman spectrometer and a smartphone-based app system, possesses practical applications primarily owing to the low cost and simple sampling technique.

Synthesis of cuprous oxide/silver (Cu2O/Ag) hybrid as surface-enhanced Raman scattering probe for trace determination of methyl orange.

Recently, there have been publications on preparing hybrid materials between noble metal and semiconductor for applications in surface-enhanced Raman scattering (SERS) substrates to detect some toxic organic dyes. However, the use of cuprous oxide/silver (Cu2O/Ag) to measure the trace amounts of methyl orange (MO) has not been reported. Therefore, in this study, the trace level of MO in water solvent was determined using a SERS substrate based on Cu2O microcubes combined with silver nanoparticles (Ag NPs). Herein, a series of Cu2O/Agx ( 1-5) hybrids with various Ag amounts was synthesized via a solvothermal method followed by a reduction process, and their SERS performance was studied in detail. X-ray diffraction (XRD) and scanning electron microscopy results confirmed that 10 nm Ag NPs were well dispersed on 200-500 nm Cu2O microcubes to form Cu2O/Ag heterojunctions. Using the as-prepared Cu2O and Cu2O/Agx as MO probe, the Cu2O/Ag5 nanocomposite showed the highest SERS activity of all samples with the limit of detection as low to 1 nM and the enhancement factor as high as 4 × 108. The logarithm of the SERS peak intensity at 1389 cm-1 increased linearly with the logarithm of the concentration of MO in the range from 1 nM to 0.1 mM.

Surface-enhanced Raman scattering (SERS) by gold nanoparticle characterizes dermal thickening by collagen in bleomycin-treated skin ex vivo.

Current skin imaging modalities, including optical, electron, and confocal microscopy, mostly require tissue fixations that could damage proteins and biological molecules. Live tissue or cell imaging such as ultrasonography and optical coherent microscope may not adequately measure the dynamic spectroscopical changes. Raman spectroscopy has been adopted for skin imaging in vivo, mostly for skin cancer imaging. However, whether the epidermal and dermal thickening in skin could be measured and distinguished by conventional Ramen spectroscopy or the surface-enhanced Raman scattering (SERS), a rapid and label-free method for noninvasive measurement remains unknown. Human skin sections from patients of atopic dermatitis and keloid, which represent epidermal and dermal thickening, respectively, were measured by conventional Ramen spectroscopy. In mice, skin sections from imiquimod (IMQ)- and bleomycin (BLE)-treated mice, which reflect the epidermal and dermal thickening, respectively, were measured by SERS, that incorporates gold nanoparticles to generate surface plasma and enhance Raman signals. Conventional Ramen spectroscopy failed to consistently show the Raman shift in human samples among the different groups. SERS successfully revealed a prominent peak around 1300cm-1 in the IMQ-treated skin; and two significant peaks around 1100and 1300cm-1 in BLE-treated group. Further quantitative analysis showed 1100cm-1 peak was significantly accentuated in the BLE-treated skin than that in control skin. SERS identified in vitro a similar 1100cm-1 peak in solutions of collagen, the major dermal biological molecules. SERS distinguishes the epidermal or dermal thickening in mouse skin with rapid and label-free measures. A prominent 1100cm-1 SERS peak in the BLE-treated skin may result from collagen. SERS might help precision diagnosis in the future.

Charge transfer effect: a new assignment of the abnormal optical absorption band of gold nanoparticles

As a significant accompanying phenomenon of surface-enhanced Raman scattering (SERS), the addition of foreign molecules to colloidal gold or silver nanoparticles results in a new abnormal optical absorption (AOA) band, which usually appears in the long-wavelength region. The assignment of this AOA band has long been debated as an important issue that is desired to be addressed in the SERS field, which is crucial for a clear understanding of the SERS enhancement mechanism and beneficial to surface plasmonics. In this study, both the calculated and measured optical absorptions of gold nanoparticle monomers and dimers as well as their interactions with adsorbed molecules, showed that the AOA band in the long-wavelength region which was assigned to the characteristic longitudinal localized surface plasmon resonance (LSPR) effect of gold nanoparticle chain aggregates in conventional SERS electromagnetic theory, should be attributed to the charge-transfer resonance absorption from gold nanoparticles to adsorbed molecules. This was further confirmed by the corresponding SERS effects. As the excitation wavelength at 785 nm was resonant with the broad AOA band centered at 750 nm, the SERS peaks of the adsorbed pyridine molecules could be dramatically enhanced due to the charge-transfer resonance effect. In contrast, under an excitation wavelength of 532 nm, the SERS peaks appeared very weak, although the excitation wavelength was resonant with the LSPR absorption band of the individual gold nanoparticles.

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 12min. 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.

Highly catalytic nanoenzyme of covalent organic framework loaded starch- surface-enhanced Raman scattering/absorption bi-mode peptide as biosensor for ultratrace determination of cadmium.

High affinity peptides (PTs) have been used in nanoanalysis, but there are no reports which combine PTs with a liquid crystal (LC) covalent organic framework (COF) supported soluble starch (SS) catalytic amplification system as a biosensor recognition element. In this study, a new, highly sensitive and selective bi-mode molecular biosensor has been developed for the determination of cadmium ion (Cd2+). Specifically, a highly catalytic and stable COF supported SS nanosol catalyst was fabricated such that a nanocatalytic indicator reaction system for HAuCl4-sodium formate was established based on surface-enhanced Raman scattering (SERS). The Au nanoparticles produced exhibited a surface plasmon resonance (SPR) absorption peak at 535 nm and a SERS peak at 1,615 cm-1. Combining the nanocatalytic amplification indicator system with the specific PTs reaction permitted a sensitive and selective SERS/absorption bi-mode platform to be developed for the determination of cadmium in rice. The linear range for SERS determination was 0.025-0.95 nmol/L and the detection limit (DL) was 0.012 nmol/L.

Glucose sandwich assay based on surface-enhanced Raman spectroscopy.

A facile and sensitive glucose sandwich assay using surface-enhanced Raman scattering (SERS) has been developed. Glucose was captured by 3-aminopheyonyl boronic acid (APBA) modified Ag nanoparticles decorated onto a polyamide surface. Then, Ag nanoparticles modified with 3-amino-6-ethynylpicolinonitrile (AEPO) and APBA were used as SERS tags. APBA forms specific cis-diol compounds with glucose molecules avoiding interference by other saccharides and biomolecules in urine enabling its selective detection. As the actual Raman reporter, AEPO exhibited a distinctive SERS peak in the Raman silent region, thus increasing the sensitivity of the glucose detection to 10-11 M. Additionally, the developed SERS assay was reusable, and its applicability in artificial urine samples demonstrated future clinical utility confirming the potential of this innovative technology as a diagnostic tool for glucose sensing.

Radiation mediated oxidation processes for the conversion of 4-aminothiophenol in to 4, 4′-dimercaptoazobenzene

Gold (Au) nanoparticles of average size ∼ 4 nm were in-situ synthesized and simultaneously embedded on SiO2 nanospheres in a single step process by irradiating a solution (HAuCl4, PVP and SiO2 nanospheres) with 6 MeV electrons at a fluence of 1.0 × 1015 e−/cm2. Substrates were prepared by coating silicon wafers with the solution of SiO2 nanospheres embedded with Au nanoparticles and used for recording Surface-enhanced Raman scattering (SERS) spectra of 4-aminothiophenol (4-ATP) and 4-ATP along with NaOH with a Raman spectrometer. The appearance of characteristic SERS peaks of both 4-ATP and 4, 4′-DMAB in Raman spectra confirmed conversion of a fraction of 4-ATP into 4, 4′-DMAB in the presence of Au nanoparticles, and the intensity of SERS peaks increased in the presence of NaOH. In the recorded SERS spectra, the prominent peaks at around 1140 cm−1, 1390 cm−1 and 1438 cm−1 were observed and assigned respectively to β(C–H), ν(NN) and ν(NN) chemical bonds, which are called b2 modes. The results of the present work revealed that in the presence of Au nanoparticles, the OH− radicals produced from NaOH play an important role in the conversion of 4-ATP into 4, 4′-DMAB, and enhancing the intensities of b2 peaks. The Au nanoparticles of size ≤5 nm have inherent nanoscale defects and therefore surface with nanoscale roughness. All these together greatly enhanced electric field which brought out the strong Raman intensity and catalytic oxidation reaction leading to conversion of 4-ATP into 4,4′-DMAB on the Au–SiO2 substrates.

Silver-loaded silica/H-ZSM-5 nanocomposite as a sensitive SERS substrate for the detection of sulfur-containing compounds in diesel fuel

Detection of sulfur-containing compounds in petroleum products is considered an important subject of research for both academia and industry. However, the utilization of surface-enhanced Raman scattering (SERS) spectroscopic methods in that direction has not been well explored. The fluorescence interference and the complexity of the constituents of oil samples represent a challenge to implement spectroanalytical SERS protocols for the characterization of sulfur contaminants. In this study, a combined nanocomposite of silver-loaded silica and H-ZSM-5 (Si/Al2 = 150) material was employed to identify different concentrations of dibenzothiophene (DBT) in commercial diesel fuel. While silver nanoparticles enhanced Raman signals associated with the DBT molecules, zeolitic materials helped in reducing the fluorescence background, and hence being able to approach a low detection level. The DBT in diesel samples could be successfully traced up to 10−7 M of concentration by interpreting the SERS peak at 1611 cm−1 which is associated with the aromatic CC stretching vibration. Full characterization of the nanocomposite substrate before and after its components being combined have been performed using FT-IR, Raman, powder-XRD, BET-N2 sorption, ICP-OES, and SEM techniques. In addition, a meaningful vibrational assignment of the SERS spectrum of DBT has been established on the basis of literature and the density-functional theory (DFT) calculation.

Rapid and quantitative detection of respiratory viruses using surface-enhanced Raman spectroscopy and machine learning

Rapid and sensitive pathogen detection is important for prevention and control of disease. Here, we report a label-free diagnostic platform that combines surface-enhanced Raman scattering (SERS) and machine learning for the rapid and accurate detection of thirteen respiratory virus species including SARS-CoV-2, common human coronaviruses, influenza viruses, and others. Virus detection and measurement have been performed using highly sensitive SiO2 coated silver nanorod array substrates, allowing for detection and identification of their characteristic SERS peaks. Using appropriate spectral processing procedures and machine learning algorithms (MLAs) including support vector machine (SVM), k-nearest neighbor, and random forest, the virus species as well as strains and variants have been differentiated and classified and a differentiation accuracy of >99% has been obtained. Utilizing SVM-based regression, quantitative calibration curves have been constructed to accurately estimate the unknown virus concentrations in buffer and saliva. This study shows that using a combination of SERS, MLA, and regression, it is possible to classify and quantify the virus in saliva, which could aid medical diagnosis and therapeutic intervention.

Study of microplastics as sorbents for rapid detection of multiple antibiotics in water based on SERS technology

Online monitoring of antibiotics in the environment attracts more and more attention. Surface-enhanced Raman scattering (SERS) is a promising technique for the detection of trace amounts of antibiotics in the environment, which is fast, non-invasive and sensitive. To investigate the enrichment of trace amounts of antibiotics in water, polyethylene microplastics (PE MPs) were prepared as sorbents to simply concentrate enrofloxacin, ciprofloxacin hydrochloride monohydrate and triclosan in water, followed by the SERS measurement of antibiotics extract washed from MPs on an AgNPs@Si SERS substrate. Limit of detection of Rhodamine 6G is 2.1 × 10-12 M achieved from the AgNPs@Si SERS, indicating a high enhancement. The detection results show that SERS peaks of the antibiotics could be observed from the spectra of the extracts eluted from MPs, indicating MPs could adsorb and desorb antibiotics from water. Besides, for enrofloxacin and triclosan, the intensity of SERS measured from the MPs extracts are higher than that of directly from the spiked water, demonstrating the proposed method could lower the detectable concentration of hydrophobic antibiotics in water. Moreover, the proposed MPs sorbents combined with SERS method was applied to detect the antibiotics in real river water, with minimal detection of 10-10 M, 10-8 M, and 10-8 M achieved for enrofloxacin, ciprofloxacin hydrochloride monohydrate and triclosan, respectively. The proposed method provides a promising simple, rapid and low reagent consuming means for monitoring antibiotics in water.

Ratiometric pH-responsive SERS strategy for glioma boundary determination

Glioma is one of the most common intracranial malignant tumors worldwide. Since the glioma is invasive and lacks a clear boundary with normal brain tissue, the neurosurgeon can only determine the extent of surgical resection based on empirical experience. Thus, accurately demarcating its boundaries has become a major challenge for surgeons. Owing to the high glycolysis metabolism of glioma cells, the acidification of the extracellular fluid has become an indicator of glioma evolution. Herein, a ratiometric pH-responsive surface-enhanced Raman scattering (SERS) strategy was developed for the rapid identification of glioma boundaries. A sensing chip composed of silver nanoparticles self-assembled film was fabricated, followed by the self-assembly of a pH-responsive SERS reporter, 4-mercaptopyridine (4-MPY). The characteristic SERS peak ratios of 4-MPY change regularly under different pH conditions. The boundary of glioma invasion was determined by measuring the pH of waterdrops infiltrated by interstitial fluids. The technology enables accurate, non-invasive, and rapid determination of local pH, thereby maximizing the removal of tumor tissue while minimizing damage to normal tissue. This technique is more rapid and simple than intraoperative pathological detection and can be possibly used for intraoperative navigation.