Sensitive and reproducible detection of SARS-CoV-2 using SERS-based microdroplet sensor.

Surface-enhanced Raman scattering (SERS)-based assays have been recently developed to overcome the low detection sensitivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SERS-based assays using magnetic beads in microtubes slightly improved the limit of detection (LoD) for SARS-CoV-2. However, the sensitivity and reproducibility of the method are still insufficient for reliable SARS-CoV-2 detection. In this study, we developed a SERS-based microdroplet sensor to dramatically improve the LoD and reproducibility of SARS-CoV-2 detection. Raman signals were measured for SERS nanotags in 140 droplets passing through a laser focal volume fixed at the center of the channel for 15 s. A comparison of the Raman signals of SERS nanotags measured in a microtube with those measured for multiple droplets in the microfluidic channel revealed that the LoD and coefficient of variation significantly improved from 36 to 0.22 PFU/mL and 21.2% to 1.79%, respectively. This improvement resulted from the ensemble average effects because the signals were measured for SERS nanotags in multiple droplets. Moreover, the total assay time decreased from 30 to 10 min. A clinical test was performed on patient samples to evaluate the clinical efficacy of the SERS-based microdroplet sensor. The assay results agreed well with those measured by the reverse transcription-polymerase chain reaction (RT-PCR) method. The proposed SERS-based microdroplet sensor is expected to be used as a new point-of-care diagnostic platform for quick and accurate detection of SARS-CoV-2 in the field.

Ti3C2Tx MXene -facilitated non-selective trapping effect: Efficient SERS detection of exosomal PD-L1.

Biosensors developed through a sandwich approach have demonstrated favorable detection performance for exosomal programmed cell death 1 ligand 1 (ExoPD-L1) detection. However, the reported PD-L1 antibodies, peptides, and aptamers utilized in these biosensors typically bind to the extracellular region, with overlapping binding sites that severely constrain the fabrication of biosensors. In this study, we present a simple approach to specifically identify and analyze ExoPD-L1 through the non-selective trapping effect of Ti3C2TX (X=-O, -F, -OH) MXene on exosomes via the formation of Ti-O-P complexation, and the selective capture of peptide-functionalized Au@MPBA (4-Mercaptophenylboronic acid) @SiO2 surface enhanced Raman scattering (SERS) tags on ExoPD-L1. The biosensor delivered a both hypersensitive and reliable performance in exosome detection with a low limit of detection (20.74 particles/mL) in the linear range of 102 to 5×106 particles/mL. Furthermore, the biosensor demonstrated excellent stability and interference resistance in detecting ExoPD-L1 in clinical serum samples, enabling the easy differentiation of breast cancer patients from healthy controls. This work provides new insights into the design of biosensors for exosome detection and can serve as a replicable template for sandwich immunoassay detection for other types of sensors, including but not limited to SERS.

Fabrication of an Ag-based SERS nanotag for histamine quantitative detection.

A crucial issue in analytical science and physiology is the detection of histamine with high sensitivity, specificity and credibility, which served as an important neurotransmitter in biofluids. Despite the high sensitivity of surface-enhanced Raman spectroscopy (SERS) at the level of single molecule, there are still challenges in providing high sensitivity for histamine with a small cross section. For the selective detection of histamine using SERS, a highly sensitive sandwich structure substrate combining Fe3O4 and an Ag-based SERS nanotag was developed. The Fe3O4@SiO2-COOH served as a capture component for enriching histamine. Upon functionalized Ag nanoparticles with glycine (Gly) and (3-Aminopheyonyl) boronic acid (APBA), they were then used to connect with histamine and serve as a SERS nanotag, respectively. A linear relationship between the Raman intensity and the histamine concentration was observed over the range 10-4-10-8 M with a limit of detection of 7.24 × 10-9 M. This methodology also exhibited good selectivity in the presence of other neurotransmitters. With our new approach, histamine can be detected sensitively and reliably in fish samples, which indicates the potential prospect of an effective method for analyzing histamine in complex specimens.

Core-shell SERS nanotags-based western blot.

Western blot (WB) is the most commonly used scheme for protein identification in life science, but it still faces great challenges in the accurate quantitative detection of low-abundance proteins. Here, we proposed a novel surface-enhanced Raman scattering-based Western blot (SERS-WB) to solve this challenge. SERS nanotags were used as quantitative labels of proteins, which were composed of gold-silver core-shell nanoparticles, and Nile blue A (NBA) molecules were anchored on the interface of the core and shell. The results show that the SERS-WB possessed excellent sensitivity with detection limit of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein of 0.15 pg, as well as wide linear dynamic range (LDR) of 382 fg to 382 ng. In addition, the target protein on nitrocellulose (NC) membrane could be directly identified by colorimetric signal due to the aggregation effect of nanoparticles, which greatly simplifies the procedure. This as-proposed strategy will bring new thoughts to technological innovation of WB.

Au@SiO2 SERS nanotags based lateral flow immunoassay for simultaneous detection of aflatoxin B1 and ochratoxin A.

Agricultural products are frequently contaminated by mycotoxins. Multiplex, ultrasensitive, and rapid determination of mycotoxins is still a challenging problem, which is of great significance to food safety and public health. Herein, a surface-enhanced Raman scattering (SERS) based lateral flow immunoassay (LFA) for the simultaneous on-site determination of aflatoxin B1 (AFB1) and ochratoxin A (OTA) on the same test line (T line) was developed, in this study. In practice, two kinds of Raman reporters 4-mercaptobenzoic acid (4-MBA), and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) encoded silica-encapsulated gold nanotags (Au4-MBA@SiO2 and AuDNTB@SiO2) were used as detection markers to identify the two different mycotoxins. Through systematic optimization of the experimental conditions, this biosensor has high sensitivity and multiplexing with the limits of detection (LODs) at 0.24 pg mL-1 for AFB1 and 0.37 pg mL-1 for OTA. These are far below the regulatory limits set by the European Commission, in which the minimum LODs for AFB1 and OTA are 2.0 and 3.0 µg kg-1. In the spiked experiment, the food matrix are corn, rice, and wheat, and the mean recoveries of the two mycotoxins ranged from 91.0% ± 6.3%-104.8% ± 5.6% for AFB1 and 87.0% ± 4.2%-112.0% ± 3.3% for OTA. These results demonstrate that the developed immunoassay has good stability, selectivity, and reliability, which can be used for routine monitoring of mycotoxin contamination.

Quantitative Detection of Mastitis Factor IL-6 in Dairy Cow Using the SERS Improved Immunofiltration Assay.

Interleukin-6 (IL-6) is generally used as a biomarker for the evaluation of inflammatory infection in humans and animals. However, there is no approach for the on-site and rapid detection of IL-6 for the monitoring of mastitis in dairy farm scenarios. A rapid and highly sensitive surface enhanced Raman scattering (SERS) immunofiltration assay (IFA) for IL-6 detection was developed in the present study. In this assay, a high sensitivity gold core silver shell SERS nanotag with Raman molecule 4-mercaptobenzoic acid (4-MBA) embedded into the gap was fabricated for labelling. Through the immuno-specific combination of the antigen and antibody, antibody conjugated SERS nanotags were captured on the test zone, which facilitated the SERS measurement. The quantitation of IL-6 was performed by the readout Raman signal in the test region. The results showed that the detection limit (LOD) of IL-6 in milk was 0.35 pg mL-1, which was far below the threshold value of 254.32 pg mL-1. The recovery of the spiking experiment was 87.0-102.7%, with coefficients of variation below 9.0% demonstrating high assay accuracy and precision. We believe the immunosensor developed in the current study could be a promising tool for the rapid assessment of mastitis by detecting milk IL-6 in dairy cows. Moreover, this versatile immunosensor could also be applied for the detection of a wide range of analytes in dairy cow healthy monitoring.

Highly sensitive detection of free testosterone assisted by magnetic nanobeads and gap-enhanced SERS nanotags.

The quantitative determination of trace free testosterone (FT) is of great significance for the diagnosis of androgen-related endocrine diseases. Herein, a fascinating detection protocol was developed for highly sensitive FT analysis through a competitive immunoassay mechanism, which was composed of magnetic nanobeads (MNBs) and gap-enhanced surface enhanced Raman scattering (SERS) nanotags. With the MNBs as detection carriers, trace FT could be enriched by simple magnetic separation. The SERS nanotag constructed with silver-gold core-shell nanoparticle was acted as quantitative label, and Raman indicators were located at the interface between silver core and gold shell. It is demonstrated that the as-proposed protocol achieves high detection sensitivity for FT of 12.11 fg mL-1, and wider linear dynamic detection range (LDR) in the concentration of 100 fg mL-1 to 100 ng mL-1 with R2 value of 0.979, which is due to the enhanced Raman signal of the gap-enhanced SERS nanotag and the high surface-to-volume ratio of the MNB, respectively. Taking advantages of such sensitivity and accuracy approach, the as-developed powerful strategy presents potential applications for rapid disease diagnosis through analyzing trace levels of FT, and can also provide guidance for the exploitation of analysis project of other analytes.

Highly sensitive surface-enhanced Raman scattering-based immunosensor incorporating half antibody-fragment for quantitative detection of Alzheimer's disease biomarker in blood.

Blood-based detection of Alzheimer's disease (AD) biomarker has become a prominent method for diagnosis of AD which can replace the complex and invasive cerebrospinal fluid (CSF)-based diagnostic method. However, the application of blood AD biomarker in actual AD diagnosis is hampered by the extremely low concentration of biomarkers in blood, as well as the existence of interfering proteins. Therefore, it is essential to develop a sensitive and specific detection platform to achieve blood-based diagnosis of AD. Here, a surface-enhanced Raman scattering (SERS)-based sensor is developed for the quantitative determination of tau protein in the plasma of AD patients. To acquire femtomolar-level detection limit, this platform involves the use of half antibody fragment immobilized onto head-flocked gold nanopillar SERS substrates and SERS-nanotags. The small size of the half antibody fragment maximizes the effect of plasmon coupling, by reducing the distance between SERS substrates and SERS-nanotags. Also, the use of half antibody fragment improves the antigen recognition ability by immobilizing the antibody with high density and efficient orientation of the antibody. The sensor using these characteristics showed a low detection limit of 3.21 fM and a wide detection range (10 fM - 1 µM). The platform was also able to accurately quantify the tau protein in the clinical plasma sample and correctly distinguish the AD patient from the healthy control. The ultrasensitive and specific SERS immunoassay platform facilitates accurate and early detection of AD biomarkers and can serve as a valuable tool for simple point-of-care testing in clinical diagnosis.

Ultrasensitive and facile detection of multiple trace antibiotics with magnetic nanoparticles and core-shell nanostar SERS nanotags.

Ultrasensitive, multiplex, rapid, and accurate quantitative determination of trace antibiotics remains a challenging issue, which is of importance to public health and safety. Herein, we presented a multiplex strategy based on magnetic nanoparticles and surface-enhanced Raman scattering (SERS) nanotags for simultaneous detection of chloramphenicol (CAP) and tetracycline (TTC). In practice, SERS nanotags based on Raman reporter probes (RRPs) encoded gold-silver core-shell nanostars were used as detection labels for identifying different types of antibiotics, and the magnetic nanoparticles could be separated simply by magnetic force, which significantly improves the detection efficiency, reduces the analysis cost, and simplifies the operation. Our results demonstrate that the as-proposed assay possesses the capacities of high sensitivity and multiplexing with the limits of detection (LODs) for CAP and TTC of 159.49 and 294.12 fg mL-1, respectively, as well as good stability and reproducibility, and high selectivity and reliability. We believe that this strategy holds a great promising perspective for the detection of trace amounts of antibiotics in microsystems, which is crucial to our life. Additionally, the assay can also be used to detect other illegal additives by altering the appropriate antibodies or aptamers.

SERS-active Au@Ag core-shell nanorod (Au@AgNR) tags for ultrasensitive bacteria detection and antibiotic-susceptibility testing.

There is a challenge to obtain an ultrasensitive and rapid approach to detect bacteria and identify resistance. As a powerful bioanalytical tool, surface-enhanced Raman scattering (SERS) in bacterial detection have attracted increasing attentions. Herein, we developed a SERS-active Au@Ag core-shell nanorod (Au@AgNR) tag platform for ultrasensitive bacteria detection and antibiotic-susceptibility testing (AST). The platform established that surface enhanced Raman scattered Rhodamine 6G (R6G) absorption at 1517 cm-1 had a good linearity (RI = 3865 + 193logC; R2 = 0.97) with logarithm of E. coli concentration over a range of 107-102 CFU (colony forming unit)/mL with limit of detection as low 102 CFU/mL. When E. coli was exposed to ampicillin at minimum inhibitory concentration (MIC, 4 µg/mL), Raman spectroscopy showed the obvious variation between ampicillin-susceptible E. coli (Amp--E. coli) and the ampicillin-resistant E. coli (Amp+-E. coli). Combined with principal component analysis (PCA) statistical analysis, the Raman intensity variation mentioned above allows to obtain rapid antibiotic resistance testing (<3.5 h). In addition, E.coli spiked into blood from C57BL/6 mice can be identified clearly, indicating the potential for point-of-care diagnostics.

Molybdenum Trioxide Nanocubes Aligned on a Graphene Oxide Substrate for the Detection of Norovirus by Surface-Enhanced Raman Scattering.

A novel biosensing system based on graphene-mediated surface-enhanced Raman scattering (G-SERS) using plasmonic/magnetic molybdenum trioxide nanocubes (mag-MoO3 NCs) has been designed to detect norovirus (NoV) via a dual SERS nanotag/substrate platform. A novel magnetic derivative of MoO3 NCs served as the SERS nanotag and the immunomagnetic separation material of the biosensor. Single-layer graphene oxide (SLGO) was adopted as the 2D SERS substrate/capture platform and acted as the signal reporter, with the ability to accommodate an additional Raman molecule as a coreporter. The developed SERS-based immunoassay achieved a signal amplification of up to ∼109-fold resulting from the combined electromagnetic and chemical mechanisms of the dual SERS nanotag/substrate system. The developed biosensor was employed for the detection of NoV in human fecal samples collected from infected patients by capturing the virus with the aid of NoV-specific antibody-functionalized magnetic MoO3 NCs. This approach enabled rapid signal amplification for NoV detection with this biosensing technology. The biosensor was tested and optimized using NoV-like particles within a broad linear range from 10 fg/mL to 100 ng/mL and a limit of detection (LOD) of ∼5.2 fg/mL. The practical applicability of the developed biosensor to detect clinical NoV subtypes in human fecal samples was demonstrated by effective detection with an LOD of ∼60 RNA copies/mL, which is ∼103-fold lower than that of a commercial enzyme-linked immunosorbent assay kit for NoV.

Simultaneous isolation and detection of single breast cancer cells using surface-enhanced Raman spectroscopy.

Nowadays, cancer is one of the most dangerous and deadly disease all around the world. Cancer that is diagnosed at early stages is more likely to be treated successfully. Treatment of progressed cancer is very difficult, and generally surviving rates are much lower. Therefore, much research has been focused on developing non-invasive methods for detection of cancer and monitoring of its progress. Within this contribution, we present a novel strategy for selective isolation and detection of breast cancer cell lines (MCF-7 and BT-20) based on surface enhanced Raman scattering (SERS). A simplified protocol based on cell-aptamer interaction has been developed in which core-shell (Au@Fe3O4) nanoparticles (CSNs) were functionalized with a mucin 1 (MUC1) specific aptamer (Apt1) to capture cells through the interaction between Apt1 and overexpressed protein (MUC1) on the surface of the tumor cells. Meanwhile, a SERS nano-tag, synthesized by the conjugation of Apt1 to the surface of BSA coated and with 4-mercaptopyridine (4-Mpy) functionalized gold nanoparticles, was used to detect the isolated cells. As a conclusion, the proposed strategy can be extended to isolate and detect cells more precisely based on the detection of different kinds of biomarkers on the surface of cancer cells, simultaneously.

Culture-Free Detection of Bacterial Pathogens on Plasmonic Nanopillar Arrays Using Rapid Raman Mapping.

We utilized a fast Raman spectral mapping technique for fast detection of bacterial pathogens. Three-dimensional (3D) plasmonic nanopillar arrays were fabricated using the nanolithography-free process consisting of maskless Ar plasma treatment of a polyethylene terephthalate substrate and subsequent metal deposition. Bacterial pathogens were immobilized on the positively charged poly(l-lysine)-coated 3D plasmonic substrate through electrostatic interactions. Then, the bacterial surfaces were selectively labeled with antibody-conjugated surface-enhanced Raman scattering (SERS) nanotags, and Raman mapping images were collected and statistically analyzed for quantitative analysis of bacteria. Salmonella typhimurium was selected as a model pathogen bacterium to confirm the efficacy of our SERS imaging technique. Minimum number of Raman mapping points with statistical reliability was determined to reduce assay time. It was possible to get a statistically reliable standard calibration curve for 529 pixels (laser spot with 60 µm interval), which required a total mapping time of 45 min to get a standard calibration curve for five different concentrations of bacteria in the 0 to 106 CFU/mL range. No amplification step was necessary for quantification because low-abundance target bacteria could be measured using the Raman spectral mapping technique. Therefore, this approach allows accurate quantification of bacterial pathogens without any culturing or enrichment process.

Gold Superparticles Functionalized with Azobenzene Derivatives: SERS Nanotags with Strong Signals.

The surface-enhanced Raman spectroscopy (SERS) nanotag was proposed as a substitute for fluorescent dye for imaging and biosensors several decades ago. However, its weak signal and poor reproducibility has hindered its application. Here, we report a new strategy to form Au superparticles (AuSPs) with high SERS enhancement via one-pot formation and self-assembly of Au nanoparticles (NPs). An azobenzene-carrying Raman reporter was synthesized to exhibit a large Raman cross-section and multiple bands. The self-assembly of the Raman reporter on AuSPs generated SERS nanotags with intense signals. A Raman reporter carrying boronic acid and azobenzene groups displayed six distinctive bands. Its corresponding SERS nanotag demonstrated a high sensing ability toward glycoprotein through aggregation-induced SERS enhancement or as a substitute for labeled antibodies in an immunoassay of the glycoprotein.

Aggregation induced Raman scattering of squaraine dye: Implementation in diagnosis of cervical cancer dysplasia by SERS imaging.

The extent of squaraine dye aggregation that reflects on surface enhanced Raman signal scattering (SERS) intensity upon adsorption on nano-roughened gold surface has been investigated. Here we have synthesized a serious of six squaraine dyes consisting of two different electron donor moiety i.e. 1,1,2-trimethyl-1H-benzo[e]indole and 2-methylbenzo[d]thiazole which modulates the chemisorptions and hydrophobicity being designated as SQ1, SQ2, SQ3, SQ4, SQ5 and SQ6. Interestingly, SQ2 (mono lipoic acid appended), SQ5 and SQ6 (conjugated with hexyl and dodecyl side chain) squaraine derivatives having more tendency of aggregation in DMSO-water mixed solvent showed significant increase of Raman scattering in the fingerprint region when chemisorbed on spherical gold nanoparticles. Two sets of SERS nanotags were prepared with colloidal gold nanoparticle (Au-NPs size: 40 nm) by incorporating Raman reporters SQ2 and SQ5 followed by thiolated PEG encapsulation (SH-PEG, SH-PEG-COOH) denoted as AuNPs-SQ2-PEG and AuNPs-SQ5-PEG. Further conjugation of these nanotag with monoclonal antibodies specific to over expressed receptors, EGFR and p16/Ki-67 in cervical cancer cell, HeLa showed prominent SERS mapping intensity and selectivity towards cell surface and nucleus. The fast and accurate recognition obtained by antibody triggered SERS-nanotag has been compared with conventional time consuming immunocytochemistry technique which prompted us to extend further investigation using real patient cervical smear sample for a non-invasive, ultrafast and accurate diagnosis.

Sensitive multiplex detection of serological liver cancer biomarkers using SERS-active photonic crystal fiber probe.

Surface-enhanced Raman scattering (SERS) spectroscopy possesses the most promising advantage of multiplex detection for biosensing applications, which is achieved due to the narrow 'fingerprint' Raman spectra from the analyte molecules. We developed an ultrasensitive platform for the multiplex detection of cancer biomarkers by combining the SERS technique with a hollow-core photonic crystal fiber (HCPCF). Axially aligned air channels inside the HCPCF provide an excellent platform for optical sensing using SERS. In addition to the flexibility of optical fibers, HCPCF provides better light confinement and a larger interaction length for the guided light and the analyte, resulting in an improvement in sensitivity to detect low concentrations of bioanalytes in extremely low sample volumes. Herein, for the first time, we demonstrate the sensitive multiplex detection of biomarkers immobilized inside the HCPCF using antibody-conjugated SERS-active nanoparticles (SERS nanotags). As a proof-of-concept for targeted multiplex detection, initially we carried out the sensing of epidermal growth factor receptor (EGFR) biomarker in oral squamous carcinoma cell lysate using three different SERS nanotags. Subsequently, we also achieved simultaneous detection of hepatocellular carcinoma (HCC) biomarkers-alpha fetoprotein (AFP) and alpha-1-antitrypsin (A1AT) secreted in the supernatant from Hep3b cancer cell line. Using a SERS-HCPCF sensing platform, we could successfully demonstrate the multiplex detection in an extremely low sample volume of ∼20 nL. In future, this study may lead to sensitive biosensing platform for the low concentration detection of biomarkers in an extremely low sample volume of body fluids to achieve early diagnosis of multiple diseases. (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).