Raman Probe Research Articles

Feasibility Study of Label-Free Raman Spectroscopy forParathyroid Gland Identification.

We aim to evaluate the feasibility of Raman spectroscopy for parathyroid gland (PG) identification during thyroidectomy. Using a novel side-viewing handheld Raman probe, a total of 324 Raman spectra of four tissue types (i.e., thyroid, lymph node, PG, and lipid) commonly encountered during thyroidectomy were rapidly (< 3 s) acquired from 80 tissue sites (thyroid [n = 10], lymph node [n = 10], PG [n = 40], lipid [n = 20]) of 10 euthanized Wistar rats. Two partial least-squares (PLS)-discriminant analysis (DA) detection models were developed, differentiating the lipid and nonlipid (i.e., thyroid, lymph node, and PG) tissues with an accuracy of 100%, and PG, lymph node, and thyroid could be detected with an accuracy of 98.4%, 93.9%, and 95.4% respectively. This work demonstrates the feasibility of Raman spectroscopy technique for PG identification and protection during thyroidectomy at the molecular level.

Real-time SERS analysis of programmed cell death patterns based on black Phosphorus–gold nanoparticles

Programmed cell death (PCD) plays a crucial role in the biological processes of living organisms and occurs in various forms, such as apoptosis, necroptosis and ferroptosis. However, traditional methods for PCD analysis are time-consuming and complex. In this paper, we propose a facile surface-enhanced Raman spectroscopy (SERS)-based strategy for the real-time analysis of three PCD patterns utilizing black phosphorus–gold nanoparticles (BP–Au NPs) as the ultrasensitive unlabeled Raman probe. BP–Au NPs, which possess excellent biocompatibility, are capable of detecting dye molecules at concentrations as low as [Formula: see text][Formula: see text]M and remain stable for at least one week in different physiological environments. In view of this, BP–Au NPs-based SERS technique can distinguish the tiny differences in the molecular fingerprints of cancer cells undergoing three PCD patterns (apoptosis, necroptosis and ferroptosis) triggered by doxorubicin, shikonin and erastin, respectively. We also have real-time monitoring of the intracellular molecular events during PCD, which spy the fluctuations of some typical SERS bands assigned to protein, DNA and lipid, revealing the unique phenotypic characteristics of each PCD pattern. This strategy provides a detailed and comprehensive analysis of the mechanisms of drug-induced PCD at the Raman level.

Raman Measurements in the Hydroformylation of Decene with Laser Powers of 1 mW in Ex Zones

AbstractA custom‐made Raman setup is improved and adjusted to allow its use in Ex zones by using a 1‐mW laser and no optical focusing elements. The application under consideration is the hydroformylation of decene to undecanal, which must take place under explosion protection. To investigate the dispersive mixture, a separation takes place in a phase separation cell in which the Raman probe performs measurements. It can be shown that, with the use of highly sensitive detectors, even very low excitation powers can lead to a processable signal, and thus the application of Raman measurement technology far below the requirements of explosion protection is possible.

A magnetic SERS-imprinted sensor for the determination of cardiac troponin I based on proteolytic peptide technology

Acute myocardial infarction is a sudden and high-mortality disease that can be accurately diagnosed by measuring the level of cardiac troponin I in the blood. Currently, cTnI commonly used clinical detection methods usually have excellent sensitivity and are suitable for large-scale sample detection analysis. However, most of these methods are operated through multiple steps of fixation, incubation, reaction and separation, and most of them require professionals to operate complex instruments, which greatly limits their applicability in real-time rapid detection. Therefore, a method that requires low professional skills and can perform rapid detection is necessary. We presents an alternative strategy to quantify cTnI in clinical serum samples using a combination of SERS and magnetic molecularly imprinted polymers (MMIP). MMIPs was synthesized under Polyvinyl Pyrrolidone (PVP) conditions without vinyl modification using characteristic peptide as template, MAA and DMAm as functional monomers. The internal Raman probe 4-MBA was connected through Ag-SH bonds in MMIPs to solve the problem that the target object had no Raman characteristic peak. MMIPs with high magnetic and adsorptive properties showed characteristic absorption peaks at the Raman shift of 1582 cm−1 after specific capture of the target templates. The Raman signals of the 4-MBA were reduced due to shielding effects and the detection range of this method was 0.001–100 ng mL−1. The recoveries and relative standard deviations (RSDs) of the spiked experiments were 103.1%–106.3 % and 3.54 %–7.38 %, respectively. In summary, this work had appropriate sensitivity and specificity, and there was no significant difference in detection results after ELISA verification. It provided a flexible and practical analysis method for detecting cTnI based on SERS technology. In addition, the imprinting materials of other disease markers can be prepared by changing the template molecules, which provides a new idea for the detection of other biomarkers.

Development of a 3D Printing-Enabled Cost-Effective Multimodal Raman Probe with High Signal-to-noise Ratio Raman Spectrum Measurements.

Raman spectroscopy has emerged as a pivotal analytical instrument, valued for its nondestructive capabilities and its capacity to provide essential material-specific insights. However, the excessive costs associated with commercially available Raman instruments present a barrier to their accessibility for many academic institutions and broader usage. Herein, we introduce an affordable and accessible approach to constructing a versatile Raman instrument capable of accommodating both spectroscopic and microscopic analyses. Through this multimodal approach that concurrently captures Raman signal and image data, we demonstrate color-based alcohol detection, showcase a high signal-to-noise ratio achieved through meticulous hardware design and signal processing, and present a cost-effective, modular design utilizing 3D printing technology. This system offers adaptability to address diverse research needs and requirements. We systematically detail the fabrication process, including the utilization of a 3D printer to produce necessary components, ultimately resulting in the assembly of a functional Raman probe system. Our experiments and subsequent analyses substantiate the accuracy and reliability of the constructed system. Specifically, we conducted experiments involving three distinct samples: water, ethanol, and methanol using the Raman probe, successfully confirming their unique Raman spectra. Furthermore, our Raman probe accurately identified ethanol concentration by assessing mixed samples with varying water-to-ethanol ratios and demonstrated a coefficient of determination value of 0.9993. This underscores the performance of the constructed Raman probe and positions it as a viable option for characterization, particularly in regions where access to conventional Raman probe may be limited.

Anti-counterfeiting labels with controllable and anti-interference coding information based on core–shell Ag@SiO2 nanomaterials for ink printing

The core–shell structured Ag@SiO2 nanomaterial integrated with surface-enhanced Raman scattering (SERS) spectroscopy promises a critical application in anti-counterfeiting. Security labels have been fabricated based on Ag@SiO2 embedded with Raman reporters. The Ag@SiO2 nanomaterial shows good stability and excellent anti-interference property for anti-counterfeiting. Multiple kinds of Raman probe molecules have been anchored in the Ag@SiO2 labels to provide specific and abundant encoding information. The flexible encoding security information could be controlled conveniently by adjusting probe molecules, which not only enrich the SERS information but also improve the level of anti-counterfeiting. Furthermore, the Ag@SiO2 shown excellent stability in organic solvent, and successfully used in ink for the anti-counterfeiting application.

Assembly and characterization of an optical fiber probe for use in a portable Raman spectrometer

This study describes the assembly and testing of an optical fiber Raman probe for use in a portable spectrometer using a 785 nm laser. The Raman probe was tested on edible vegetable oils (extra virgin olive, canola, sunflower, corn, and soybean), with the distal end of the probe immersed in each sample. The spectra of a dipyrone tablet and a mothball were also obtained. The silica peaks were subtracted by taking a spectrum with the probe tip immersed in water. The spectra of the samples exhibited characteristic peaks from vibrations of unsaturated fatty acids between 900 and 1800 cm−1, as well as characteristic peaks of silica from 600 to 1200 cm−1. The spectra of the samples after subtraction of the silica spectrum (probe in water) showed characteristic peaks without alterations in position or relative intensities compared without the probe. Exploratory analysis by principal component analysis was able to identify peaks of the constituent fatty acids in the oils and associate them with the oil type. A classification model based on partial least squares regression allowed for the classification of oil spectra with an accuracy of 77%. This study demonstrated the feasibility of coupling a probe to a portable Raman spectrometer with the goal of monitoring and controlling industrial processes.

Invited: Mimicking Biological Synapse Activity with LDH-Based Memristor

Memristors, mimicking biological synapse response, are believed to have a great potential for use as an adaptive memory unit in future neuromorphic computing systems. Here we report on fabrication of two-dimensional memristor comprised of layered double hydroxide (LDH) material, covered with graphene layer, serving as a terminal for connecting to electrodes. We demonstrated nonvolatile resistive switching of LDH memristor. Device performance is studied upon replacing graphene semimetal with alternative 2D materials. Comprehensive characterization of the memristor materials was performed with correlated Raman, Electron, and Scanning Probe Microscopy methods.ACKNOWLEDGMENTThis work was supported by the US Department of Defense (Contract #W911QY2220006), 2DCC-MIP NSF cooperative agreement DMR-2039351. Work was performed at JSNN, a member of the Southeastern Nanotechnology Infrastructure Corridor (SENIC) and National Nanotechnology Coordinated Infrastructure (NNCI), which is supported by the NSF (ECCS-1542174)

Phenylboronic Acid-Functionalized Ratiometric Surface-Enhanced Raman Scattering Nanoprobe for Selective Tracking of Hg2+ and CH3Hg+ in Aqueous Media and Living Cells.

The development of appropriate molecular tools to monitor different mercury speciation, especially CH3Hg+, in living organisms is attractive because its persistent accumulation and toxicity are very harmful to human health. Herein, we develop a novel activity-based ratiometric SERS nanoprobe to selectively monitor Hg2+ and CH3Hg+ in aqueous media and in vivo. In this nanoprobe, a new bifunctional Raman probe bis-s-s'-[(s)-(4-(ethylcarbamoyl)phenyl)boronic acid] (b-(s)-EPBA) was synthesized and immobilized on the surface of gold nanoparticles via a Au-S bond, in which the phenylboronic acid group was employed as the recognition unit for Hg2+ and CH3Hg+ based on the Hg-promoted transmetalation reaction. In the presence of Hg2+ and CH3Hg+, a new surface-enhanced Raman scattering (SERS) peak aroused from of C-Hg appeared at 1080 cm-1, and the SERS intensity at 1002 cm-1 belonged to the B-O symmetric stretching decreased simultaneously. The quantitative tracking of Hg2+ and CH3Hg+ was realized based on the SERS intensity ratio (I1080/I1303) with rapid response (∼4 min) and high sensitivity, with detection limits of 10.05 and 25.13 nM, respectively. Moreover, the SERS sensor was used for the quantitative detection of Hg2+ and CH3Hg+ in four actual water samples with a high accuracy and excellent recovery. More importantly, cell imaging experiments showed that AuNPs@b-(s)-EPBA could quantitatively detect intracellular CH3Hg+ and had a good concentration dependence in ratiometric SERS imaging. Meanwhile, we demonstrated that AuNPs@b-(s)-EPBA could detect and image CH3Hg+ in zebrafish. We anticipate that AuNPs@b-(s)-EPBA could potentially be used to study the physiological functions related to CH3Hg+ in the future.

Covalent Organic Framework-Structured Raman Probes for Ultrasensitive In Vivo Bioimaging.

Organic Raman probes, including polymers and small molecules, have attracted great attention in biomedical imaging owing to their excellent biocompatibility. However, the development of organic Raman probes is usually hindered by a mismatch between their absorption spectra and wavelength-fixed excitation, which makes it difficult to achieve resonance excitation necessary to obtain strong Raman signals. Herein, we introduce a covalent organic framework (COF) into the fine absorption spectrum regulation of organic Raman probes, resulting in their significant Raman signal enhancement. In representative examples, a polymer poly(diketopyrrolopyrrole-p-phenylenediamine) (DPP-PD) and a small molecule azobenzene are transformed into the corresponding COF-structured Raman probes. Their absorption peaks show an accurate match of less than 5 nm with the NIR excitation. As such, the COF-structured Raman probes acquire highly sensitive bioimaging capabilities compared to their precursors with negligible signals. By further mechanism studies, we discover that the crystallinity and size of COFs directly affect the π-conjugation degree of Raman probes, thus changing their bandgaps and absorption spectra. Our study offers a universal and flexible method for improving the signal performance of organic Raman probes without changing their structural units, making it more convenient to obtain the highly sensitive organic Raman probes for in vivo bioimaging.

Microlens Hollow-Core Fiber Probes for Operando Raman Spectroscopy.

We introduce a flexible microscale all-fiber-optic Raman probe which can be embedded into devices to enable operando in situ spectroscopy. The facile-constructed probe is composed of a nested antiresonant nodeless hollow-core fiber combined with an integrated high refractive index barium titanate microlens. Pump laser 785 nm excitation and near-infrared collection are independently characterized, demonstrating an excitation spot of full-width-half-maximum 1.1 μm. Since this is much smaller than the effective collection area, it has the greatest influence on the collected Raman scattering. Our characterization scheme provides a suitable protocol for testing the efficacy of these fiber probes using various combinations of fiber types and microspheres. Raman measurements on a surface-enhanced Raman spectroscopy sample and a copper battery electrode demonstrate the viability of the fiber probe as an alternative to bulk optic Raman microscopes, giving comparable collection to a 10 objective, thus paving the way for operando Raman studies in applications such as lithium battery monitoring.

Combined Electrochemical Deposition and Photo-Reduction to Fabricate SERS-Active Silver Substrates: Characterization and Application for Malachite Green Detection in Aquaculture Water.

This research introduces a novel approach using silver (Ag) nanostructures generated through electrochemical deposition and photo-reduction of Ag on fluorine-doped tin oxide glass substrates (denoted as X-Ag-AgyFTO, where 'X' and 'y' represent the type of light source and number of deposited cycles, respectively) for surface-enhanced Raman spectroscopy (SERS). This study used malachite green (MG) as a Raman probe to evaluate the enhancement factors (EFs) in SERS-active substrates under varied fabrication conditions. For the substrates produced via electrochemical deposition, we determined a Raman EF of 6.15 × 104 for the Ag2FTO substrate. In photo-reduction, the impact of reductant concentration, light source, and light exposure duration were examined on X-Ag nanoparticle formation to achieve superior Raman EFs. Under optimal conditions (9.0 mM sodium citrate, 460 nm blue-LED at 10 W for 90 min), the combination of blue-LED-reduced Ag (B-Ag) and an Ag2FTO substrate (denoted as B-Ag-Ag2FTO) exhibited the best Raman EF of 2.79 × 105. This substrate enabled MG detection within a linear range of 0.1 to 1.0 µM (R2 = 0.98) and a detection limit of 0.02 µM. Additionally, the spiked recoveries in aquaculture water samples were between 90.0% and 110.0%, with relative standard deviations between 3.9% and 6.3%, indicating the substrate's potential for fungicide detection in aquaculture.

Facile modification method for the controlled synthesis of dumbbell-like gold nanoparticles (AuNDBs) for application in detecting glucose using the SERS method

Dumbbell-like nanoparticles, previously known for their excellent light absorption and dispersion, have diverse applications, especially in SERS. Herein, we have explored a fast and novel controlled synthesis approach to achieve a high formation of AuNDBs. Precise control of precursor amounts and seed addition order halted seed growth at the AuNDB formation stage, preventing further elongation into rod-like structures. The AuNDBs were functionalized with 4-mercaptobenzoic acid (4-MBA) to create SERS nanosubstrates and deposited on the glass slide through 3-mercaptopropyl (dimethoxy)methylsilane by the spin-coating technique. The detection of d-glucose was achieved by measuring the signal decrease of the 4-MBA Raman probe molecules on the nanosubstrates at different trace concentrations of the analyte. These novel SERS substrates have the potential to provide an enhancement factor (EF) of 2.41 × 107 and a limit of detection (LOD) as low as 1.17 nmol/L. The electromagnetic field distribution of AuNDBs with many arrangements surrounded by air and d-glucose solutions was also determined by using the finite-difference time-domain technique (FDTD). The FDTD results confirmed that d-glucose on the SERS substrates caused the local electromagnetic field to degrade, resulting in the degree of the Raman signal. Our findings suggest that dumbbell-shaped SERS substrates can effectively detect very low levels of glucose, making them ideal for designing novel anisotropic nanoparticle-functionalized surfaces with ease.

Gold-Hydrogel Nanocomposites for High-Resolution Laser-Based 3D Printing of Scaffolds with SERS-Sensing Properties.

Although visible light-based stereolithography (SLA) represents an affordable technology for the rapid prototyping of 3D scaffolds for in vitro support of cells, its potential could be limited by the lack of functional photocurable biomaterials that can be SLA-structured at micrometric resolution. Even if innovative photocomposites showing biomimetic, bioactive, or biosensing properties have been engineered by loading inorganic particles into photopolymer matrices, main examples rely on UV-assisted extrusion-based low-resolution processes. Here, SLA-printable composites were obtained by mixing a polyethylene glycol diacrylate (PEGDA) hydrogel with multibranched gold nanoparticles (NPs). NPs were engineered to copolymerize with the PEGDA matrix by implementing a functionalization protocol involving covalent grafting of allylamine molecules that have C═C pendant moieties. The formulations of gold nanocomposites were tailored to achieve high-resolution fast prototyping of composite scaffolds via visible light-based SLA. Furthermore, it was demonstrated that, after mixing with a polymer and after laser structuring, gold NPs still retained their unique plasmonic properties and could be exploited for optical detection of analytes through surface-enhanced Raman spectroscopy (SERS). As a proof of concept, SERS-sensing performances of 3D printed plasmonic scaffolds were successfully demonstrated with a Raman probe molecule (e.g., 4-mercaptobenzoic acid) from the perspective of future extensions to real-time sensing of cell-specific markers released within cultures. Finally, biocompatibility tests preliminarily demonstrated that embedded NPs also played a key role by inducing physiological cell-cytoskeleton rearrangements, further confirming the potentialities of such hybrid nanocomposites as groundbreaking materials in laser-based bioprinting.

Label-Free Fiber-Optic Raman Spectroscopy for Intravascular Coronary Atherosclerosis and Plaque Detection.

The rupture of atherosclerotic plaques remains one of the leading causes of morbidity and mortality worldwide. The plaques have certain pathological characteristics including a fibrous cap, inflammation, and extensive lipid deposition in a lipid core. Various invasive and noninvasive imaging techniques can interrogate structural aspects of atheroma; however, the composition of the lipid core in coronary atherosclerosis and plaques cannot be accurately detected. Fiber-optic Raman spectroscopy has the capability of in vivo rapid and accurate biomarker detection as an emerging omics technology. Previous studies demonstrated that an intravascular Raman spectroscopic technique may assess and manage the therapeutic and medication strategies intraoperatively. The Raman spectral information identified plaque depositions consisting of lipids, triglycerides, and cholesterol esters as the major components by comparing normal region and early plaque formation region with histology. By focusing on the composition of plaques, we could identify the subgroups of plaques accurately and rapidly by Raman spectroscopy. Collectively, this fiber-optic Raman spectroscopy opens up new opportunities for coronary atherosclerosis and plaque detection, which would assist optimal surgical strategy and instant postoperative decision-making. In this paper, we will review the advancement of label-free fiber-optic Raman probe spectroscopy and its applications of coronary atherosclerosis and atherosclerotic plaque detection.

Validating the utility of heavy water (Deuterium Oxide) as a potential Raman spectroscopic probe for identification of antibiotic resistance

The impact of microbial infections is increasing over time, and it is one of the major reasons for death in both developed and developing countries. colistin is considered as the antibiotic of last choice for infections brought by major multidrug-resistant (MDR), gram-negative bacteria such as Enterobacter species, Acinetobacter species, and Pseudomonas aeruginosa. Existing approaches to diagnose these resistant species are relatively slow and take up to 2 to 3 days. In this work, we propose a novel interdisciplinary method based on Raman spectroscopy and heavy water to identify colistin-resistant microbes. Our hypothesis is based on the fact that resistant bacteria will be metabolically active in the culture medium containing antibiotics and heavy water, and these bacteria will take up deuterium instead of hydrogen to newly synthesized lipids and proteins. This effect will generate a ‘C − D’ bond-specific Raman spectral marker. Successful identification of this band in the spectral profile can confirm the presence of colistin-resistant bacteria. We have validated the efficacy of this approach in identifying colistin-resistant bacteria spiked in artificial urine and have compared sensitivity at different bacterial concentrations. Overall findings suggest that heavy water can potentially serve as a suitable Raman probe for identifying metabolically active colistin-resistant bacteria via urine under clinically implementable time and can be used in clinical settings after validation.

Surface-enhanced raman scattering properties of Au@TiO2 plasma exciton array structures

Surface Enhanced Raman Scattering (SERS) is an ultrasensitive spectroscopic analysis technique widely used for molecular detection. SERS is characterized by rapid analysis, trace detection, ultra-high sensitivity, non-destructive, etc., and can analyze the concentration, composition, and structure of the detected substances. It has a significant potential for application in many fields, such as biomedicine, trace detection, food safety, and environmental protection. However, fabrication of homogeneous, stable, and ultrasensitive SERS substrates remains a challenge for the practical application of SERS technology. Precious metals and semiconductors have been proven attractive and versatile for SERS detection. In this work, three-dimensional Au@TiO2 substrates were prepared using a facile strategy, and two different types of Raman probe molecules were used to demonstrate the versatility of the design. Using 4-MBA as the probe molecule, the enhancement factor of the TiO2 substrate could reach 1.52×104, and the detection limit was as low as 10-16 M. The results indicate that the Au@TiO2 substrate is simple to prepare, inexpensive, and serves as an excellent SERS substrate structure. 4-MBA and R6G dual probe molecules demonstrate the universality of the Au@TiO2 nanocavity array structure. In the meantime, the results of the simulations match the experimental results. It also demonstrates that the three-dimensional Au@TiO2 has the potential to be an excellent SERS substrate.

Expanding the horizons: Raman probe development and spectra preprocessing evaluation for recognition of large hydroxyapatite‐based samples

AbstractThis paper addresses the challenge posed by the size of certain objects that do not conform to the constraints of microscope‐coupled Raman spectrometers, making sample collection impossible due to their inherent value or nature. Specifically, materials like hydroxyapatite‐based substances used in artistic and ornamental carvings, such as bone or ivory, fall within this problematic category. The focus of this study is the enhancement of analytical capabilities in the context of large objects using a Raman microscope system. The study details the innovation involving a remote probe integrated with fiber optics, elaborating on the design and performance aspects, and emphasizing the appropriateness of chosen components in the analysis of ivory artifacts belonging to private collectors. In order to assess the robustness of our discriminative approaches, an archaeological bone and the exposed dentine in a human tooth were also evaluated and compared. Results showed that using an 805‐nm longpass dichroic mirror successfully directed the near‐infrared laser onto the samples and significantly suppressed the Rayleigh scattering contribution to the spectrum. Regarding the preprocessing methods to spectra evaluation essayed, the most promising approach was the use of principal component analysis for dimension reduction followed by k‐means cluster analysis. By leveraging the complementary strengths of PCA and k‐means clustering, the robustness and interpretability of clustering analyses are enhanced.

Novel SERS Probe Based on Ag Nanobean/Cu Foam for Deep-Sea Extreme Environment Biomolecule Detection.

Here, we report on progress made in coupling advances in surface-enhanced Raman scattering (SERS) techniques with a deep-ocean deployable Raman spectrometer. Our SERS capability is provided by development of a Cu foam-loaded silver-nanobean (Ag/Cu foam) which we have successfully coupled to the tip of a Raman probe head capable of insertion into deep-sea sediments and associated fluids. Our purpose is to expand the range of molecular species which can be detected in deep-sea biogeochemical environments, and our initial targets are a series of amino acids reportedly found in pore waters of seep locations. Our work has progressed to the point of a full dock-based end-to-end test of the essential ship tether-ROV-deep-sea Raman system. We show here the initial results from this test as the essential requirement before at sea full ocean depth deployment. We describe in detail the procedures for preparing the Ag/Cu foam bean and demonstrate in our end-to-end test that this, when coupled to the spectrometer probe tip, yields a SERS signal enhancement of 1.2 × 106 for test molecules and detection of amino acids at 10-6 M levels consistent with reported levels of natural occurrence. Each nanobean unit is for single-use sensing since invasion of the sample fluid into the Ag/Cu foam matrix is not reversible. We describe techniques for bean rotation/replacement at depth to allow for multiple analyses at several locations during each ROV dive.

Flexible SERS substrates with gradient porous Cu structure dealloying from the thermal diffusion couples of Al/Cu stacking foils

Accurate and sensitive surface-enhanced Raman scattering (SERS) substrates are urgently demanded for the various analytic techniques. The SERS substrates with multimodal and gradient porous structure, utilized for the Rhodamine 6G (R6G) detection, have been designed and successfully fabricated from the thermal diffusion couples of Al and Cu foils via chemical dealloying. The structural inheritance of the initial microstructure of thermal diffusion couple precursors governs the formation of the final layer-by-layer gradient porous Cu with different characteristic pore sizes and morphology. An outmost surface layer of disordered lipstick-like porous structure on the SERS substrates enhances SERS effects as has been expected. The SERS activity of R6G Raman probes on gradient porous Cu is evaluated as a SERS enhancement factor of 6.63 × 1012 and limit of detection of 2.10 × 10−17 mol/L, higher than other porous SERS substrates. Meanwhile, the present SERS substrates are flexible and exhibit good bending properties due to the unreacted Cu layer as a supporting skeleton and other gradient porous Cu layers. The superior SERS performances are considered to result from the synergetic effects of the surface scattering of porous Cu rods and enhancements of the high electromagnetic fields between the small gaps between the nanopores.