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Bacterial infections are a leading cause of death globally. The detection of DNA sequences correlated to the causative pathogen has become a vital tool in medical diagnostics. In practice, PCR-based assays for the simultaneous detection of multiple pathogens currently rely on probe-based quantitative strategies that require expensive equipment but have limited sensitivity or multiplexing capabilities. Hence, novel approaches to address the limitations of the current gold standard methods are still in high demand. In this study, we propose a simple multiplex PCR/SERS assay for the simultaneous detection of four bacterial pathogens, namely P. aeruginosa, S. aureus, S. epidermidis, and M. smegmatis. Wherein, specific primers for amplifying each target gDNA were applied, followed by applying SERS nanotags functionalized with complementary DNA probes and Raman reporters for specific identification of the target bacterial pathogens. The PCR/SERS assay showed high specificity and sensitivity for genotyping bacterial pathogen gDNA, whereby as few as 100 copies of the target gDNA could be detected. With high sensitivity and the convenience of standard PCR amplification, the proposed assay shows great potential for the sensitive detection of multiple pathogen infections to aid clinical decision-making. [ABSTRACT FROM AUTHOR]

Surface-enhanced Raman scattering (SERS) nanotags functionalized with lectins as the biological recognition element can be used to target the carbohydrate portion of carbohydrate-carrying molecules (glycoconjugates). An investigation of the optical stability of such functionalized SERS nanotags is an essential initial step before future application and quantification of surface glycan biomarkers on cells and extracellular vesicles. Herein, we report an innovative approach to evaluate the SERS stability of lectin-conjugated nanotags by investigating any possible interfering lectin–lectin interactions in a mixture of different lectin-conjugated SERS nanotags, as well as an assessment of lectin–glycan interaction by mixing wheat germ agglutinin (WGA)-conjugated SERS nanotags with different glycoproteins. No lectin cross-reactivity was found in the mixture of lectin-conjugated SERS nanotags, evidenced by the constant SERS intensity. Additionally, the results showed that the lectins conjugated to SERS nanotags retain their ability to interact with glycans, as evidenced by the changes in the nanotag color and extinction spectra. Their SERS intensity remained constant as supported by finite-element method (FEM) simulation results, demonstrating a high SERS stability and selectivity of lectin-conjugated nanotags towards multiplex applications. [ABSTRACT FROM AUTHOR]

Small extracellular vesicles (sEVs) are lipid bilayer vesicles that carry key molecules (e.g., proteins, DNAs, RNAs, and lipids) for cell-to-cell communication, being regarded as promising biomarkers for cancer diagnosis. However, the detection of sEVs is still challenging due to their unique characteristics such as size and phenotype heterogeneity. The surface-enhanced Raman scattering (SERS) assay is a promising tool for sEV analysis as it shows the advantages of robustness, high sensitivity, and specificity. Previous studies proposed different "sandwich" immunocomplex assembling strategies and various capturing probes for sEV detection by the SERS assay. However, no studies have reported the effect of immunocomplex assembling strategies and capturing probes on the analysis of sEVs using this assay. Hence, to achieve the highest performance of the SERS assay for analysing ovarian cancer-derived sEVs, we first assessed the presence of ovarian cancer markers such as EpCAM on cancer cells and sEVs by using flow cytometry and immunoblotting. We found that cancer cells and their derived sEVs present EpCAM and therefore EpCAM was used to functionalise SERS nanotags for the comparison study of "sandwich" immunocomplex assembling strategies. Then, we compared three types of capturing probes (magnetic beads conjugated with anti-CD9, CD63, or CD81 antibodies) for sEV detection. Our study showed the strategy of pre-mixing of sEVs with SERS nanotags and the anti-CD9 capturing probe would achieve the best performance with the minimum detection of sEVs down to 1.5 × 105 particles per μL and with high specificity in distinguishing sEVs from different ovarian cancer cell lines. We further profiled the surface protein biomarkers (EpCAM, CA125, and CD24) on ovarian cancer-derived sEVs in both PBS and plasma (sEVs spiked in healthy plasma) using the improved SERS assay, showing high sensitivity and specificity. As such, we anticipate that our improved SERS assay has the potential to be used clinically as one of the effective detection methods of ovarian cancer. [ABSTRACT FROM AUTHOR]

The molecular diagnosis of KRAS mutations has become crucial for clinical decision-making in colorectal cancer (CRC) treatments. Currently, the common methods for detecting mutations are based on quantitative PCR, DNA sequencing and droplet digital PCR (ddPCR), which require expensive specialized equipment and testing reagents. Herein, we propose a simple and specific strategy by integrating asymmetric PCR with surface-enhanced Raman spectroscopy (Asy-PCR/SERS) for the detection of KRAS G12V mutation, one of the most common driver mutations in CRC. To discriminate mutant targets from non-targets, Asy-PCR was applied to obtain single-stranded DNA (ssDNA) with unequal amounts of forward and reverse primers, subsequently, detection of the target mutant ssDNA amplicons was attempted by hybridization with Raman reporter-coded and allele-specific oligonucleotide-functionalized gold nanoparticles (SERS nanotags). The oligo encoding of the KRAS G12V mutant sequence could be identified by using a portable Raman spectrometer where the characteristic spectra of SERS nanotags indicate the presence of mutant targets. The Asy-PCR/SERS method showed high specificity and sensitivity for identifying as few as 0.1% mutant alleles of KRAS G12V mutation from non-target sequences. Using colorectal polyp biopsies, we demonstrated that Asy-PCR/SERS assay could distinguish KRAS G12V (c.35G > T) and KRAS G12D (c.35G > A) which occur at the same nucleotide location. As KRAS G12V is a driver oncogene in other cancers including lung, pancreatic, ovarian and endometrial cancers, the proposed assay shows great potential for application in additional tumor streams. [ABSTRACT FROM AUTHOR]

Methicillin-resistant Staphylococcus aureus (MRSA) is considered to be one of the important hospital-acquired pathogens. MRSA is also commonly associated with hospital-acquired infections and mortality. Quantitative and precise detection of MRSA is essential for rapid diagnosis and subsequent effective disease management strategies. We herein developed a highly specific method for rapid MRSA detection that combines surface-enhanced Raman spectroscopy (SERS) nanotags and polymerase chain reaction (PCR). SERS provided the sensitivity and spectral multiplexing capability while PCR provided the specificity required for the assay. The method was tested by the simultaneous detection of two MRSA specific genes (mecA and femA) amplified from genomic DNA isolated from clinical specimens. Magnetic isolation and rapid duplex detection were performed to obtain a detectable signal down to 104 input copies within 80 min. This demonstrated the potential of the SERS–PCR based approach for the accurate identification of MRSA at an early-diagnosis stage. This study also provides an alternative approach to the existing methods for detecting clinical targets without compromising sensitivity and selectivity, and with minimal handling steps. We thus believe that this approach will find a broad application in clinical applications. [ABSTRACT FROM AUTHOR]

Circulating tumor cells (CTCs) are a promising biomarker for monitoring cancer metastasis and therapy response. However, the rarity and inherent fragility of CTCs pose great challenges for CTC detection. Currently, CTC detection requires advanced instruments that might be inaccessible in most hospitals or labs. To allow simple and rapid CTC detection, we propose a sensor platform that relies only on the intrinsic properties of magnetic nanoparticles (MNPs) including rapid separation as well as nanozyme activity. The detection of melanoma CTC, which causes most skin cancer-related deaths, is demonstrated based on bifunctional MNPs. The magnetic property of MNPs enables melanoma CTC isolation and enrichment within 5 min from blood. The nanozyme activity of MNPs allows naked-eye detection of melanoma CTC by catalyzing the oxidation of colourless 3,3′,5,5′-tetramethylbenzidine (TMB) into blue coloured products. Melanoma CTC enumeration can be further achieved by UV-vis measurement. Our platform has successfully detected 13 melanoma CTCs per mL within 50 min. We thus believe this platform could find broad applications both in the clinic and in research. [ABSTRACT FROM AUTHOR]

SERS labels are a new class of nanotags for optical detection based on Raman scattering. Central advantages include their spectral multiplexing capacity due to the small line width of vibrational Raman bands, quantification based on spectral intensities, high photostability, minimization of autofluorescence from biological specimens via red to near-infrared (NIR) excitation, and the need for only a single laser excitation line. Current concepts for the rational design and synthesis of SERS labels are summarized in this review. Chemical constituents of SERS labels are the plasmonically active metal colloids for signal enhancement upon resonant laser excitation, organic Raman reporter molecules for adsorption onto the metal surface for identification, and an optional protective shell. Different chemical approaches towards the synthesis of rationally designed SERS labels are highlighted, including also their subsequent bioconjugation. [ABSTRACT FROM AUTHOR]

Rationally designed gold/silver nanoshells (Au/Ag-NS) with plasmon resonances optimized for red laser excitation in order to minimize autofluorescence from clinical samples exhibit scattering cross-sections, which are ca. one order of magnitude larger compared with solid quasi-spherical gold nanoparticles (Au-NPs) of the same size. Hydrophilic stabilization and sterical accessibility for subsequent bioconjugation of Au/Ag-NS is achieved by coating their surface with a self-assembled monolayer (SAM) of rationally designed Raman reporter molecules comprising terminal mono- and tri-ethylene glycol (EG) spacers, respectively. The stability of the hydrophilically stabilized metal colloid was tested under different conditions. In contrast to metal colloids coated with a SAM without terminal EG spacers, the hydrophilically stabilized SERS particles do not aggregate under physiologically relevant conditions, i.e., buffer solutions with high ionic strength. Using these rationally designed SERS particles in conjunction with a microspectroscopic acquisition scheme, a sandwich immunoassay for the sensitive detection of interleukin-6 (IL-6) was developed. Several control experiments demonstrate the high specificity of the assay towards IL-6, with a lowest detectable concentration of ca. 1 pg mL−1. The signal strength of the Au/Ag-NS is at least one order of magnitude higher compared with hydrophilically stabilized, non-aggregated solid quasi-spherical Au-NPs of the same size. [ABSTRACT FROM AUTHOR]