Osmosensor-mediated control of Ca2+ spiking in pollen germination.

Higher plants survive terrestrial water deficiency and fluctuation by arresting cellular activities (dehydration) and resuscitating processes (rehydration). However, how plants monitor water availability during rehydration is unknown. Although increases in hypo-osmolarity-induced cytosolic Ca2+ concentration (HOSCA) have long been postulated to be the mechanism for sensing hypo-osmolarity in rehydration1,2, the molecular basis remains unknown. Because osmolarity triggers membrane tension and the osmosensing specificity of osmosensing channels can only be determined in vivo3-5, these channels have been classified as a subtype of mechanosensors. Here we identify bona fide cell surface hypo-osmosensors in Arabidopsis and find that pollen Ca2+ spiking is controlled directly by water through these hypo-osmosensors-that is, Ca2+ spiking is the second messenger for water status. We developed a functional expression screen in Escherichia coli for hypo-osmosensitive channels and identified OSCA2.1, a member of the hyperosmolarity-gated calcium-permeable channel (OSCA) family of proteins6. We screened single and high-order OSCA mutants, and observed that the osca2.1/osca2.2 double-knockout mutant was impaired in pollen germination and HOSCA. OSCA2.1 and OSCA2.2 function as hypo-osmosensitive Ca2+-permeable channels in planta and in HEK293 cells. Decreasing osmolarity of the medium enhanced pollen Ca2+ oscillations, which were mediated by OSCA2.1 and OSCA2.2 and required for germination. OSCA2.1 and OSCA2.2 convert extracellular water status into Ca2+ spiking in pollen and may serve as essential hypo-osmosensors for tracking rehydration in plants.

Plant cell-surface GIPC sphingolipids sense salt to trigger Ca2+ influx.

Salinity is detrimental to plant growth, crop production and food security worldwide. Excess salt triggers increases in cytosolic Ca2+ concentration, which activate Ca2+-binding proteins and upregulate the Na+/H+ antiporter in order to remove Na+. Salt-induced increases in Ca2+ have long been thought to be involved in the detection of salt stress, but the molecular components of the sensing machinery remain unknown. Here, using Ca2+-imaging-based forward genetic screens, we isolated the Arabidopsis thaliana mutant monocation-induced [Ca2+]i increases 1 (moca1), and identified MOCA1 as a glucuronosyltransferase for glycosyl inositol phosphorylceramide (GIPC) sphingolipids in the plasma membrane. MOCA1 is required for salt-induced depolarization of the cell-surface potential, Ca2+ spikes and waves, Na+/H+ antiporter activation, and regulation of growth. Na+ binds to GIPCs to gate Ca2+ influx channels. This salt-sensing mechanism might imply that plasma-membrane lipids are involved in adaption to various environmental salt levels, and could be used to improve salt resistance in crops.

Dual-Modal Colorimetric and Surface-Enhanced Raman Scattering (SERS)-Based Lateral Flow Immunoassay for Ultrasensitive Detection of SARS-CoV-2 Using a Plasmonic Gold Nanocrown.

The 2019 coronavirus disease (COVID-19) outbreak created an unprecedented need for rapid, sensitive, and cost-effective point-of-care diagnostic tests to prevent and mitigate the spread of the SARS-CoV-2 virus. Herein, we demonstrated an advanced lateral flow immunoassay (LFIA) platform with dual-functional [colorimetric and surface-enhanced Raman scattering (SERS)] detection of the spike 1 (S1) protein of SARS-CoV-2. The nanosensor was integrated with a specially designed core-gap-shell morphology consisting of a gold shell decorated with external nanospheres, a structure referred to as gold nanocrown (GNC), labeled with a Raman reporter molecule 1,3,3,1',3',3'-hexamethyl-2,2'-indotricarbocyanine iodide (HITC) to produce a strong colorimetric signal as well as an enhanced SERS signal. Among the different plasmonics-active GNC nanostructures, the GNC-2 morphology, which has a shell decorated with an optimum number and size of nanospheres, produces an intense dark-blue colorimetric signal and ultrahigh SERS signal. The limit of detection (LOD) of the S1 protein via colorimetric detection LFIA was determined to be 91.24 pg/mL. On the other hand, the LOD for the SERS LFIA method was more than three orders of magnitude lower at 57.21 fg/mL. Furthermore, we analyzed the performance of the GNC-2 nanosensor for directly analyzing the S1 protein spiked in saliva samples without any sample pretreatment and achieving the LOD as low as 39.65 fg/mL using SERS-based plasmonics-enhanced LFIA, indicating ultrahigh detection sensitivity. Overall, our GNC nanosensor showed excellent sensitivity, reproducibility, and rapid detection of the SARS-CoV-2 S1 protein, demonstrating excellent potential as a promising point-of-care platform for the early detection of respiratory virus infections.

A simple low-cost flexible plasmonic patch based on spiky gold nanostars for ultra-sensitive SERS sensing.

Recently, transparent and flexible surface-enhanced Raman scattering (SERS) substrates have received great interest for direct point-of-care detection of analytes on irregular nonplanar surfaces. In this study, we proposed a simple cost-effective strategy to develop a flexible SERS patch utilizing multibranched sharp spiked gold nanostars (GNS) decorated on a commercially available adhesive Scotch Tape for achieving ultra-high SERS sensitivity. The experimental SERS measurements were correlated with theoretical finite element modeling (FEM), which indicates that the GNS having a 2.5 nm branch tip diameter (GNS-4) exhibits the strongest SERS enhancement. Using rhodamine 6G (R6G) as a model analyte, the SERS performance of the flexible SERS patch exhibited a minimum detection limit of R6G as low as 1 pM. The enhancement factor of the SERS patch with GNS-4 was calculated as 6.2 × 108, which indicates that our flexible SERS substrate has the potential to achieve ultra-high sensitivity. The reproducibility was tested with 30 different spots showing a relative standard deviation (RSD) of SERS intensity of about 5.4%, indicating good reproducibility of the SERS platform. To illustrate the usefulness of the flexible SERS sensor patch, we investigated the detection of a carcinogenic compound crystal violet (CV) on fish scales, which is often used as an effective antifungal agent in the aquaculture industry. The results realized the trace detection of CV with the minimum detection limit as low as 1 pM. We believe that our transparent, and flexible SERS patch based on GNS-4 has potential as a versatile, low-cost platform for real-world SERS sensing applications on nonplanar surfaces.

Rapid SERS assay for determination of the opioid fentanyl using silver-coated sharply branched gold nanostars.

A high-throughput surface-enhanced Raman scattering (SERS)-sensing platform is presented for FNT detection in human urine without any sample preparation. The sensing platform is based on plasmonics-active silver-coated sharply branched gold nanostars (SGNS). The effect of silver thickness was investigated experimentally and theoretically, and the results indicated that SERS enhancement was maximum at an optimum silver thickness of 45 nm on the sharply spiked SGNS. The proposed high-throughput SERS platform exhibited ultrahigh sensitivity and excellent enhancement uniformity for a model analyte, i.e., crystal violet. Moreover, the SERS-sensing platform demonstrated good sensitivity of FNT spiked in human urine samples with two differential linear response ranges of 2 to 0.2 µg/mL and 0.1 µg/mL to 100 pg/mL, respectively,  with a detection limit as low as 10.02 pg/mL. The spiked human urine samples show satisfactory recovery values from 92.5 to 102% with relative standard deviations (RSD) of less than 10%. In summary, the high-throughput performance of the proposed microplate-based SERS platform demonstrated great potential for rapid low-cost SERS-based sensing applications.

Xenorecognition and costimulation of porcine endothelium-derived extracellular vesicles in initiating human porcine-specific T cell immune responses.

Porcine vascular endothelial cells (PECs) form a mechanistic centerpiece of xenograft rejection. Here, we determined that resting PECs release swine leukocyte antigen class I (SLA-I) but not swine leukocyte antigen class-II DR (SLA-DR) expressing extracellular vesicles (EVs) and investigated whether these EVs proficiently initiate xenoreactive T cell responses via direct xenorecognition and costimulation. Human T cells acquired SLA-I+ EVs with or without direct contact to PECs, and these EVs colocalized with T cell receptors. Although interferon gamma-activated PECs released SLA-DR+ EVs, the binding of SLA-DR+ EVs to T cells was sparse. Human T cells demonstrated low levels of proliferation without direct contact to PECs, but marked T cell proliferation was induced following exposure to EVs. EV-induced proliferation proceeded independent of monocytes/macrophages, suggesting that EVs delivered both a T cell receptor signal and costimulation. Costimulation blockade targeting B7, CD40L, or CD11a significantly reduced T cell proliferation to PEC-derived EVs. These findings indicate that endothelial-derived EVs can directly initiate T cell-mediated immune responses, and suggest that inhibiting the release of SLA-I EVs from organ xenografts has the potential to modify the xenograft rejection. We propose a secondary-direct pathway for T cell activation via xenoantigen recognition/costimulation by endothelial-derived EVs.

Solution-Based Ultra-Sensitive Surface-Enhanced Raman Scattering Detection of the Toxin Bacterial Biomarker Pyocyanin in Biological Fluids Using Sharp-Branched Gold Nanostars.

There is a critical need for sensitive rapid point-of-care detection of bacterial infection biomarkers in complex biological fluids with minimal sample preparation, which can improve early-stage diagnosis and prevent several bacterial infections and fatal diseases. A solution-based surface-enhanced Raman scattering (SERS) detection platform has long been sought after for low cost, rapid, and on-site detection of analyte molecules, but current methods still exhibit poor sensitivity. In this study, we have tuned the morphology of the surfactant-free gold nanostars (GNSs) to achieve sharp protruding spikes for maximum SERS enhancement. We have controlled the GNS spike morphologies and optimized SERS performance in the solution phase using para-mercaptobenzoic acid as an SERS probe. To illustrate the potential for point-of-care applications, we have utilized a portable Raman instrument for measurements. For pathogenic agent sensing applications, we demonstrated rapid and sensitive detection of the toxin biomarker pyocyanin (PYO) used as the bacterial biomarker model system. Pyocyanin is a toxic compound produced and secreted by the common water-borne Gram-negative bacterium Pseudomonas aeruginosa, a pathogen known for advanced antibiotic resistance and association with serious diseases such as ventilator-associated pneumonia and cystic fibrosis. The limit of detection (LOD) achieved for PYO was 0.05 nM using sharp branched GNSs. Furthermore, as a proof of strategy, this SERS detection of PYO was performed directly in drinking water, human saliva, and human urine without any sample treatment pre-purification, achieving an LOD of 0.05 nM for drinking water and 0.4 nM for human saliva and urine. This work provides a proof-of-principle demonstration for the high sensitivity detection of the bacterial toxin biomarker with minimal sample preparation: the "mix and detect" detection of the GNS platform is simple, robust, and rapid, taking only 1-2 min for each measurement. Overall, our SERS detection platform shows great potential for point-of-need sensing and point-of-care diagnostics in biological fluids.

A hybrid plasmonic nanoprobe using polyvinylpyrrolidone-capped bimetallic silver-gold nanostars for highly sensitive and reproducible solution-based SERS sensing.

Practical solution-based assays using surface-enhanced Raman spectroscopy (SERS) with portable instrumentation are currently of particular interest for rapid, efficient, and low-cost detection of analytes. However, current assays still have limited applicability due to their poor sensitivity and reproducibility. Herein, we demonstrate highly stable polyvinylpyrrolidone (PVP)-capped bimetallic silver-coated gold nanostars (BGNS-Ag-PVP) as a solution-based SERS nanoprobe that is capable of producing a strong, uniform, and reproducible SERS signal using a portable Raman instrument. The developed hybrid BGNS-Ag-PVP nanostructure shows tunable optical properties with improved SERS sensitivity and reproducibility as compared to gold nanostars. We have synthesized bimetallic nanoprobes BGNS-Ag-PVP having three different silvers, referred to as BGNS-Ag-PVP-1, BGNS-Ag-PVP-2, and BGNS-Ag-PVP-3. The SERS performance of BGNS-Ag-PVP was studied using methylene blue (Meb) as a probe molecule, and we achieved a detection limit of up to 10 nM indicating the high sensitivity of the solution-based SERS platform. The application of such bimetallic nanoparticles is demonstrated via the sensitive detection of the antithyroid drug methimazole (Mz) used as a model analyte system. We have achieved a detection limit of 1 nM for Mz spiked with human urine indicating three orders of magnitude lower than previously reported solution-based SERS detection methods. Furthermore, the SERS performance was reproducible over 3 months indicating excellent stability and repeatability. The result illustrates the potential of this solution-based SERS detection platform as a promising sensing tool for analytes such as illicit drugs, and biomarkers that have affinity to bind on nanoprobes.

Multiplex SERS detection of polycyclic aromatic hydrocarbon (PAH) pollutants in water samples using gold nanostars and machine learning analysis.

Polycyclic aromatic hydrocarbons (PAHs) have attracted a lot of environmental concern because of their carcinogenic and mutagenic properties, and the fact they can easily contaminate natural resources such as drinking water and river water. This study presents a simple and sensitive point-of-care SERS detection of PAHs combined with machine learning algorithms to predict the PAH content more precisely and accurately in real-life samples such as drinking water and river water. We first synthesized multibranched sharp-spiked surfactant-free gold nanostars (GNSs) that can generate strong surface-enhanced Raman scattering (SERS) signals, which were further coated with cetyltrimethylammonium bromide (CTAB) for long-term stability of the GNSs as well as to trap PAHs. We utilized CTAB-capped GNSs for solution-based 'mix and detect' SERS sensing of various PAHs including pyrene (PY), nitro-pyrene (NP), anthracene (ANT), benzo[a]pyrene (BAP), and triphenylene (TP) spiked in drinking water and river water using a portable Raman module. Very low limits of detection (LOD) were achieved in the nanomolar range for the PAHs investigated. More importantly, the detected SERS signal was reproducible for over 90 days after synthesis. Furthermore, we analyzed the SERS data using artificial intelligence (AI) with machine learning algorithms based on the convolutional neural network (CNN) model in order to discriminate the PAHs in samples more precisely and accurately. Using a CNN classification model, we achieved a high prediction accuracy of 90% in the nanomolar detection range and an f1 score (harmonic mean of precision and recall) of 94%, and using a CNN regression model, achieved an RMSEconc = 1.07 × 10-1 µM. Overall, our SERS platform can be effectively and efficiently used for the accurate detection of PAHs in real-life samples, thus opening up a new, sensitive, selective, and practical approach for point-of-need SERS diagnosis of small molecules in complex practical environments.

OSCA1 is an osmotic specific sensor: a method to distinguish Ca2+ -mediated osmotic and ionic perception.

Genetic mutants defective in stimulus-induced Ca2+ increases have been gradually isolated, allowing the identification of cell-surface sensors/receptors, such as the osmosensor OSCA1. However, determining the Ca2+ -signaling specificity to various stimuli in these mutants remains a challenge. For instance, less is known about the exact selectivity between osmotic and ionic stresses in the osca1 mutant. Here, we have developed a method to distinguish the osmotic and ionic effects by analyzing Ca2+ increases, and demonstrated that osca1 is impaired primarily in Ca2+ increases induced by the osmotic but not ionic stress. We recorded Ca2+ increases induced by sorbitol (osmotic effect, OE) and NaCl/CaCl2 (OE + ionic effect, IE) in Arabidopsis wild-type and osca1 seedlings. We assumed the NaCl/CaCl2 total effect (TE) = OE + IE, then developed procedures for Ca2+ imaging, image analysis and mathematic fitting/modeling, and found osca1 defects mainly in OE. The osmotic specificity of osca1 suggests that osmotic and ionic perceptions are independent. The precise estimation of these two stress effects is applicable not only to new Ca2+ -signaling mutants with distinct stimulus specificity but also the complex Ca2+ signaling crosstalk among multiple concurrent stresses that occur naturally, and will enable us to specifically fine tune multiple signal pathways to improve crop yields.

Ultra-high SERS detection of consumable coloring agents using plasmonic gold nanostars with high aspect-ratio spikes.

Solution-based SERS detection by using a portable Raman instrument has emerged as an important tool due to its simplicity, and flexibility for rapid and on-site screening of analyte molecules. However, this method has several shortcomings, including poor sensitivity especially for weak-affinity analyte molecules, where there is no close contact between the plasmonic metal surface and analyte molecule. Examples of weak-affinity molecules include pigment molecules that are commonly used as a consumable coloring agent, such as allura red (AR), and sunset yellow (SY). As high consumption of colorant agents has been shown to cause adverse effects on human health, there is a strong need to develop a simple and practical sensing system with high sensitivity for these agents. Here we present a novel, highly sensitive solution-based SERS detection method for AR, and SY by using CTAC capped gold nanostars (GNS) having different aspect ratios (GNS-2, GNS-4, and GNS-5) without utilizing any aggregating agents which can enhance SERS signal however it reduces batch to batch reproducibility. The influence of the aspect ratio of GNS on SERS properties was investigated. We have achieved a limit of detection (LOD) of AR and SY as low as 0.5 and 1 ppb, respectively by using GNS-5 with the advantages of minimal sample preparation by just mixing the analyte solution into a well plate containing GNS solution. In addition, excellent colloidal stability and reproducibility have further enhanced the applicability in real-world samples. Overall, our results evidence that the solution-based SERS detection platform using high aspect-ratio GNS can be applied for practical application to detect pigment molecules in real samples with satisfactory results.

Intravital optical imaging for immune cell tracking after photoimmunotherapy with plasmonic gold nanostars.

Bladder cancer has been ranked as one of the most commonly occurring cancers in men and women with approximately half of the diagnoses being the late stage and/or metastatic diseases. We have developed a novel cancer treatment by combining gold nanostar-mediated photothermal therapy with checkpoint inhibitor immunotherapy to treat bladder cancer. Experiment results with a murine animal model demonstrated that our developed photoimmunotherapy therapy is more efficacious than any individual studied treatment. In addition, we used intravital optical imaging with a dorsal skinfold window chamber animal model to study immune responses and immune cell accumulation in a distant tumor following our photoimmunotherapy. The mice used have the CX3CR1-GFP receptor on monocytes, natural killer cells, and dendritic cells allowing us to dynamically track their presence by fluorescence imaging. Our proof-of-principle study results showed that the photoimmunotherapy triggered anti-cancer immune responses to generate anti-cancer immune cells which accumulate in metastatic tumors. Our study results illustrate that intravital optical imaging is an efficient and versatile tool to investigate immune responses and mechanisms of photoimmunotherapy in future studies.

Flg22-induced Ca2+ increases undergo desensitization and resensitization.

The flagellin epitope flg22, a pathogen-associated molecular pattern (PAMP), binds to the receptor-like kinase FLAGELLIN SENSING2 (FLS2), and triggers Ca2+ influx across the plasma membrane (PM). The flg22-induced increases in cytosolic Ca2+ concentration ([Ca2+ ]i ) (FICA) play a crucial role in plant innate immunity. It's well established that the receptor FLS2 and reactive oxygen species (ROS) burst undergo sensitivity adaptation after flg22 stimulation, referred to as desensitization and resensitization, to prevent over responses to pathogens. However, whether FICA also mount adaptation mechanisms to ensure appropriate and efficient responses against pathogens remains poorly understood. Here, we analysed systematically [Ca2+ ]i increases upon two successive flg22 treatments, recorded and characterized rapid desensitization but slow resensitization of FICA in Arabidopsis thaliana. Pharmacological analyses showed that the rapid desensitization might be synergistically regulated by ligand-induced FLS2 endocytosis as well as the PM depolarization. The resensitization of FICA might require de novo FLS2 protein synthesis. FICA resensitization appeared significantly slower than FLS2 protein recovery, suggesting additional regulatory mechanisms of other components, such as flg22-related Ca2+ permeable channels. Taken together, we have carefully defined the FICA sensitivity adaptation, which will facilitate further molecular and genetic dissection of the Ca2+ -mediated adaptive mechanisms in PAMP-triggered immunity.

Plasmonic Gold Nanostar-Mediated Photothermal Immunotherapy.

Cancer is among the leading cause of death around the world, causing close to 10 million deaths each year. Significant efforts have been devoted to developing novel technologies that can detect and treat cancer early and effectively to reduce cancer recurrences, treatment costs, and mortality. Gold nanoparticles (GNP) have been given particular attention for its use with photo-induced hyperthermia coupled with novel immunotherapy methods to provide a new platform for highly selective and less invasive cancer treatment. Among the various GNP platforms, gold nanostars (GNS) have a unique star-shaped geometric structure that allows superior light absorption and photothermal heating. This photothermal effect have also been found to amplify the anti-tumor immune response and can be exploited with adjuvant treatments using immune checkpoint inhibitors. This combination treatment known as Synergistic Immuno Photo Nanotherapy (SYMPHONY) has been shown to reverse tumor-mediated immunosuppression and has led to effective and long-lasting immunity against not only primary tumors but also cancer metastasis. This overview highlights the development and applications of GNS-mediated therapy developed in our laboratory for cancer treatment. This paper also presents recent results of experimental studies to illustrate the superior performance of GNS for photothermal treatment applications.

Smartphone-Based Device for Colorimetric Detection of MicroRNA Biomarkers Using Nanoparticle-Based Assay.

The detection of microRNAs (miRNAs) is emerging as a clinically important tool for the non-invasive detection of a wide variety of diseases ranging from cancers and cardiovascular illnesses to infectious diseases. Over the years, miRNA detection schemes have become accessible to clinicians, but they still require sophisticated and bulky laboratory equipment and trained personnel to operate. The exceptional computing ability and ease of use of modern smartphones coupled with fieldable optical detection technologies can provide a useful and portable alternative to these laboratory systems. Herein, we present the development of a smartphone-based device called Krometriks, which is capable of simple and rapid colorimetric detection of microRNA (miRNAs) using a nanoparticle-based assay. The device consists of a smartphone, a 3D printed accessory, and a custom-built dedicated mobile app. We illustrate the utility of Krometriks for the detection of an important miRNA disease biomarker, miR-21, using a nanoplasmonics-based assay developed by our group. We show that Krometriks can detect miRNA down to nanomolar concentrations with detection results comparable to a laboratory-based benchtop spectrophotometer. With slight changes to the accessory design, Krometriks can be made compatible with different types of smartphone models and specifications. Thus, the Krometriks device offers a practical colorimetric platform that has the potential to provide accessible and affordable miRNA diagnostics for point-of-care and field applications in low-resource settings.

3D-printed phantoms for characterizing SERS nanoparticle detectability in turbid media.

Recent advances in plasmonic nanoparticle synthesis have enabled extremely high per-particle surface-enhanced Raman scattering (SERS) efficiencies. This has led to the development of SERS tags for in vivo applications (e.g. tumor targeting and detection), providing high sensitivity and fingerprint-like molecular specificity. While the SERS enhancement factor is a major contributor to SERS tag performance, in practice the throughput and excitation-collection geometry of the optical system can significantly impact detectability. Test methods to objectively quantify SERS particle performance under realistic conditions are necessary to facilitate clinical translation. Towards this goal, we have developed 3D-printed phantoms with tunable, biologically-relevant optical properties. Phantoms were designed to include 1 mm-diameter channels at different depths, which can be filled with SERS tag solutions. The effects of channel depth and particle concentration on the detectability of three different SERS tags were evaluated using 785 nm laser excitation at the maximum permissible exposure for skin. Two of these tags were commercially available, featuring gold nanorods as the SERS particle, while the third tag was prepared in-house using silver-coated gold nanostars. Our findings revealed that the measured SERS intensity of tags in solution is not always a reliable predictor of detectability when applied in a turbid medium such as tissue. The phantoms developed in this work can be used to assess the suitability of specific SERS tags and instruments for their intended clinical applications and provide a means of optimizing new SERS device-tag combination products.

Plasmonic nanobiosensors for detection of microRNA cancer biomarkers in clinical samples.

MicroRNAs (miRNAs) play an important role in the regulation of biological processes and have demonstrated great potential as biomarkers for the early detection of various diseases, including esophageal adenocarcinoma (EAC) and Barrett's esophagus (BE), the premalignant metaplasia associated with EAC. Herein, we demonstrate the direct detection of the esophageal cancer biomarker, miR-21, in RNA extracted from 17 endoscopic tissue biopsies using the nanophotonics technology our group has developed, termed the inverse molecular sentinel (iMS) nanobiosensor, with surface-enhanced Raman scattering (SERS) detection. The potential of this label-free, homogeneous biosensor for cancer diagnosis without the need for target amplification was demonstrated by discriminating esophageal cancer and Barrett's esophagus from normal tissue with notable diagnostic accuracy. This work establishes the potential of the iMS nanobiosensor for cancer diagnostics via miRNA detection in clinical samples without the need for target amplification, validating the potential of this assay as part of a new diagnostic strategy. Combining miRNA diagnostics with the nanophotonics technology will result in a paradigm shift in achieving a general molecular analysis tool that has widespread applicability for cancer research as well as detection of cancer. We anticipate further development of this technique for future use in point-of-care testing as an alternative to histopathological diagnosis as our method provides a quick result following RNA isolation, allowing for timely treatment.

Direct SERDS sensing of molecular biomarkers in plants under field conditions.

Molecular biomarkers such as microRNAs (miRNAs) play important roles in regulating various developmental processes in plants. Understanding these pathways will help bioengineer designing organisms for efficient biomass accumulation. Current methods for RNA analysis require sample extraction and multi-step sample analysis, hindering work in field studies. Recent work in the incorporation of nanomaterials for plant bioengineering research is leading the way of an agri-tech revolution. As an example, surface-enhanced Raman scattering (SERS)-based sensors can be used to monitor RNA in vivo. However, the use of SERS in the field has been limited due to issues with observing Raman signal over complex background. To this end, shifted-excitation Raman difference spectroscopy (SERDS) offers an effective solution to extract the SERS signal from high background based on a physical approach. In this manuscript, we report the first application of SERDS on SERS sensors. We investigated this technique on SERS sensor developed for the detection of a microRNA biomarker, miR858. We tested the technique on in vitro samples and validated the technique by detecting the presence of exogenous miR858 in plants directly under ambient light in a growth chamber. The possibility of moving the detection of nucleic acid targets outside the constraints of laboratory setting enables numerous important bioengineering applications. Such applications can revolutionize biofuel production and agri-tech through the use of nanotechnology-based monitoring of plant growth, plant health, and exposure to pollution and pathogens.

SERS in Plain Sight: A Polarization Modulation Method for Signal Extraction.

Surface-enhanced Raman spectroscopy (SERS) is a powerful analytical spectroscopy offering advantages ranging from "vibrational fingerprints" to multiplexed detection. However, the use of this technique in real-world applications has been limited due to difficulties in detecting inherently weak Raman signals often embedded in strong interfering background signals. A variety of plasmonics-active platforms have been developed to increase Raman signals but are not sufficient to extract weak SERS signals from intense interfering background signals. Herein, we describe a practical method, referred to as polarization modulation-SERS (PM-SERS), which utilizes the polarization dependence of anisotropic SERS-active nanostructures to modulate the plasmonic effect to extract SERS signals and remove background. The modulation is obtained by switching the polarization of the excitation source at a specific frequency involving addition of only few optical components such as liquid crystal polarizers to a typical Raman setup. In this work, we characterized the polarization-dependent response of the SERS substrates fabricated using the oblique angle evaporation (OAV) technique and their response under laser excitation using a polarization modulated source. We demonstrated that the PM-SERS method can extract the analyte weak SERS signals from the strong interfering background signal in different situations, involving a fluorescent sample and a strong background light, and we show the possibility of using PM-SERS at a quasi-real time rate (0.5 Hz). We believe that the PM-SERS method will help expand the translation of applications that utilize SERS-substrates to real-world settings.

Inverse Molecular Sentinel-Integrated Fiberoptic Sensor for Direct and in Situ Detection of miRNA Targets.

Molecular advances have been made in analysis systems for a wide variety of applications ranging from biodiagnostics, biosafety, bioengineering, and biofuel research applications. There are, however, limited practical tools necessary for in situ and accurate detection of nucleic acid targets during field work. New technology is needed to translate these molecular advances from laboratory settings into the real-life practical monitoring realm. The exquisite characteristics (e.g., sensitivity and adaptability) of plasmonic nanosensors have made them attractive candidates for field-ready sensing applications. Herein, we have developed a fiber-based plasmonic sensor capable of direct detection (i.e., no washing steps required) of nucleic acid targets, which can be detected simply by immerging the sensor in the sample solution. This sensor is composed of an optical fiber that is decorated with plasmonic nanoprobes based on silver-coated gold nanostars (AuNS@Ag) to detect target nucleic acids using the surface-enhanced Raman scattering (SERS) sensing mechanism of nanoprobes referred to as inverse molecular sentinels (iMS). These fiber-optrodes can be reused for several detection-regeneration cycles (>6). The usefulness and applicability of the iMS fiber-sensors was tested by detecting target miRNA in extracts from leaves of plants that were induced to have different expression levels of miRNA targets. These fiber-optrodes enable direct detection of miRNA in plant tissue extract without the need for complex assays by simply immersing the fiber in the sample solution. The results indicate the fiber-based sensors developed herein have the potential to be a powerful tool for field and in situ analysis of nucleic acid samples.