FARSB Facilitates Hepatocellular Carcinoma Progression by Activating the mTORC1 Signaling Pathway.

Hepatocellular carcinoma (HCC) is a common malignant tumor with high mortality. Human phenylalanine tRNA synthetase (PheRS) comprises two α catalytic subunits encoded by the FARSA gene and two ß regulatory subunits encoded by the FARSB gene. FARSB is a potential oncogene, but no experimental data show the relationship between FARSB and HCC progression. We found that the high expression of FARSB in liver cancer is closely related to patients' low survival and poor prognosis. In liver cancer cells, the mRNA and protein expression levels of FARSB are increased and promote cell proliferation and migration. Mechanistically, FARSB activates the mTOR complex 1 (mTORC1) signaling pathway by binding to the component Raptor of the mTORC1 complex to play a role in promoting cancer. In addition, we found that FARSB can inhibit erastin-induced ferroptosis by regulating the mTOR signaling pathway, which may be another mechanism by which FARSB promotes HCC progression. In summary, FARSB promotes HCC progression and is associated with the poor prognosis of patients. FARSB is expected to be a biomarker for early screening and treatment of HCC.

Nuclear respiratory factor 1 drives hepatocellular carcinoma progression by activating LPCAT1-ERK1/2-CREB axis.

BACKGROUND: Nuclear respiratory factor 1 (NRF1) is a transcription factor that participates in several kinds of tumor, but its role in hepatocellular carcinoma (HCC) remains elusive. This study aims to explore the role of NRF1 in HCC progression and investigate the underlying mechanisms. RESULTS: NRF1 was overexpressed and hyperactive in HCC tissue and cell lines and high expression of NRF1 indicated unfavorable prognosis of HCC patients. NRF1 promoted proliferation, migration and invasion of HCC cells both in vitro and in vivo. Mechanistically, NRF1 activated ERK1/2-CREB signaling pathway by transactivating lysophosphatidylcholine acyltransferase 1 (LPCAT1), thus promoting cell cycle progression and epithelial mesenchymal transition (EMT) of HCC cells. Meanwhile, LPCAT1 upregulated the expression of NRF1 by activating ERK1/2-CREB signaling pathway, forming a positive feedback loop. CONCLUSIONS: NRF1 is overexpressed in HCC and promotes HCC progression by activating LPCAT1-ERK1/2-CREB axis. NRF1 is a promising therapeutic target for HCC patients.

A Metasurface Plasmonic Analysis Platform Combined with Gold Nanoparticles for Ultrasensitive Quantitative Detection of Small Molecules.

Food safety related to drug residues in food has become a widespread public concern. Small-molecule drug residue analysis often relies on mass spectrometry, thin-layer chromatography, or enzyme-linked immunosorbent assays (ELISA). Some of these techniques have limited sensitivity and accuracy, while others are time-consuming, costly, and rely on specialized equipment that requires skilled operation. Therefore, the development of a sensitive, fast, and easy-to-operate biosensor could provide an accessible alternative to conventional small-molecule analysis. Here, we developed a nanocup array-enhanced metasurface plasmon resonance (MetaSPR) chip coupled with gold nanoparticles (AuNPs) (MSPRAN) to detect small molecules. As sulfamethazine drug residues in poultry eggs may cause health issues, we selected this as a model to evaluate the feasibility of using MSPRAN for small-molecule detection. The MSPRAN biosensor employed competitive immunoassay technology for sulfamethazine detection. The limit of detection was calculated as 73 pg/mL, with sensitivity approximately twice that of previously reported detection methods. Additionally, the recovery rate of the biosensor, tested in egg samples, was similar to that measured using ELISA. Overall, this newly developed MSPRAN biosensor platform for small-molecule detection provides fast and reliable results, facile operation, and is relatively cost-effective for application in food safety testing, environmental monitoring, or clinical diagnostics.

Ultrasensitive Detection of C-Reactive Protein by a Novel Nanoplasmonic Immunoturbidimetry Assay.

Nanotechnology has attracted much attention, and may become the key to a whole new world in the fields of food, agriculture, building materials, machinery, medicine, and electrical engineering, because of its unique physical and chemical properties, including high surface area and outstanding electrical and optical properties. The bottom-up approach in nanofabrication involves the growth of particles, and we were inspired to propose a novel nanoplasmonic method to detect the formation of nanoparticles in real time. This innovative idea may contribute to the promotion of nanotechnology development. An increase in nanometer particle size leads to optical extinction or density (OD)-value changes in our nanosensor chip at a specific wavelength measured in a generic microplate reader. Moreover, in applying this method, an ultrasensitive nanoplasmonic immunoturbidimetry assay (NanoPITA) was carried out for the high-throughput quantification of hypersensitive C-reactive protein (CRP), a well-known biomarker of cardiovascular, inflammatory, and tumor diseases. The one-step detection of the CRP concentration was completed in 10 min with high fidelity, using the endpoint analysis method. The new NanoPITA method not only produced a linear range from 1 ng/mL to 500 ng/mL CRP with the detection limit reduced to 0.54 ng/mL, which was an improvement of over 1000 times, with respect to regular immunoturbidity measurement, but was also effective in blood detection. This attractive method, combined with surface plasmon resonance and immunoturbidimetry, may become a new technology platform in the application of biological detection.

Applying Machine Learning with Localized Surface Plasmon Resonance Sensors to Detect SARS-CoV-2 Particles.

The sudden outbreak of COVID-19 rapidly developed into a global pandemic, which caused tens of millions of infections and millions of deaths. Although SARS-CoV-2 is known to cause COVID-19, effective approaches to detect SARS-CoV-2 using a convenient, rapid, accurate, and low-cost method are lacking. To date, most of the diagnostic methods for patients with early infections are limited to the detection of viral nucleic acids via polymerase chain reaction (PCR), or antigens, using an enzyme-linked immunosorbent assay or a chemiluminescence immunoassay. This study developed a novel method that uses localized surface plasmon resonance (LSPR) sensors, optical imaging, and artificial intelligence methods to directly detect the SARS-CoV-2 virus particles without any sample preparation. The virus concentration can be qualitatively and quantitatively detected in the range of 125.28 to 106 vp/mL through a few steps within 12 min with a limit of detection (LOD) of 100 vp/mL. The accuracy of the SARS-CoV-2 positive or negative assessment was found to be greater than 97%, and this was demonstrated by establishing a regression machine learning model for the virus concentration prediction (R2 > 0.95).

Application of Machine Learning and Weighted Gene Co-expression Network Algorithm to Explore the Hub Genes in the Aging Brain.

Aging is a major risk factor contributing to neurodegeneration and dementia. However, it remains unclarified how aging promotes these diseases. Here, we use machine learning and weighted gene co-expression network (WGCNA) to explore the relationship between aging and gene expression in the human frontal cortex and reveal potential biomarkers and therapeutic targets of neurodegeneration and dementia related to aging. The transcriptional profiling data of the human frontal cortex from individuals ranging from 26 to 106 years old was obtained from the GEO database in NCBI. Self-Organizing Feature Map (SOM) was conducted to find the clusters in which gene expressions downregulate with aging. For WGCNA analysis, first, co-expressed genes were clustered into different modules, and modules of interest were identified through calculating the correlation coefficient between the module and phenotypic trait (age). Next, the overlapping genes between differentially expressed genes (DEG, between young and aged group) and genes in the module of interest were discovered. Random Forest classifier was performed to obtain the most significant genes in the overlapping genes. The disclosed significant genes were further identified through network analysis. Through WGCNA analysis, the greenyellow module is found to be highly negatively correlated with age, and functions mainly in long-term potentiation and calcium signaling pathways. Through step-by-step filtering of the module genes by overlapping with downregulated DEGs in aged group and Random Forest classifier analysis, we found that MAPT, KLHDC3, RAP2A, RAP2B, ELAVL2, and SYN1 were co-expressed and highly correlated with aging.

Printed Electrode for Measuring Phosphate in Environmental Water.

Phosphate is a major nonpoint source pollutant in both the Louisiana local streams as well as in the Gulf of Mexico coastal waters. Phosphates from agricultural run-off have contributed to the eutrophication of global surface waters. Phosphate environmental dissemination and eutrophication problems are not yet well understood. Thus, this study aimed to monitor phosphate in the local watershed to help identify potential hot spots in the local community (Mississippi River, Louisiana) that may contribute to nutrient loading downstream (in the Gulf of Mexico). An electrochemical method using a physical vapor deposited cobalt microelectrode was utilized for phosphate detection using cyclic voltammetry and amperometry. The testing results were utilized to evaluate the phosphate distribution in river water and characterize the performance of the microsensor. Various characterizations, including the limit of detection, sensitivity, and reliability, were conducted by measuring the effect of interferences, including dissolved oxygen, pH, and common ions. The electrochemical sensor performance was validated by comparing the results with the standard colorimetry phosphate detection method. X-ray photoelectron spectroscopy (XPS) measurements were performed to understand the phosphate sensing mechanism on the cobalt electrode. This proof-of-concept sensor chip could be utilized for on-field monitoring using a portable, hand-held potentiostat.

Heterogeneous nuclear ribonucleoprotein L facilitates recruitment of 53BP1 and BRCA1 at the DNA break sites induced by oxaliplatin in colorectal cancer.

Although oxaliplatin is an effective chemotherapeutic drug for treatment of colorectal cancer (CRC), tumor cells can develop mechanisms to evade oxaliplatin-induced cell death and show high tolerance and acquired resistance to this drug. Heterogeneous nuclear ribonucleoprotein L (hnRNP L) has been proved to play a critical role in DNA repair during IgH class switch recombination (CSR) in B lymphocytes, while, its role in CRC and chemotherapeutic resistance remain unknown. Our study aims to uncover an unidentified mechanism of regulating DNA double-strand breaks (DSBs) by hnRNP L in CRC cells treated by oxaliplatin. In present study, we observed that knockdown of hnRNP L enhanced the level of DNA breakage and sensitivity of CRC cells to oxaliplatin. The expression of key DNA repair factors (BRCA1, 53BP1, and ATM) was unaffected by hnRNP L knockdown, thereby excluding the likelihood of hnRNP L mediation via mRNA regulation. Moreover, we observed that phosphorylation level of ATM changed oppositely to 53BP1 and BRCA1 in the CRC cells (SW620 and HCT116) which exhibit synergistic effect by oxaliplatin plus hnRNP L impairment. And similar phenomenon was observed in the foci formation of these critical repair factors. We also found that hnRNP L binds directly with these DNA repair factors through its RNA-recognition motifs (RRMs). Analysis of cell death indicated that the RRMs of hnRNP L are required for cell survival under incubation with oxaliplatin. In conclusion, hnRNP L is critical for the recruitment of the DNA repair factors in oxaliplatin-induced DSBs. Targeting hnRNP L is a promising new clinical approach that could enhance the effectiveness of current chemotherapeutic treatment in patients with resistance to oxaliplatin.

Protein binding kinetics quantification via coupled plasmonic-photonic resonance nanosensors in generic microplate reader.

Almost no analytical assays, either colorimetric or fluorescence assays, for generic microplate readers is capable of dynamic measurements of protein-protein binding or the quantification of kinetic association and dissociation constants of protein interactions. On the other hand, protein binding kinetics quantification can be uniquely done on special expensive surface plasmon resonance (SPR) sensing equipment. Here we report the integration of coupled plasmonic-photonic resonance nanosensors in standard 96-well plate format and by using which, for the very first time, the demonstration of label-free dynamic SPR-like protein binding measurement and kinetics quantification in a generic microplate reader. Our low-cost label-free nanosensor plate enables very sensitive detection of immobilized protein interactions based on the transmission optical density (OD) value changes at specific wavelengths measured in a generic microplate reader. The relative end-point OD value changes show a good linear response with protein concentrations (from 0.05 to 50 µg/ml). And the protein quantification in serum results are consistent with the concurrent hospital lab tests. Most importantly, the kinetic association and dissociation constants of protein interactions in our sensor plate wells are determined by time-lapse dynamic OD value measurement in the generic microplate reader. Enabled by our unique nanosensor plate, SPR-like measurement of protein binding kinetics is now available using generic microplate reader ubiquitous in many chemistry and biomedical research labs.

C-Reaction Protein Detection in Human Saliva by Nanoplasmonic Color Imaging.

A label-free and highly sensitive imaging sensor based on plasmonic-photonic interaction in a gold-titanium dioxidegold metal-insulator-metal (MIM) plasmonic nanocup array is reported. The sensor can detect proteins and exhibits superior performance in visible light sensing. This device enables very sensitive detection of an increase in superstrate refractive index based on changes in the red channel intensity of color imaging. Compared to other conventional plasmonic sensors, our device achieves transmission imaging detection by using normal white light and minimizes instrumentation requirement. In this study, we used the device to detect C-reaction protein (CRP) level in saliva, which is widely tested to help make clinical decisions in different diseases and disorders. The intensity imaging showed a good linear response between CRP concentration (from 5 to 100 ng/mL) and relative intensity change in the device. The lowest concentration of CRP that could be detected was 5 ng/mL. Moreover, it could achieve a positive detection in saliva from patients when the CRP level in serum was 3.2 µg/mL. Owing to the high performance of the MIM plasmonic nanocup array, the proposed device is promising for future portable optical sensing with visible light illumination and imaging.

3D Plasmon Coupling Assisted Sers on Nanoparticle-Nanocup Array Hybrids.

Unique colorimetric optical properties of nanomaterials can effectively influence the light absorption or emission of molecules. Here, we design plasmonic substrate for surface-enhanced Raman scattering (SERS) by inducing three-dimensional (3D) hot spots on the sensing surface. The 3D hot spots are formed by the self-assembly of plasmonic nanoparticles (NPs) on a 3D plasmonic nanocup array structure. This 3D hot spot formation on the periodic nanocup arrays achieves much higher SERS enhancement factor than the 2D NP arrays, which have been conventionally sought SERS substrates. We also utilize the colorimetric properties of the nanocup arrays for an additional degree of SERS enhancement. Colorimetry, achieved by tunable plasmon resonance wavelength by controlling dielectric property on the nanocup array surface, eases the modulation of the plasmonic resonance condition without modifying the nanostructure design. By continuously monitoring the shifts of the plasmon resonance condition and its effect on the light absorption and emission of the nearby molecules, we verify that larger SERS enhancement is achieved when the plasmon resonance wavelength is matched with the Raman excitation wavelength. The ease of plasmon resonance tuning of this nanocup array-nanoparticle hybrid structure allows versatile SERS enhancement for a variety of different Raman measurement conditions.

Spectrometer-Free Plasmonic Biosensing with Metal-Insulator-Metal Nanocup Arrays.

The development of high performing and accessible sensors is crucial to future point-of-care diagnostic sensing systems. Here, we report on a gold-titanium dioxide-gold metal-insulator-metal plasmonic nanocup array device for spectrometer-free refractometric sensing with a performance exceeding conventional surface plasmon resonance sensors. This device shows distinct spectral properties such that a superstrate refractive index increase causes a transmission intensity increase at the peak resonance wavelength. There is no spectral shift at this peak and there are spectral regions with no transmission intensity change, which can be used as internal device references. The sensing mechanism, plasmon-cavity coupling optimization, and material properties are studied using electromagnetic simulations. The optimal device structure is determined using simulation and experimental parameter sweeps to tune the cavity confinement and the resonance coupling. An experimental sensitivity of 800 ΔT%/RIU is demonstrated. Spectrometer-free, imaged-based detection is also carried out for the cancer biomarker carcinoembryonic antigen with a 10 ng/mL limit of detection. The high performance and distinct spectral features of this metal-insulator-metal plasmonic nanocup array make this device promising for future portable optical sensing systems with minimal instrumentation requirements.

Plasmonic nanohole array for enhancing the SERS signal of a single layer of graphene in water.

We numerically design and experimentally test a SERS-active substrate for enhancing the SERS signal of a single layer of graphene (SLG) in water. The SLG is placed on top of an array of silver-covered nanoholes in a polymer and is covered with water. Here we report a large enhancement of up to 2 × 105 in the SERS signal of the SLG on the patterned plasmonic nanostructure for a 532 nm excitation laser wavelength. We provide a detailed study of the light-graphene interactions by investigating the optical absorption in the SLG, the density of optical states at the location of the SLG, and the extraction efficiency of the SERS signal of the SLG. Our numerical calculations of both the excitation field and the emission rate enhancements support the experimental results. We find that the enhancement is due to the increase in the confinement of electromagnetic fields on the location of the SLG that results in enhanced light absorption in the graphene at the excitation wavelength. We also find that water droplets increase the density of optical radiative states at the location of the SLG, leading to enhanced spontaneous emission rate of graphene at its Raman emission wavelengths.

Marangoni Convection Assisted Single Molecule Detection with Nanojet Surface Enhanced Raman Spectroscopy.

Many single-molecule (SM) label-free techniques such as scanning probe microscopies (SPM) and magnetic force spectroscopies (MFS) provide high resolution surface topography information, but lack chemical information. Typical surface enhanced Raman spectroscopy (SERS) systems provide chemical information on the analytes, but lack spatial resolution. In addition, a challenge in SERS sensors is to bring analytes into the so-called "hot spots" (locations where the enhancement of electromagnetic field amplitude is larger than 103). Previously described methods of fluid transport around hot spots like thermophoresis, thermodiffusion/Soret effect, and electrothermoplasmonic flow are either too weak or detrimental in bringing new molecules to hot spots. Herein, we combined the resonant plasmonic enhancement and photonic nanojet enhancemnet of local electric field on nonplanar SERS structures, to construct a stable, high-resolution, and below diffraction limit platform for single molecule label-free detection. In addition, we utilize Marangoni convection (mass transfer due to surface tension gradient) to bring new analytes into the hotspot. An enhancement factor of ∼3.6 × 1010 was obtained in the proposed system. Rhodamine-6G (R6G) detection of up to a concentration of 10-12 M, an improvement of two orders of magnitude, was achieved using the nanojet effect. The proposed system could provide a simple, high throughput SERS system for single molecule analysis at high spatial resolution.

Application of an advanced maximum likelihood estimation restoration method for enhanced-resolution and contrast in second-harmonic generation microscopy.

Second-harmonic generation (SHG) microscopy has gained popularity because of its ability to perform submicron, label-free imaging of noncentrosymmetric biological structures, such as fibrillar collagen in the extracellular matrix environment of various organs with high contrast and specificity. Because SHG is a two-photon coherent scattering process, it is difficult to define a point spread function (PSF) for this modality. Hence, compared to incoherent two-photon processes like two-photon fluorescence, it is challenging to apply the various PSF-engineering methods to improve the spatial resolution to be close to the diffraction limit. Using a synthetic PSF and application of an advanced maximum likelihood estimation (AdvMLE) deconvolution algorithm, we demonstrate restoration of the spatial resolution in SHG images to that closer to the theoretical diffraction limit. The AdvMLE algorithm adaptively and iteratively develops a PSF for the supplied image and succeeds in improving the signal to noise ratio (SNR) for images where the SHG signals are derived from various sources such as collagen in tendon and myosin in heart sarcomere. Approximately 3.5 times improvement in SNR is observed for tissue images at depths of up to ∼480 nm, which helps in revealing the underlying helical structures in collagen fibres with an ∼26% improvement in the amplitude contrast in a fibre pitch. Our approach could be adapted to noisy and low resolution modalities such as micro-nano CT and MRI, impacting precision of diagnosis and treatment of human diseases.

Monitoring the electrochemical responses of neurotransmitters through localized surface plasmon resonance using nanohole array.

In this study, a novel spectroelectrochemical method was proposed for neurotransmitters detection. The central sensing device was a hybrid structure of nanohole array and gold nanoparticles, which demonstrated good conductivity and high localized surface plasmon resonance (LSPR) sensitivity. By utilizing such specially-designed nanoplasmonic sensor as working electrode, both electrical and spectral responses on the surface of the sensor could be simultaneously detected during the electrochemical process. Cyclic voltammetry was implemented to activate the oxidation and recovery of dopamine and serotonin, while transmission spectrum measurement was carried out to synchronously record to LSPR responses of the nanoplasmonic sensor. Coupling with electrochemistry, LSPR results indicated good integrity and linearity, along with promising accuracy in qualitative and quantitative detection even for mixed solution and in brain tissue homogenates. Also, the detection results of other negatively-charged neurotransmitters like acetylcholine demonstrated the selectivity of our detection method for transmitters with positive charge. When compared with traditional electrochemical signals, LSPR signals provided better signal-to-noise ratio and lower detection limits, along with immunity against interference factors like ascorbic acid. Taking the advantages of such robustness, the coupled detection method was proved to be a promising platform for point-of-care testing for neurotransmitters.

Self-Referenced Smartphone-Based Nanoplasmonic Imaging Platform for Colorimetric Biochemical Sensing.

Colorimetric sensors usually suffer due to errors from variation in light source intensity, the type of light source, the Bayer filter algorithm, and the sensitivity of the camera to incoming light. Here, we demonstrate a self-referenced portable smartphone-based plasmonic sensing platform integrated with an internal reference sample along with an image processing method to perform colorimetric sensing. Two sensing principles based on unique nanoplasmonics enabled phenomena from a nanostructured plasmonic sensor, named as nanoLCA (nano Lycurgus cup array), were demonstrated here for colorimetric biochemical sensing: liquid refractive index sensing and optical absorbance enhancement sensing. Refractive indices of colorless liquids were measured by simple smartphone imaging and color analysis. Optical absorbance enhancement in the colorimetric biochemical assay was achieved by matching the plasmon resonance wavelength with the chromophore's absorbance peak wavelength. Such a sensing mechanism improved the limit of detection (LoD) by 100 times in a microplate reader format. Compared with a traditional colorimetric assay such as urine testing strips, a smartphone plasmon enhanced colorimetric sensing system provided 30 times improvement in the LoD. The platform was applied for simulated urine testing to precisely identify the samples with higher protein concentration, which showed potential point-of-care and early detection of kidney disease with the smartphone plasmonic resonance sensing system.

White blood cell counting on smartphone paper electrochemical sensor.

White blood cell (WBC) analysis provides rich information in rapid diagnosis of acute bacterial and viral infections as well as chronic disease management. For patients with immune deficiency or leukemia WBC should be persistently monitored. Current WBC counting method relies on bulky instrument and trained personnel and is time consuming. Rapid, low-cost and portable solution is in highly demand for point of care test. Here we demonstrate a label-free smartphone based electrochemical WBC counting device on microporous paper with patterned gold microelectrodes. WBC separated from whole blood was trapped by the paper with microelectrodes. WBC trapped on the paper leads to the ion diffusion blockage on microelectrodes, therefore cell concentration is determined by peak current on the microelectrodes measured by a differential pulse voltammeter and the quantitative results are collected by a smartphone wirelessly within 1min. We are able to rapidly quantify WBC concentrations covering the common physiological and pathological range (200-20000µL-1) with only 10µL sample and high repeatability as low as 10% in CoV (Coefficient of Variation). The unique smartphone paper electrochemical sensor ensures fast cell quantification to achieve rapid and low-cost WBC analysis at the point-of-care under resource limited conditions.

Large-area, lithography-free, low-cost SERS sensor with good flexibility and high performance.

Cost-effective, sensitive and bio-compatible surface-enhanced Raman spectroscopy (SERS) substrate has been in high demand since the Raman spectrum was designated as a significant tool for analyzing the composition of liquids, gases and solids in 1998 [1]. In this research, we presented the design, fabrication and characterization of an improved gold-based SERS substrate. With fine tuning of the SiO2 thickness we achieved a 3.391 times improvement and achieved an enhancement factor of 1.55 * 10(7) which is 15 times better than the current gold-standard Klarite substrate. Such improvement is ascribed to the localized surface plasmon resonance (SPR) and propagating SPR, which is proved by full-wave finite-difference time-domain simulations.

Sensitivity Tuning through Additive Heterogeneous Plasmon Coupling between 3D Assembled Plasmonic Nanoparticle and Nanocup Arrays.

Plasmonic substrates have fixed sensitivity once the geometry of the structure is defined. In order to improve the sensitivity, significant research effort has been focused on designing new plasmonic structures, which involves high fabrication costs; however, a method is reported for improving sensitivity not by redesigning the structure but by simply assembling plasmonic nanoparticles (NPs) near the evanescent field of the underlying 3D plasmonic nanostructure. Here, a nanoscale Lycurgus cup array (nanoLCA) is employed as a base colorimetric plasmonic substrate and an assembly template. Compared to the nanoLCA, the NP assembled nanoLCA (NP-nanoLCA) exhibits much higher sensitivity for both bulk refractive index sensing and biotin-streptavidin binding detection. The limit of detection of the NP-nanoLCA is at least ten times smaller when detecting biotin-streptavidin conjugation. The numerical calculations confirm the importance of the additive plasmon coupling between the NPs and the nanoLCA for a denser and stronger electric field in the same 3D volumetric space. Tunable sensitivity is accomplished by controlling the number of NPs in each nanocup, or the number density of the hot spots. This simple yet scalable and cost-effective method of using additive heterogeneous plasmon coupling effects will benefit various chemical, medical, and environmental plasmon-based sensors.