Advancing COVID-19 Diagnosis: Enhancement in SERS-PCR with 30-nm Au Nanoparticle-Internalized Nanodimpled Substrates.

Real-time polymerase chain reaction (RT-PCR) with fluorescence detection is the gold standard for diagnosing coronavirus disease 2019 (COVID-19) However, the fluorescence detection in RT-PCR requires multiple amplification steps when the initial deoxyribonucleic acid (DNA) concentration is low. Therefore, this study has developed a highly sensitive surface-enhanced Raman scattering-based PCR (SERS-PCR) assay platform using the gold nanoparticle (AuNP)-internalized gold nanodimpled substrate (AuNDS) plasmonic platform. By comparing different sizes of AuNPs, it is observed that using 30 nm AuNPs improves the detection limit by approximately ten times compared to 70 nm AuNPs. Finite-difference time-domain (FDTD) simulations show that multiple hotspots are formed between AuNPs and the cavity surface and between AuNPs when 30 nm AuNPs are internalized in the cavity, generating a strong electric field. With this 30 nm AuNPs-AuNDS SERS platform, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ribonucleic acid (RNA)-dependent RNA polymerase (RdRp) can be detected in only six amplification cycles, significantly improving over the 25 cycles required for RT-PCR. These findings pave the way for an amplification-free molecular diagnostic system based on SERS.

Interior Hotspot Engineering in Ag-Au Bimetallic Nanocomposites by In Situ Galvanic Replacement Reaction for Rapid and Sensitive Surface-Enhanced Raman Spectroscopy Detection.

Engineering of interior hotspots provides a paradigm shift from traditional surface-enhanced Raman spectroscopy (SERS), in which the detection sensitivity depends on the positioning of adsorbed molecules. In the present work, we developed an Ag-Au bimetallic nanocomposite (SGBMNC) SERS platform with interior hotspots through facile chemical syntheses. Ag nanoparticles replaced by Au via the galvanic replacement reaction (GRR) provided hotspot regions inside the SGBMNC that remarkably enhanced the plasmonic activity compared to the conventional SERS platforms without the internal hotspots. The diffusion of analytes into the proposed interior hotspots during the GRR process enabled sensitive detections within 10 s. The SERS behaviors of the SGBMNC platform were investigated using methylene blue (MB) as a Raman probe dye. A quantitative study revealed excellent detection performance, with a limit of detection (LOD) of 42 pM for MB dye and a highly linear correlation between peak intensity and concentration (R2 ≥ 0.91). The SGBMNC platform also enabled the detection of toxic benzyl butyl phthalate with a sufficient LOD of 0.09 ppb (i.e., 280 pM). Therefore, we believe that the proposed methodology can be used for SERS assays of hazardous materials in practical fields.

Hydrogel-Assisted 3D Volumetric Hotspot for Sensitive Detection by Surface-Enhanced Raman Spectroscopy.

Effective hotspot engineering with facile and cost-effective fabrication procedures is critical for the practical application of surface-enhanced Raman spectroscopy (SERS). We propose a SERS substrate composed of a metal film over polyimide nanopillars (MFPNs) with three-dimensional (3D) volumetric hotspots for this purpose. The 3D MFPNs were fabricated through a two-step process of maskless plasma etching and hydrogel encapsulation. The probe molecules dispersed in solution were highly concentrated in the 3D hydrogel networks, which provided a further enhancement of the SERS signals. SERS performance parameters such as the SERS enhancement factor, limit-of-detection, and signal reproducibility were investigated with Cyanine5 (Cy5) acid Raman dye solutions and were compared with those of hydrogel-free MFPNs with two-dimensional hotspots. The hydrogel-coated MFPNs enabled the reliable detection of Cy5 acid, even when the Cy5 concentration was as low as 100 pM. We believe that the 3D volumetric hotspots created by introducing a hydrogel layer onto plasmonic nanostructures demonstrate excellent potential for the sensitive and reproducible detection of toxic and hazardous molecules.

A cyclodextrin-decorated plasmonic gold nanosatellite substrate for selective detection of bipyridylium pesticides.

A cyclodextrin-decorated gold nanosatellite (AuNSL) substrate was developed as a surface-enhanced Raman scattering sensor for the selective sensing of bipyridylium pesticides such as paraquat (PQ), diquat (DQ), and difenzoquat (DIF). The AuNSL structure was fabricated via vacuum deposition of gold nanoparticles (AuNPs) on a gold nanopillar substrate, and a large density of hot-spots was formed for Raman signal enhancement. Thiolated ß-cyclodextrin (SH-CD) was surface-modified on the AuNSL as a chemical receptor. The detection limit of PQ, DQ, and DIF on the SH-CD-coated AuNSL (CD-AuNSL) was 0.05 ppm for each, and showed linear correlation in a concentration range of 10 ppm-0.05 ppm. Then, selective bipyridylium pesticide detection was performed by comparing the Raman intensity of each pesticide with and without the washing step. After the washing step, 90% of the PQ, DQ, and DIF Raman signals were maintained on the CD-AuNSL substrate with a uniform selectivity in a mapping area of 200 µm × 200 µm. Furthermore, selective pesticide detection was performed using a ground-apple solution without pretreatment. Raman signals were clearly observed after the washing step and they showed a limit of detection down to a concentration of 0.05 ppm for each pesticide. Principal component analysis (PCA) of the binary and ternary mixtures of PQ, DQ, and DIF showed that each component could be easily identified via the typical Raman fingerprint analysis. The developed CD-AuNSL is expected to be applied for various chemical sensors, especially for pyridine-containing toxic substances in the environment and metabolite biomarkers in biofluids.

Microfluidic Designing Microgels Containing Highly Concentrated Gold Nanoparticles for SERS Analysis of Complex Fluids.

Surface-enhanced Raman scattering (SERS) is one of the most promising methods to detect small molecules for point-of-care analysis as it is rapid, nondestructive, label-free, and applicable for aqueous samples. Here, microgels containing highly concentrated yet evenly dispersed gold nanoparticles are designed to provide SERS substrates that simultaneously achieve contamination-free metal surfaces and high signal enhancement and reproducibility. With capillary microfluidic devices, water-in-oil-in-water (W/O/W) double-emulsion drops are prepared to contain gold nanoparticles and hydrogel precursors in innermost drop. Under hypertonic condition, water is selectively pumped out from the innermost drops. Therefore, gold nanoparticles are gently concentrated without forming aggregates, which are then captured by hydrogel matrix. The resulting microgels have a concentration of gold nanoparticles ≈30 times higher and show Raman intensity two orders of magnitude higher than those with no enrichment. In addition, even distribution of gold nanoparticles results in uniform Raman intensity, providing high signal reproducibility. Moreover, as the matrix of the microgel serves as a molecular filter, large adhesive proteins are rejected, which enables the direct detection of small molecules dissolved in the protein solution. It is believed that this advanced SERS platform is useful for in situ detection of toxic molecules in complex mixtures such as biological fluids, foods, and cosmetics.

3D nanoporous plasmonic chips for extremely sensitive NO2 detection.

The detection of toxic gas molecules using the surface-enhanced Raman spectroscopy (SERS) technique is very challenging due to the low affinity of gas molecules. Here, we report extremely sensitive SERS-based NO2 gas sensors based on 3D nanoporous Au nanostructures with a high affinity for NO2 gas molecules and high density of hotspots.

SERS-Active-Charged Microgels for Size- and Charge-Selective Molecular Analysis of Complex Biological Samples.

Surface-enhanced Raman scattering (SERS) provides a dramatic increase of Raman intensity for molecules adsorbed on nanogap-rich metal nanostructures, serving as a promising tool for molecular analysis. However, surface contamination caused by protein adsorption and low surface concentration of small target molecules reduce the sensitivity, which severely restricts the use of SERS in many applications. Here, charged microgels containing agglomerates of gold nanoparticles (Au NPs) are designed using droplet-based microfluidics to provide a reliable SERS substrate with molecular selectivity and high sensitivity. The limiting mesh size of hydrogel enables the autonomous exclusion of large proteins and the charged matrix concentrates oppositely charged small molecules through electrostatic attraction. As nanogaps among Au NPs in the agglomerates enhance Raman intensity, Raman spectrum of the adsorbed molecules is selectively measured with high sensitivity in the absence of interruption from adhesive proteins. Therefore, the SERS-active-charged microgels can be used for direct analysis of pristine biological samples without the pretreatment steps of separation and concentration, which are commonly a prerequisite for Raman analysis. For the purpose of demonstration, a direct detection of fipronil sulfone with partial negative charges, a metabolite of toxic insecticide, dissolved in eggs using the positively charged microgels without any pretreatment of the samples, is shown.

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.

Highly sensitive and on-site NO2 SERS sensors operated under ambient conditions.

Nitrogen dioxide (NO2) produced by hydrocarbon combustion has a significant adverse impact on human health and the environment. In the current work, we developed a high-performance (limit of detection: 0.1 ppm), on-site, rapid NO2 gas sensor that could be operated under ambient conditions by combining a highly sensitive 3D porous SERS substrate and a handheld Raman spectrometer.

Uniform Microgels Containing Agglomerates of Silver Nanocubes for Molecular Size-Selectivity and High SERS Activity.

Surface-enhanced Raman scattering (SERS) is a promising technique for molecular analysis as the molecular fingerprints (Raman spectra) are amplified to detectable levels compared with common spectroscopy. Metal nanostructures localize electromagnetic field on their surfaces, which can lead to dramatic increase of Raman intensity of molecules adsorbed. However, the metal surfaces are prone to contamination, thereby requiring pretreatment of samples to remove adhesive molecules. To avoid the pretreatment and potentially achieve point-of-care (POC) analysis, we have developed SERS-active microgels using the droplet-microfluidic system. As the microgels are composed of water-swollen network with consistent mesh size, they selectively allow diffusion of molecules smaller than the mesh, thereby excluding large adhesives. To render the microgels highly SERS-active, we destabilize silver nanocubes to form agglomerates, which are embedded in the matrix of microgels. The nanogaps in the agglomerates provide high sensitivity in Raman measurement and size-selective permeability of the microgel matrix obviates the pretreatment of samples. To validate the functions, we demonstrate the direct detection of Aspirin dissolved in whole blood without any pretreatment.

Cancer screening through surface-enhanced Raman spectroscopy fingerprinting analysis of urinary metabolites using surface-carbonized silver nanowires on a filter membrane.

BACKGROUND: Label-free surface-enhanced Raman spectroscopy (SERS)-based metabolic profiling has great potential for early cancer diagnosis, but further advancements in analytical methods and clinical evidence studies are required for clinical applications. To improve the cancer diagnostic accuracy of label-free SERS spectral analysis of complex biological fluids, it is necessary to obtain specifically enhanced SERS signals of cancer-related metabolites present at low concentrations. RESULTS: This study presents a novel 3D SERS sensor, comprising a surface-carbonized silver nanowire (AgNW)-stacked filter membrane, alongside an optimized urine/methanol/chloroform extraction technique, which specifically changes the molecular adsorption and orientation of aromatic metabolites onto SERS substrates. By analyzing the pretreated urine samples on the surface-carbonized AgNW 3D SERS sensor, distinct and highly enhanced SERS peaks derived from semi-polar aromatic metabolites were observed for pancreatic cancer and prostate cancer samples compared with normal controls. Urine metabolite analysis using SERS fingerprinting successfully differentiated pancreatic cancer and prostate cancer groups from normal control group: normal control (n = 56), pancreatic cancer (n = 40), and prostate cancer (n = 39). SIGNIFICANCE AND NOVELTY: We confirmed the clinical feasibility of performing fingerprint analysis of urinary metabolites based on the surface-carbonized AgNW 3D SERS sensor and methanol/chloroform extraction for noninvasive cancer screening. This technology holds potential for large-scale screening owing to its high accuracy, and cost effective, simple and rapid detection method.

Metal-enhanced fluorescence biosensor integrated in capillary flow-driven microfluidic cartridge for highly sensitive immunoassays.

Point-of-care testing (POCT) for low-concentration protein biomarkers remains challenging due to limitations in biosensor sensitivity and platform integration. This study addresses this gap by presenting a novel approach that integrates a metal-enhanced fluorescence (MEF) biosensor within a capillary flow-driven microfluidic cartridge (CFMC) for the ultrasensitive detection of the Parkinson's disease biomarker, aminoacyl-tRNA synthetase complex interacting multi-functional protein 2 (AIMP-2). Crucial point to this approach is the orientation-controlled immobilization of capture antibody on a nanodimple-structured MEF substrate within the CFMC. This strategy significantly enhances fluorescence signals without quenching, enabling accurate quantification of low-concentration AIMP-2 using a simple digital fluorescence microscope with a light-emitting diode excitation source and a digital camera. The resulting platform exhibits exceptional sensitivity, achieving a limit of detection in the pg/mL range for AIMP-2 in human serum. Additionally, the CFMC design incorporates a capillary-driven passive sample transport mechanism, eliminating the need for external pumps and further simplifying the detection process. Overall, this work demonstrates the successful integration of MEF biosensing with capillary microfluidics for point-of-care applications.

Fabrication of three-dimensional nanostructured titania materials by prism holographic lithography and the sol-gel reaction.

We present a simple, easy method for fabricating high-quality titania inverted replicas of 3D holographically featured structures. A combination of single-prism holographic lithography and sol-gel chemistry was used to prepare 3D titania inverse structures with flat and completely open surfaces without the use of additional postprocessing steps, such as reactive ion etching, ion-beam milling, and/or polishing steps. A hydrophobic, stable liquid titania precursor facilitated the complete infiltration of the precursor into the hydrophobic 3D SU-8 polymer template, which produced very uniform high-quality titania inverse structures. Although the degree of film shrinkage during the calcination process was large (∼34%), the optical strength of the 3D titania inverse photonic crystals doubled because of the high-refractive-index contrast. Compared to titania inverse opal structures, the filling fraction (∼27%) of titania materials has been doubled. This is the first work to fabricate titania inverse photonic crystals with a high filling fraction by utilizing prism holographic lithography and the sol-gel chemistry reaction of a stable titania precursor. The X-ray diffraction patterns indicated the presence of a crystalline anatase or rutile phase depending on the calcination temperature.

Highly efficient hybrid thin-film solar cells using a solution-processed hole-blocking layer.

We report the origin of the improvement of the power conversion efficiency (PCE) of hybrid thin-film solar cells when a soluble C(60) derivative, [6,6]-phenyl-C(61)-butyric acid methyl ester (PCBM), is introduced as a hole-blocking layer. The PCBM layer could establish better interfacial contact by decreasing the reverse dark-saturation current density, resulting in a decrease in the probability of carrier recombination. The PCE of this optimized device reached a maximum value of 8.34% and is the highest yet reported for hybrid thin-film solar cells.

Micromolding-Assisted Production of SERS-Active Microcylinders for Size- and Charge-Selective Molecular Detection.

Surface-enhanced Raman scattering (SERS) is an effective technique for amplifying the Raman signal of molecules by using metal nanostructures. However, these metal surfaces are susceptible to contamination by undesirable adhesives in complex mixtures, typically necessitating a time-consuming and costly sample pretreatment. In order to circumvent this, metal nanoparticles have been uniformly embedded within microgels by using microfluidics. In this work, we introduce a simple, scalable micromolding method for creating SERS-active cylindrical microgels designed to eliminate the need for pretreatment. These microcylinders are created through the simultaneous photoreduction and photo-cross-linking of precursor solutions. These solutions are optimized for consistent, high-intensity Raman signals as well as molecular size and charge selectivity. A sequential micromolding method is employed to design dual-compartment microcylinders, offering additional functionalities such as optical encoding, magnetoresponsiveness, and dual-charge selectivity. These SERS-active microcylinders provide robust Raman signals of small molecules, even in the presence of adhesive proteins, without compromising sensitivity. To demonstrate this capability, we directly detect pyocyanin in saliva and tartrazine in whole milk without any need for sample pretreatment.

In-situ fabrication of 3D interior hotspots templated with a protein@Au core-shell structure for label-free and on-site SERS detection of viral diseases.

Nanoscale plasmonic hotspots play a critical role in the enhancement of molecular Raman signals, enabling the sensitive and reliable trace analysis of biomedical molecules via surface-enhanced Raman spectroscopy (SERS). However, effective and label-free SERS diagnoses in practical fields remain challenging because of clinical samples' random adsorption and size mismatch with the nanoscale hotspots. Herein, we suggest a novel SERS strategy for interior hotspots templated with protein@Au core-shell nanostructures prepared via electrochemical one-pot Au deposition. The cytochrome c and lysates of SARS-CoV-2 (SLs) embedded in the interior hotspots were successfully functionalized to confine the electric fields and generate their optical fingerprint signals, respectively. Highly linear quantitative sensitivity was observed with the limit-of-detection value of 10-1 PFU/mL. The feasibility of detecting the targets in a bodily fluidic environment was also confirmed using the proposed templates with SLs in human saliva and nasopharyngeal swabs. These interior hotspots templated with the target analytes are highly desirable for early and on-site SERS diagnoses of infectious diseases without any labeling processes.

Dual-Enhanced Plasmonic Biosensing for Point-of-Care Sepsis Detection.

Rapid, sensitive, simultaneous quantification of multiple biomarkers in point-of-care (POC) settings could improve the diagnosis and management of sepsis, a common, potentially life-threatening condition. Compared to high-end commercial analytical systems, POC systems are often limited by low sensitivity, limited multiplexing capability, or low throughput. Here, we report an ultrasensitive, multiplexed plasmonic sensing technology integrating chemifluorescence signal enhancement with plasmon-enhanced fluorescence detection. Using a portable imaging system, the dual chemical and plasmonic amplification enabled rapid analysis of multiple cytokine biomarkers in 1 h with sub-pg/mL sensitivities. Furthermore, we also developed a plasmonic sensing chip based on nanoparticle-spiked gold nanodimple structures fabricated by wafer-scale batch processes. We used the system to detect six cytokines directly from clinical plasma samples (n = 20) and showed 100% accuracy for sepsis detection. The described technology could be employed in rapid, ultrasensitive, multiplexed plasmonic sensing in POC settings for myriad clinical conditions.

Label-free detection and discrimination of respiratory pathogens based on electrochemical synthesis of biomaterials-mediated plasmonic composites and machine learning analysis.

Seasonal outbreaks of respiratory viral infections remain a global concern, with increasing morbidity and mortality rates recorded annually. Timely and false responses contribute to the widespread of respiratory pathogenic diseases owing to similar symptoms at an early stage and subclinical infection. The prevention of emerging novel viruses and variants is also a big challenge. Reliable point-of-care diagnostic assays for early infection diagnosis play a critical role in the response to threats of epidemics or pandemics. We developed a facile method for specifically identifying different viruses based on surface-enhanced Raman spectroscopy (SERS) with pathogen-mediated composite materials on Au nanodimple electrodes and machine learning (ML) analyses. Virus particles were trapped in three-dimensional plasmonic concave spaces of the electrode via electrokinetic preconcentration, and Au films were simultaneously electrodeposited, leading to the acquisition of intense and in-situ SERS signals from the Au-virus composites for ultrasensitive SERS detection. The method was useful for rapid detection analysis (<15 min), and the ML analysis for specific identification of eight virus species, including human influenza A viruses (i.e., H1N1 and H3N2 strains), human rhinovirus, and human coronavirus, was conducted. The highly accurate classification was achieved using the principal component analysis-support vector machine (98.9%) and convolutional neural network (93.5%) models. This ML-associated SERS technique demonstrated high feasibility for direct multiplex detection of different virus species for on-site applications.

3D plasmonic coral nanoarchitecture paper for label-free human urine sensing and deep learning-assisted cancer screening.

Practical human biofluid sensing requires a sensor device to differentiate patients from the normal group with high sensitivity and specificity. Label-free molecular identification from human biofluids allows direct classification of abnormal samples, providing insights for disease diagnosis and finding of new biomarkers. Here, we introduce a label-free surface-enhanced Raman scattering sensor based on a three-dimensional plasmonic coral nanoarchitecture (3D-PCN), which has strong electromagnetic field enhancement through multiple hot spots. The 3D-PCN was synthesized on a paper substrate via direct one-step gold reduction, forming a coral-like nanoarchitecture with high absorption property for biofluids. This was fabricated as a urine test strip and then integrated with a handheld Raman system to develop an on-site urine diagnostic platform. The developed platform successfully classified the human prostate and pancreatic cancer urines in a label-free method supported by two types of deep learning networks, with high clinical sensitivity and specificity. Our technology has the potential to be utilized not only for urinary cancer diagnosis but also for various human biofluid sensing systems as a future point-of-care testing platform.