Biological imaging of HEK293 cells expressing PLCgamma1 using surface-enhanced Raman microscopy

Surface-enhanced Raman scattering (SERS) imaging has been used for the targeting and imaging of specific cancer markers in live cells. For this purpose, Au/Ag core-shell nanoparticles, conjugated with monoclonal antibodies, were prepared. The procedures to label live cells with those bimetallic nanoprobes have been developed and used for highly sensitive SERS imaging of live cells. In the present study, live HEK293 cells expressing PLCgamma1 have been used as the optical imaging target. Our results demonstrate the potential feasibility of SERS imaging technology for the highly sensitive imaging of cancer biomarkers in live cells.     

Ag Nanoparticle‐Grafted PAN‐Nanohump Array Films with 3D High‐Density Hot Spots as Flexible and Reliable SERS Substrates

A facile fabrication approach of large‐scale flexible films is reported, with one surface side consisting of Ag‐nanoparticle (Ag‐NP) decorated polyacrylonitrile (PAN) nanohump (denoted as Ag‐NPs@PAN‐nanohump) arrays. This is achieved via molding PAN films with ordered nanohump arrays on one side and then sputtering much smaller Ag‐NPs onto each of the PAN‐nanohumps. Surface‐enhanced Raman scattering (SERS) activity of the Ag‐NPs@PAN‐nanohump array films can be improved by curving the flexible PAN film with ordered nanohump arrays during the Ag‐sputtering process to increase the density of the Ag‐NPs on the sidewalls of the PAN‐nanohumps. More 3D hot spots are thus achieved on a large‐scale. The Ag‐NPs@PAN‐nanohump array films show high SERS activity with good Raman signal reproducibility for Rhodamine 6G probe molecules. To trial their practical application, the Ag‐NPs@PAN‐nanohump array films are employed as SERS substrates for trace detection of trinitrotoluene and a congener of polychlorinated biphenyls. A lower detection limit of 10⁻¹²m and 10⁻⁵m can be achieved, respectively. Furthermore, the flexible Ag‐NPs@PAN‐nanohump array films can also be utilized as swabs to probe traces of methyl parathion on the surface of fruits such as apples. The as‐fabricated SERS substrates therefore have promising potential for applications in rapid safety inspection and environmental protection.     

High‐Yield Synthesis of Hollow Octahedral Silver Nanocages with Controllable Pack Density and Their High‐Performance Sers Application

Nanoparticle‐assembled octahedral Ag nanocages with sharp edges have been successfully synthesized through a Cu₂O‐based template‐assisted strategy. In the reaction system, Ag nanoparticles can be self‐assembled on the surface of Cu₂O octahedrons, which is accomplished by the reduction of Ag⁺ by NaBH₄ in the presence of sodium citrate as a capping agent. The hollow octahedral Ag nanocages are obtained after removing the inner Cu₂O cores with acetic acid. According to the scanning electron microscopy (SEM) and transmission electron microscopy characterization, the Ag nanocages are weaved by small nanoparticles, the rough surfaces are bestrewed with pores and sharp edges. It is found that the pack density of Ag nanoparticles strongly affects the surface enhanced Raman scattering (SERS) activities. The as‐prepared 1.05‐Ag cages with optimal pack density have suitable interparticle distance and suitable size of pores, which significantly enhance SERS signals. The SERS signals of rhodamine 6G (R6G) molecules can be detected at an ultralow concentration of 10^?14 m when 1.05‐Ag cages are used as substrates. In addition to sensitivity, 1.05‐Ag cages also exhibit good reproducibility. It is expected that the ultrahigh sensitivity will endow the Ag nanocages to become a promising candidate as high‐performance SERS‐based chemical sensor.     

Multiplexed Molecular Imaging of Fresh Tissue Surfaces Enabled by Convection‐Enhanced Topical Staining with SERS‐Coded Nanoparticles

There is a need for intraoperative imaging technologies to guide breast‐conserving surgeries and to reduce the high rates of re‐excision for patients in which residual tumor is found at the surgical margins during postoperative pathology analyses. Feasibility studies have shown that utilizing topically applied surface‐enhanced Raman scattering (SERS) nanoparticles (NPs), in conjunction with the ratiometric imaging of targeted versus untargeted NPs, enables the rapid visualization of multiple cell‐surface biomarkers of cancer that are overexpressed at the surfaces of freshly excised breast tissues. In order to reliably and rapidly perform multiplexed Raman‐encoded molecular imaging of large numbers of biomarkers (with five or more NP flavors), an enhanced staining method has been developed in which tissue surfaces are cyclically dipped into an NP‐staining solution and subjected to high‐frequency mechanical vibration. This dipping and mechanical vibration (DMV) method promotes the convection of the SERS NPs at fresh tissue surfaces, which accelerates their binding to their respective biomarker targets. By utilizing a custom‐developed device for automated DMV staining, this study demonstrates the ability to simultaneously image four cell‐surface biomarkers of cancer at the surfaces of fresh human breast tissues with a mixture of five flavors of SERS NPs (four targeted and one untargeted control) topically applied for 5 min and imaged at a spatial resolution of 0.5 mm and a raster‐scanned imaging rate of >5 cm² min^?1.     

A Droplet‐Based High‐Throughput SERS Platform on a Droplet‐Guiding‐Track‐Engraved Superhydrophobic Substrate

A novel droplet‐based surface‐enhanced Raman scattering (SERS) sensor for high‐throughput real‐time SERS monitoring is presented. The developed sensors are based on a droplet‐guiding‐track‐engraved superhydrophobic substrate covered with hierarchical SERS‐active Ag dendrites. The droplet‐guiding track with a droplet stopper is designed to manipulate the movement of a droplet on the superhydrophobic substrate. The superhydrophobic Ag dendritic substrates are fabricated through a galvanic displacement reaction and subsequent self‐assembled monolayer coating. The optimal galvanic reaction time to fabricate a SERS‐active Ag dendritic substrate for effective SERS detection is determined, with the optimized substrate exhibiting an enhancement factor of 6.3 × 10⁵. The height of the droplet stopper is optimized to control droplet motion, including moving and stopping. Based on the manipulation of individual droplets, the optimized droplet‐based real‐time SERS sensor shows high resistance to surface contaminants, and droplets containing rhodamine 6G, Nile blue A, and malachite green are successively controlled and detected without spectral interference. This noble droplet‐based SERS sensor reduces sample preparation time to a few seconds and increased detection rate to 0.5 µ L s^?1 through the simple operation mechanism of the sensor. Accordingly, our sensor enables high‐throughput real‐time molecular detection of various target analytes for real‐time chemical and biological monitoring.     

High‐Throughput Block Optical DNA Sequence Identification

Optical techniques for molecular diagnostics or DNA sequencing generally rely on small molecule fluorescent labels, which utilize light with a wavelength of several hundred nanometers for detection. Developing a label‐free optical DNA sequencing technique will require nanoscale focusing of light, a high‐throughput and multiplexed identification method, and a data compression technique to rapidly identify sequences and analyze genomic heterogeneity for big datasets. Such a method should identify characteristic molecular vibrations using optical spectroscopy, especially in the “fingerprinting region” from ≈400–1400 cm^?1. Here, surface‐enhanced Raman spectroscopy is used to demonstrate label‐free identification of DNA nucleobases with multiplexed 3D plasmonic nanofocusing. While nanometer‐scale mode volumes prevent identification of single nucleobases within a DNA sequence, the block optical technique can identify A, T, G, and C content in DNA k‐mers. The content of each nucleotide in a DNA block can be a unique and high‐throughput method for identifying sequences, genes, and other biomarkers as an alternative to single‐letter sequencing. Additionally, coupling two complementary vibrational spectroscopy techniques (infrared and Raman) can improve block characterization. These results pave the way for developing a novel, high‐throughput block optical sequencing method with lossy genomic data compression using k‐mer identification from multiplexed optical data acquisition.     

Split‐GFP: SERS Enhancers in Plasmonic Nanocluster Probes

The assembly of plasmonic metal nanoparticles into hot spot surface‐enhanced Raman scattering (SERS) nanocluster probes is a powerful, yet challenging approach for ultrasensitive biosensing. Scaffolding strategies based on self‐complementary peptides and proteins are of increasing interest for these assemblies, but the electronic and the photonic properties of such hybrid nanoclusters remain difficult to predict and optimize. Here, split‐green fluorescence protein (sGFP) fragments are used as molecular glue and the GFP chromophore is used as a Raman reporter to assemble a variety of gold nanoparticle (AuNP) clusters and explore their plasmonic properties by numerical modeling. It is shown that GFP seeding of plasmonic nanogaps in AuNP/GFP hybrid nanoclusters increases near‐field dipolar couplings between AuNPs and provides SERS enhancement factors above 10⁸. Among the different nanoclusters studied, AuNP/GFP chains allow near‐infrared SERS detection of the GFP chromophore imidazolinone/exocyclic C?C vibrational mode with theoretical enhancement factors of 10⁸–10⁹. For larger AuNP/GFP assemblies, the presence of non‐GFP seeded nanogaps between tightly packed nanoparticles reduces near‐field enhancements at Raman active hot spots, indicating that excessive clustering can decrease SERS amplifications. This study provides rationales to optimize the controlled assembly of hot spot SERS nanoprobes for remote biosensing using Raman reporters that act as molecular glue between plasmonic nanoparticles.     

Composite‐Scattering Plasmonic Nanoprobes for Label‐Free,Quantitative Biomolecular Sensing

Biosensing based on localized surface plasmon resonance (LSPR) relies on concentrating light to a nanometeric spot and leads to a highly enhanced electromagnetic field near the metal nanostructure. Here, a design of plasmonic nanostructures based on rationally structured metal–dielectric combinations is presented, called composite scattering probes (CSPs), to generate an integrated multimodal biosensing platform featuring LSPR and surface‐enhanced Raman spectroscopy (SERS). Specifically, CSP configurations are proposed, which have several prominent resonance peaks enabling higher tunability and sensitivity for self‐referenced multiplexed analyte sensing. Using electron‐beam evaporation and thermal dewetting, large‐area, uniform, and tunable CSPs are fabricated, which are suitable for label‐free LSPR and SERS measurements. The CSP prototypes are used to demonstrate refractive index sensing and molecular analysis using albumin as a model analyte. By using partial least squares on recorded absorption profiles, differentiation of subtle changes in refractive index (as low as 0.001) in the CSP milieu is demonstrated. Additionally, CSPs facilitate complementary untargeted plasmon‐enhanced Raman measurements from the sample's compositional contributors. With further refinement, it is envisioned that the method may lead to a sensitive, versatile, and tunable platform for quantitative concentration determination and molecular fingerprinting, particularly where limited a priori information of the sample is available.     

Tracking Drug‐Induced Epithelial–Mesenchymal Transition in Breast Cancer by a Microfluidic Surface‐Enhanced Raman Spectroscopy Immunoassay

Epithelial–mesenchymal transition (EMT) is a primary mechanism for cancer metastasis. Detecting the activation of EMT can potentially convey signs of metastasis to guide treatment management and improve patient survival. One of the classic signatures of EMT is characterized by dynamic changes in cellular expression levels of E‐cadherin and N‐cadherin, whose soluble active fragments have recently been reported to be biomarkers for cancer diagnosis and prognosis. Herein, a microfluidic immunoassay (termed “SERS immunoassay”) based on sensitive and simultaneous detection of soluble E‐cadherin (sE‐cadherin) and soluble N‐cadherin (sN‐cadherin) for EMT monitoring in patients' plasma is presented. The SERS immunoassay integrates in situ nanomixing and surface‐enhanced Raman scattering readout to enable accurate detection of sE‐cadherin and sN‐cadherin from as low as 10 cells mL^?1. This assay enables tracking of a concurrent decrease in sE‐cadherin and increase in sN‐cadherin in breast cancer cells undergoing drug‐induced mesenchymal transformation. The clinical potential of the SERS immunoassay is further demonstrated by successful detection of sE‐cadherin and sN‐cadherin in metastatic stage IV breast cancer patient plasma samples. The SERS immunoassay can potentially sense the activation of EMT to provide early indications of cancer invasions or metastasis.     

Hierarchical 3D SERS Substrates Fabricated by Integrating Photolithographic Microstructures and Self‐Assembly of Silver Nanoparticles

Most of the surface‐enhanced Raman scattering (SERS) substrates are 2D planar systems, which limits the SERS active area to a single Cartesian plane. Here, we fabricate 3D SERS substrates with the aim to break the traditional 2D SERS active area limitation, and to extend the SERS hotspots into the third dimension along the z‐axis. Our 3D SERS substrates are tailored with increased SERS hotspots in the z‐direction from tens of nanometers to tens of micrometers, increasing the hotspots in the z‐direction by at least an order of magnitude larger than the confocal volume (～1 μm) of most Raman spectrometers. Various hierarchical 3D SERS‐active microstructures are fabricated by combining 3D laser photolithography with Langmuir‐Blodgett nanoparticle assembly. 3D laser photolithography creates microstructured platforms required to extend the SERS‐active area into 3D, and the self‐assembly of Ag nanoparticles ensures homogeneous coating of SERS‐active Ag nanoparticles over the entire microstructured platforms. Large‐area 3D Raman imaging demonstrates that homogeneous signals can be collected throughout the entire 3D SERS substrates. We vary the morphology, height, and inclination angles of the 3D microstructures, where the inclination angle is found to exhibit strong influence on the SERS signals. We also demonstrate a potential application of this hierarchical 3D SERS substrate in information tagging, storage and encryption as SERS micro‐barcodes, where multiple micro‐barcodes can be created within a single set of microstructures.     

Controlled Electrodeposition of Gold on Graphene: Maximization of the Defect‐Enhanced Raman Scattering Response

A reliable method to prepare a surface‐enhanced Raman scattering (SERS) active substrate is developed herein, by electrodeposition of gold nanoparticles (Au NPs) on defect‐engineered, large area chemical vapour deposition graphene (GR). A plasma treatment strategy is used in order to engineer the structural defects on the basal plane of large area single‐layer graphene. This defect‐engineered Au functionalized GR, offers reproducible SERS signals over the large area GR surface. The Raman data, along with X‐ray photoelectron spectroscopy and analysis of the water contact angle are used to rationalize the functionalization of the graphene layer. It is found that Au NPs functionalization of the “defect‐engineered” graphene substrates permits detection of concentrations as low as 10^?16 m for the probe molecule Rhodamine B, which offers an outstanding molecular sensing ability. Interestingly, a Raman signal enhancement of up to ≈10⁸ is achieved. Moreover, it is observed that GR effectively quenches the fluorescence background from the Au NPs and molecules due to the strong resonance energy transfer between Au NPs and GR. The results presented offer significant direction for the design and fabrication of ultra‐sensitive SERS platforms, and also open up possibilities for novel applications of defect engineered graphene in biosensors, catalysis, and optoelectronic devices.     

Highly Surface‐roughened “Flower‐like” Silver Nanoparticles for Extremely Sensitive Substrates of Surface‐enhanced Raman Scattering

Surface‐enhanced Raman scattering (SERS) is a new optical spectroscopic analysis technique with potential for highly sensitive detection of molecules. Recently, many efforts have been made to find SERS substrates with high sensitivity and reproducibility. In this Research News article, we provide a focused review on the synthesis of monodispersed silver particles with a novel, highly roughened, “flower‐like” morphology by reducing silver nitrate with ascorbic acid in aqueous solutions. The nanometer‐scale surface roughness of the particles can provide several hot spots on a single particle, which significantly increases SERS enhancement. The incident polarization‐dependent SERS of individual particles is also studied. Although the different “hot spots” on a single particle can have a strong polarization dependency, the total Raman signals from an individual particle usually have no obvious polarization dependency. Moreover, these flower‐like silver particles can be measured by SERS with high enhancement several times, which indicates the high stability of the hot spots. Hence, the flower‐like silver particles here can serve as highly sensitive and reproducible SERS substrates.     

Antibody‐Mimetic Peptoid Nanosheet for Label‐Free Serum‐Based Diagnosis of Alzheimer's Disease

Alzheimer's disease (AD) is the most common form of dementia characterized by progressive cognitive decline. Current diagnosis of AD is based on symptoms, neuropsychological tests, and neuroimaging, and is usually evident years after the pathological process. Early assessment at the preclinical or prodromal stage is in a great demand since treatment after the onset can hardly stop or reverse the disease progress. However, early diagnosis of AD is challenging due to the lack of reliable noninvasive approaches. Here, an antibody‐mimetic self‐assembling peptoid nanosheet containing surface‐exposed Aβ42‐recognizing loops is constructed, and a label‐free sensor for the detection of AD serum is developed. The loop‐displaying peptoid nanosheet is demonstrated to have high affinity to serum Aβ42, and to be able to identify AD sera with high sensitivity. The dense distribution of molecular recognition loops on the robust peptoid nanosheet scaffold not only mimics the architecture of antibodies, but also reduces the nonspecific binding in detecting multicomponent samples. This antibody‐mimetic 2D material holds great potential toward the blood‐based diagnosis of AD, and meanwhile provides novel insights into the antibody alternative engineering and the universal application in biological and chemical sensors.     

Ultrasensitive Detection of Prostate‐Specific Antigen and Thrombin Based on Gold‐Upconversion Nanoparticle Assembled Pyramids

Self‐assembled nanostructures have been used for the detection of numerous cancer biomarkers. In this study, a gold‐upconversion‐nanoparticle (Au‐UCNP) pyramid based on aptamers is fabricated to simultaneously detect thrombin and prostate‐specific antigen (PSA) using surface‐enhanced Raman scattering (SERS) and fluorescence, respectively. The higher the concentration of thrombin, the lower the intensity of SERS. PSA connected with the PSA aptamer leads to an increase in fluorescence intensity. The limit of detection of thrombin and PSA reaches 57 × 10^?18 and 0.032 × 10^?18m , respectively. In addition, the pyramid also exhibits great target specificity. The results of human serum target detection demonstrate that the Au‐UCNP pyramid is an excellent choice for the quantitative determination of cancer biomarkers, and is feasible for the early diagnosis of cancer.     

Sensitive and Reproducible Immunoassay of Multiple Mycotoxins Using Surface‐Enhanced Raman Scattering Mapping on 3D Plasmonic Nanopillar Arrays

A surface‐enhanced Raman scattering‐based mapping technique is reported for the highly sensitive and reproducible analysis of multiple mycotoxins. Raman images of three mycotoxins, ochratoxin A (OTA), fumonisin B (FUMB), and aflatoxin B1 (AFB1) are obtained by rapidly scanning the surface‐enhanced Raman scattering (SERS) nanotags‐anchoring mycotoxins captured on a nanopillar plasmonic substrate. In this system, the decreased gap distance between nanopillars by their leaning effects as well as the multiple hot spots between SERS nanotags and nanopillars greatly enhances the coupling of local plasmonic fields. This strong enhancement effect makes it possible to perform a highly sensitive detection of multiple mycotoxins. In addition, the high uniformity of the densely packed nanopillar substrate minimizes the spot‐to‐spot fluctuations of the Raman peak intensity in the scanned area when Raman mapping is performed. Consequently, this makes it possible to gain a highly reproducible quantitative analysis of mycotoxins. The limit of detections (LODs) are determined to be 5.09, 5.11, and 6.07 pg mL^?1 for OTA, FUMB, and AFB1, and these values are approximately two orders of magnitude more sensitive than those determined by the enzyme‐linked immunosorbent assays. It is believed that this SERS‐based mapping technique provides a facile tool for the sensitive and reproducible quantification of various biotarget molecules.     

Synergistic Modulation of Surface Interaction to Assemble Metal Nanoparticles into Two‐Dimensional Arrays with Tunable Plasmonic Properties

A simple strategy based on the synergistic modulation of inter‐particle and substrate‐particle interaction is applied for the large‐scale fabrication of two‐dimensional (2D) Au and Ag nanoparticle arrays. The surface charge of the substrate is used to redistribute the double layer electric charges on the particles and to modulate the inter‐particle distance within the 2D nanoparticle arrays on the substrate. The resultant arrays, with a wide range of inter‐particle distances, display tunable plasmonic properties. It can be foreseen that such 2D nanoparticle arrays possess potential applications as multiplexed colorimetric sensors, integrated devices and antennas. Herein, it is demonstrated that these arrays can be employed as wavelength‐selective substrates for multiplexed acquisition of surface‐enhanced Raman scattering (SERS) spectra. This simple one step process provides an attractive and low cost strategy to produce high quality and large area 2D ordered arrays with tunable properties.     

Native MicroRNA Targets Trigger Self‐Assembly of Nanozyme‐Patterned Hollowed Nanocuboids with Optimal Interparticle Gaps for Plasmonic‐Activated Cancer Detection

The modernized use of nucleic acid (NA) sequences to drive nanostructure self‐assembly has given rise to a new class of designed nanomaterials with controllable plasmonic functionalities for broad surface‐enhanced Raman scattering (SERS)‐based bioanalysis applications. Herein, dual usage of microRNAs (miRNAs) as both valuable cancer biomarkers and direct self‐assembly triggers is identified and capitalized upon for custom‐designed plasmonic nanostructures. Through strict NA hybridization of miRNA targets, Au nanospheres selectively self‐assemble onto hollowed Au/Ag alloy nanocuboids with ideal interparticle distances (≈2.3 nm) for optimal SERS signaling. The intrinsic material properties of the self‐assembled nanostructures further elevate miRNA detection performance via nanozyme catalytic SERS signaling cascades. This enables fM‐level miR‐107 detection limit within a clinically‐relevant range without any molecular target amplification. The miRNA‐triggered nanostructure self‐assembly approach is further applied in clinical patient samples, and showcases the potential of miR‐107 as a non‐invasive prostate cancer diagnostic biomarker. The use of miRNA targets to drive nanostructure self‐assembly holds great promise as a practical tool for miRNA detection in disease applications.     

Design of Magnetic‐Plasmonic Nanoparticle Assemblies via Interface Engineering of Plasmonic Shells for Targeted Cancer Cell Imaging and Separation

Magnetic‐plasmonic nanoparticles have received considerable attention for widespread applications. These nanoparticles (NPs) exhibiting surface‐enhanced Raman scattering (SERS) activities are developed due to their potential in bio‐sensing applicable in non‐destructive and sensitive analysis with target‐specific separation. However, it is challenging to synthesize these NPs that simultaneously exhibit low remanence, maximized magnetic content, plasmonic coverage with abundant hotspots, and structural uniformity. Here, a method that involves the conjugation of a magnetic template with gold seeds via chemical binding and seed‐mediated growth is proposed, with the objective of obtaining plasmonic nanostructures with abundant hotspots on a magnetic template. To obtain a clean surface for directly functionalizing ligands and enhancing the Raman intensity, an additional growth step of gold (Au) and/or silver (Ag) atoms is proposed after modifying the Raman molecules on the as‐prepared magnetic‐plasmonic nanoparticles. Importantly, one‐sided silver growth occurred in an environment where gold facets are blocked by Raman molecules; otherwise, the gold growth is layer‐by‐layer. Moreover, simultaneous reduction by gold and silver ions allowed for the formation of a uniform bimetallic layer. The enhancement factor of the nanoparticles with a bimetallic layer is approximately 10⁷. The SERS probes functionalized cyclic peptides are employed for targeted cancer‐cell imaging and separation.     

Improved SERS Intensity from Silver‐Coated Black Silicon by Tuning Surface Plasmons

An economical method of fabricating large‐area (up to a 100‐mm wafer) silver (Ag)‐coated black silicon (BS) substrates is demonstrated by cryogenic deep reactive ion etching with inductively coupled plasma. This method enables a simple adjustment of the spike structure (e.g., height, width, sidewall slope and density of the spikes) on the silicon substrate, which thus offers the advantages of accurate tuning the density and amplitude of the localized surface plasmons after Ag coating. Using this method, an enhancement factor of 10⁹ is achieved for the probe molecule of rhodamine 6G (around two orders of magnitude higher than previous results based on Ag‐coated BS) in surface‐enhanced Raman scattering (SERS) measurement. The presented results pave the way to make Ag‐coated BS substrates as economic and large‐area platforms for diverse surface plasmon related applications (such as SERS and surface plasmon based biosensors).