"Three-in-One" Plasmonic Au@PtOs Nanocluster Driven Lateral Flow Assay for Multimodal Cancer Exosome Biosensing.

Exploiting the multiple properties of nanozymes for the multimode lateral flow assay (LFA) is urgently required to improve the accuracy and versatility. Herein, we developed a novel plasmonic Au nanostar@PtOs nanocluster (Au@PtOs) as a multimode signal tag for LFA detection. Based on the PtOs bimetallic nanocluster doping strategy, Au@PtOs can indicate both excellent SERS enhancement and nanozyme catalytic activity. Meanwhile, Au@PtOs displays a better photothermal effect than that of Au nanostars. Therefore, catalytic colorimetric/SERS/temperature three-mode signals can be read out based on the Au@PtOs nanocomposite. The Au@PtOs was combined with LFA and applied for breast cancer exosome detection. The detection limit for the colorimetric/SERS/temperature mode was 2.6 × 103/4.1 × 101/4.6 × 102 exosomes/µL, respectively, which was much superior to the common Au nanoparticles LFA (∼105 exosomes/µL). Moreover, based on the fingerprint molecular recognition ability of the SERS mode, exosome phenotypes derived from different breast cancer cell lines can be discriminated easily. Based on the convenient visual colorimetric mode and sensitive SERS/temperature quantitative modes, Au@PtOs driven LFA can satisfy the requirements of accurate and flexible multimodal sensing in different application scenarios.

Portable fluorescent lateral flow assay for ultrasensitive point-of-care analysis of acute myocardial infarction related microRNA.

Point-of-care quantitative analysis of tracing microRNA disease-biomarkers remains a great challenge in the clinical diagnosis. In this paper, we developed a portable fluorescent lateral flow assay for ultrasensitive quantified detection of acute myocardial infarction related microRNAs in bio-samples. SiO2@DQD (bilayer quantum dots assembly with SiO2 core) based fluorescent lateral flow strip was fabricated as the analysis tool. In order to quantify the tracing microRNA in biosamples, a catalytic hairpin assembly and CRISPR/Cas12a cascade amplification method was performed and combined with the fabricated SiO2@DQD lateral flow strip. Thus, our platform gathered double advantages of portability and ultrasensitive quantification. Based on our strips, target myocardial biomarker microRNA-133a can be detected with a detection limit of 0.32 fM, which was almost 1000-fold sensitive compared with previous reported microRNAs-lateral flow strips. Significantly, this portable fluorescent strip can directly detect microRNAs in serum without any pretreatment and PCR amplification steps. When spiked in serum samples, a recovery of 99.65 %-102.38 % can be obtained. Therefore, our method offers a potential tool for ultrasensitive quantification of diseases related microRNA in the point-of-care diseases diagnosis field.

Electrostatic Adsorption of Dense AuNPs onto Silica Core as High-Performance SERS Tag for Sensitive Immunochromatographic Detection of Streptococcus pneumoniae.

Streptococcus pneumoniae (S. pneumoniae) is a prominent pathogen of bacterial pneumonia and its rapid and sensitive detection in complex biological samples remains a challenge. Here, we developed a simple but effective immunochromatographic assay (ICA) based on silica-Au core-satellite (SiO2@20Au) SERS tags to sensitively and quantitatively detect S. pneumoniae. The high-performance SiO2@20Au tags with superior stability and SERS activity were prepared by one-step electrostatic adsorption of dense 20 nm AuNPs onto 180 nm SiO2 core and introduced into the ICA method to ensure the high sensitivity and accuracy of the assay. The detection limit of the proposed SERS-ICA reached 46 cells/mL for S. pneumoniae and was 100-fold more sensitive than the traditional AuNPs-based colorimetric ICA method. Further, considering its good stability, specificity, reproducibility, and easy operation, the SiO2@20Au-SERS-ICA developed here has great potential to meet the demands of on-site and accurate detection of respiratory pathogens.

Split aptamer regulated CRISPR/Cas12a biosensor for 17ß-estradiol through a gap-enhanced Raman tags based lateral flow strategy.

It is significant to exploit the full potential of CRISPR/Cas based biosensor for non-nucleic-acid targets. Here, we developed a split aptamer regulated CRISPR/Cas12a and gap-enhanced Raman tags based lateral flow biosensor for small-molecule target, 17ß-estradiol. In this assay, one split aptamer of 17ß-estradiol was designed to complement with crRNA of Cas12a so that the trans-cleavage ability of CRISPR/Cas12a can be regulated by the competitive binding of 17ß-estradiol and split aptamers. Through integration of the signal amplification ability of CRISPR/Cas12a and the ultra-sensitive gap-enhanced Raman tags based lateral flow assay, a visible-SERS dual mode determination of 17ß-estradiol can be established. 17ß-estradiol can be visibly recognized as low as 10 pM and accurately quantified with a detection limit of 180 fM by SERS signals, which is at least 103-fold lower than that of the previous immunoassay lateral flow strategies. Our assay provides a novel perspective to develop split aptamer regulated CRISPR/Cas12a coupling with SERS lateral flow strips for ultrasensitive and easy-to-use non-nucleic-acid targets detection.

Ultrasensitive multichannel immunochromatographic assay for rapid detection of foodborne bacteria based on two-dimensional film-like SERS labels.

Rapid and sensitive detection of multiple foodborne bacteria without DNA amplification is still challenging. Here, we proposed an immunochromatographic assay (ICA) with multiplex analysis ability and high sensitivity for direct detection of bacteria in real food samples, based on an improved surface-enhanced Raman scattering (SERS) sensing strategy. Multifunctional Au shell-coated graphene oxide nanosheets (GO@Au) were fabricated and for the first time introduced into the ICA system as a two-dimensional film-like SERS label, which possessed huge surface area, excellent stability, and superior SERS activity. Different from the conventional spherical nanotags, the antibody-conjugated GO@Au nanosheet effectively and rapidly adhered to bacterial cells, improved the dispersibility of bacteria-nanolabel complexes on the ICA strips, and provided numerous stable hotspots for SERS signal enhancement. The combination of GO@Au labels and the ICA system achieved the multiplex and ultrasensitive determination of three major foodborne pathogens, namely, Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella typhimurium, in a single test, with low detection limits (8, 10, and 10 cells/mL) and short detection time (20 min). The proposed biosensor demonstrated high stability and good accuracy in various food samples and is thus a promising tool for the rapid identification of bacteria.

In Situ Exosomal MicroRNA Determination by Target-Triggered SERS and Fe3O4@TiO2-Based Exosome Accumulation.

Exosomal microRNAs (miRNAs) have been proved to be important biomarkers for the early diagnosis of cancers. However, the accurate quantification of exosomal miRNAs is hampered either by laborious exosome isolation and lysis or by RNA extraction and the amplification process. Here, we reported an in situ platform for direct exosomal miRNAs from serum samples. First, locked nucleic acid (LNA)-modified Au@DTNB (DTNB is the Raman reporter molecule 5,5'-dithiobis-(2-nitrobenzoic acid)) was synthesized as surface-enhanced Raman scattering (SERS) tags to enter into exosomes and assemble with target miRNAs to induce hot-spot SERS signals. Second, Fe3O4@TiO2 nanoparticles were added to enrich the exosomes through affinity interaction of the TiO2 shell for further SERS detection. Based on the platform, target miRNAs can be directly qualified in situ with a detection limit of 0.21 fM, which is better or comparable with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and other in situ methods reported before. Moreover, neither capture antibody nor ultracentrifugation pretreatment was needed in the whole detection procedure. Using exosomal miRNA-10b as a proof of concept, pancreatic ductal adenocarcinoma (PDAC) patients can be recognized from normal controls (NCs) with an accuracy of 99.6%. The simple and sensitive in situ exosomal miRNA detection assay can be seen as a noninvasive liquid biopsy assay for clinical cancer diagnostic adaption.

Personalized detection of circling exosomal PD-L1 based on Fe3O4@TiO2 isolation and SERS immunoassay.

Circling exosomal PD-L1 can be expected as a predictor for the clinical responds of anti-PD-1/PD-L1 therapy. Here, we present a simple method integrating capture and analysis of exosomal PD-L1 directly from serum. Firstly, Fe3O4@TiO2 nanoparticles were used to enrich exosomes through the binding of TiO2 shell and hydrophilic phosphate head of the exosome phospholipids. Model exosomes can be enriched and separated from solution within 5 min with a capture efficiency of 96.5%. Secondly, anti-PD-L1 antibody modified Au@Ag@MBA SERS tags were added to label the exosomal PD-L1 for quantification. The whole process can be finished within 40 min with a detection limit of 1 PD-L1+exosome/µL. Furthermore, this method was used for personalized exosomal PD-L1quantification by using a 4 µL clinical serum sample individually. Based on the personalized SERS signal analysis, NSCLC patients can be distinguished from the healthy controls easily. More important, the advantage of clearly individual quantification may help the doctor to discover the relationship of exosomal PD-L1 and the immnuotherapy responds in individual level.

Dual-recognition surface-enhanced Raman scattering(SERS)biosensor for pathogenic bacteria detection by using vancomycin-SERS tags and aptamer-Fe3O4@Au.

Rapid and reliable detection of pathogenic bacteria is vital to prevent and control bacterial diseases. In this study, we present a magnetically assisted surface-enhanced Raman scattering (SERS) biosensor based on the dual-recognition of bacterial cell by aptamer and antibiotic molecules. Aptamer-Fe3O4@Au magnetic nanoparticles (AuMNPs) were synthesized as magnetic and SERS activated substrate for specific bacteria enrichment, vancomycin-SERS tags (Au@MBA) were prepared for the sensitive quantification of pathogenic bacteria. Due to the Au-shell based dual-SERS enhancement and aptamer/vancomycin based dual-recognition ability, a detection limit of 3 cells/mL with a wide dynamic linear range from 10 to 107 cells/mL can be achieved within 50 min without other non-target bacteria interference. When applied in real samples, the approach shows recoveries from 95.0% to 106.4% with relative standard derivation (RSD) less than 5.3%. The SERS strategy could be used to detect a broad range of bacteria by using different aptamers, moreover, the simple operation and precise quantification ability empower this assay great potential in the application of food safety and infectious disease point-of-care diagnosis.

Dual-SERS biosensor for one-step detection of microRNAs in exosome and residual plasma of blood samples for diagnosing pancreatic cancer.

MicroRNAs have been proved to be the biomarker for early detection of pancreatic cancer and the precisely quantitation of the MicroRNA-10b in the blood samples even can distinguish pancreatic cancer from chronic pancreatitis (CP) and normal controls (NC). In this study, we developed a DSN-assisted dual-SERS biosensor for microRNA-10b in exosome and residual plasma of blood samples detection based on the Fe3O4 @Ag-DNA-Au@Ag@DTNB (SERS tag) conjugates. In presence of target microRNA, it can hybridized with the complementary DNA probes. DSN enzyme was then added to selectively cleaves the DNA probe of the DNA/microRNA duplex, SERS tags can be released from the Fe3O4@Ag and SERS intensity quenching can be triggered, the released microRNA can enter the cycle to decluster other DNA and SERS tags. Due to the dual-SERS enhancement of the Fe3O4@Ag-SERS tag conjugates and the recycling signal amplification, a detection limit of 1 aM with single-base recognition can be performed by one step. The target microRNA in plasma-derived exosome and residual supernatant plasma of blood samples from pancreatic ductal adenocarcinoma (PDAC), chronic pancreatitis (CP) and normal controls (NC) were directly quantified and significant SERS signal distinction can be found among them. The precise quantitation, one-step and one-pot operation can ensure this assay a promising future for point-of-care cancer diagnosis technology.

A novel strategy to evaluate the degradation of quantum dots: identification and quantification of CdTe quantum dots and corresponding ionic species by CZE-ICP-MS.

In view of the significance and urgency of the speciation analysis of quantum dots (QDs) and their degradation products for clarifying their degradation rules and toxicity mechanisms, a method for the identification and quantification of CdTe QDs and corresponding ionic species in complex matrices was developed using capillary zone electrophoresis (CZE) coupled to inductively coupled plasma-mass spectrometry (ICP-MS). The quality assessment of commercial CdTe QDs and serum pharmacokinetics of synthesized CdTe QDs in rats were successfully undertaken using the developed CZE-ICP-MS method.

Combined use of vancomycin-modified Ag-coated magnetic nanoparticles and secondary enhanced nanoparticles for rapid surface-enhanced Raman scattering detection of bacteria.

BACKGROUND: Pathogenic bacteria have always been a significant threat to human health. The detection of pathogens needs to be rapid, accurate, and convenient. METHODS: We present a sensitive surface-enhanced Raman scattering (SERS) biosensor based on the combination of vancomycin-modified Ag-coated magnetic nanoparticles (Fe3O4@Ag-Van MNPs) and Au@Ag nanoparticles (NPs) that can effectively capture and discriminate bacterial pathogens from solution. The high-performance Fe3O4@Ag MNPs were modified with vancomycin and used as bacteria capturer for magnetic separation and enrichment. The modified MNPS were found to exhibit strong affinity with a broad range of Gram-positive and Gram-negative bacteria. After separating and rinsing bacteria, Fe3O4@Ag-Van MNPs and Au@Ag NPs were synergistically used to construct a very large number of hot spots on bacteria cells, leading to ultrasensitive SERS detection. RESULTS: The dominant merits of our dual enhanced strategy included high bacterial-capture efficiency (>65%) within a wide pH range (pH 3.0-11.0), a short assay time (<30 min), and a low detection limit (5×102 cells/mL). Moreover, the spiked tests show that this method is still valid in milk and blood samples. Owing to these capabilities, the combined system enabled the sensitive and specific discrimination of different pathogens in complex solution, as verified by its detection of Gram-positive bacterium Escherichia coli, Gram-positive bacterium Staphylococcus aureus, and methicillin-resistant S. aureus. CONCLUSION: This method has great potential for field applications in food safety, environmental monitoring, and infectious disease diagnosis.

Dual-Selective and Dual-Enhanced SERS Nanoprobes Strategy for Circulating Hepatocellular Carcinoma Cells Detection.

The detection of hepatocellular carcinoma (HCC) circulating tumor cells (CTCs) from a blood sample can be a very powerful noninvasive approach for the early detection and therapy of liver cancer. However, the extreme rarity of tumor cells in blood containing billions of other cells makes the capture and identification of CTCs with sufficient sensitivity and specificity a real challenge. Here, a magnetically assisted surface-enhanced Raman scattering (SERS) biosensor for HCC CTC detection is reported for the first time. The biosensor consists of two basic elements: anti-ASGPR antibody-Fe3 O4 @Ag magnetic nanoparticles and anti-GPC3 antibody-Au@Ag@DTNB nanorods. According to the dual-selectivity of the anti-ASGPR and anti-GPC3 antibodies and the dual-enhancement SERS signal of the MNPs silver shell and the Au@Ag NRs SERS tags, a limit of detection of 1 cell mL-1 for HCC CTC in human peripheral blood samples with a linear relationship from 1 to 100 cells mL-1 can be obtained. The system shows good performance in real serum, which suggests it may be a promising tool for HCC clinical diagnosis.

Dual signal amplification strategy for high-sensitivity detection of copper species in bio-samples with a tunable dynamic range.

A facile and sensitive method with a tunable dynamic range has been proposed for the detection of Cu2+ based on the self-cleavage of Cu2+-specific DNAzyme and the Cu2+-based inhibition of HRP activity, and this method was applied to evaluate the copper species in healthy people and WD patients.

Fe3O4@Ag magnetic nanoparticles for microRNA capture and duplex-specific nuclease signal amplification based SERS detection in cancer cells.

A functionalized Fe3O4@Ag magnetic nanoparticle (NP) biosensor for microRNA (miRNA) capture and ultrasensitive detection in total RNA extract from cancer cells was reported in this paper. Herein, Raman tags-DNA probes modified Fe3O4@Ag NPs were designed both as surface-enhanced Raman scattering (SERS) SERS and duplex-specific nuclease signal amplification (DSNSA) platform. Firstly, target miRNAs were captured to the surface of Fe3O4@Ag NPs through DNA/RNA hybridization. In the presence of endonuclease duplex specific nuclease (DSN), one target miRNA molecule could rehybrid thousands of DNA probes to trigger the signal-amplifying recycling. Base on the superparamagnetic of Fe3O4@Ag NPs, target miRNA let-7b can be captured, concentrated and direct quantified within a PE tube without any PCR preamplification treatment. The detection limit was 0.3fM (15 zeptomole, 50µL), nearly 3 orders of magnitude lower than conventional fluorescence based DSN biosensors for miRNA(∼100fM), even single-base difference between the let-7 family members can be discriminated. The result provides a novel proposal to combine the perfect single-base recognition and signal-amplifying ability of the endonuclease DSN with cost-effective SERS strategy for miRNA point-of-care (POC) clinical diagnostics.

Polyethylenimine-interlayered core-shell-satellite 3D magnetic microspheres as versatile SERS substrates.

Precise fabrication of subtle nanogaps amid individual nanoparticles or between adjacent ones to obtain the highest SERS enhancement is still a challenge. Here, we reported a novel approach for fabricating core-shell-satellite 3D magnetic microspheres (CSSM), that easily form a porous 1.5 nm PEI interlayer to accommodate molecules and create sufficient hotspots between the inner Fe3O4@Ag core and outer assembled Au@Ag satellites. Experiments and finite-difference time-domain (FDTD) simulation demonstrated that the enhancement factor (EF) was about 2.03 × 10(8) and 6.25 × 10(6), respectively. In addition, the micro-scale magnetic core endowed the CSSM with a superior magnetic nature, which enabled easy separation and further enhanced Raman signals due to enrichment of targeted analytes and abundant interparticle hotspots created by magnetism-induced aggregation. Our results further demonstrated that the CSSM is expected to be a versatile SERS substrate, which has been verified by the detection of the adsorbed pesticide thiram and the non-adsorbed pesticide paraquat with a detection limit as low as 5 × 10(-12) M and 1 × 10(-10) M, respectively. The novel CSSM can overcome the long-standing limitations of SERS for the trace characterization of various analytes in different solutions and promises to transform SERS into a practical analytical technique.

"Turn on" and label-free core-shell Ag@SiO2 nanoparticles-based metal-enhanced fluorescent (MEF) aptasensor for Hg(2+).

A turn on and label-free fluorescent apasensor for Hg(2+) with high sensitivity and selectivity has been demonstrated in this report. Firstly, core-shell Ag@SiO2 nanoparticles (NPs) were synthetized as a Metal-Enhanced Fluorescent (MEF) substrate, T-rich DNA aptamers were immobilized on the surface of Ag@SiO2 NPs and thiazole orange (TO) was selected as fluorescent reporter. After Hg(2+) was added to the aptamer-Ag@SiO2 NPs and TO mixture buffer solution, the aptamer strand can bind Hg(2+) to form T-Hg(2+)-T complex with a hairpin structure which TO can insert into. When clamped by the nucleic acid bases, the fluorescence quanta yield of TO will be increased under laser excitation and emitted a fluorescence emission. Furthermore, the fluorescence emission can be amplified largely by the MEF effect of the Ag@SiO2 NPs. The whole experiment can be finished within 30 min and the limit of detection is 0.33 nM even with interference by high concentrations of other metal ions. Finally, the sensor was applied for detecting Hg(2+) in different real water samples with satisfying recoveries over 94%.

A fluorescent aptasensor for H5N1 influenza virus detection based-on the core-shell nanoparticles metal-enhanced fluorescence (MEF).

A fluorescent aptasensor system has been designed for the sensitive detection of recombinant hemagglutinin (rHA) protein of the H5N1 influenza virus in human serum. Guanine-richen anti-rHA aptamers by SELEX were immobilized on the surface of the Ag@SiO2 nanoparticles which performed as a metal-enhanced fluorescence (MEF) sensing platform. Thiazole orange (TO) was used as fluorescent tag which reported to the G-quadruplex secondary structural induced by aptamer-rHA binding event. In the absence of rHA protein, TO was free in the solution with almost no fluorescence emission. When rHA protein was added to the solution, the aptamer strand bound rHA protein to form a stable G-quadruplex complex, which can bind TO and excite the fluorescence emission of TO. Moreover, the excited-state TO captured by the G-quadruplex complex was forced to the surface of the Ag@SiO2 nanoparticles and could experience a surface plasmon resonance enhancement which can be transformed into more efficient fluorescence emission signals, therefore, the fluorescence signal of TO can be amplified largely. This system does not require covalent labeling with fluorophores to the aptamer and the background noise is very low. The detection of rHA protein of the H5N1 influenza virus could be operated both in aqueous buffer and human serum with the detection limit of 2 and 3.5ng/mL respectively. More important, the whole detection process can be finished in a PE tube within 30min, which makes it suitable as a self-contained diagnostic kit for H5N1 influenza virus point-of-care (POC) diagnostic.

SERS molecular sentinel for the RNA genetic marker of PB1-F2 protein in highly pathogenic avian influenza (HPAI) virus.

We have developed a simple and sensitive assay for the detection of the RNA genetic marker associated with high pathogenicity influenza (HPAI) virus. The assay constituted of an array of Raman label tagged hairpin-DNA immobilized on a surface-enhanced Raman scattering (SERS) active substrate as the molecular sentinel (MS) reporter. Upon incubation of the assay with the target RNA, the structure of the hairpin-DNA probe changed from stem-loop configuration (closed state) to DNA/RNA hybridization configuration (open state) so that the Raman label tag will be physically separated from the SERS substrate and induce a decrease of Raman scattering intensity. A metal film over nanosphere (MFON) substrate was developed with a SERS enhancement of about 1.7 × 10(5). Based on this MS-modified substrate, the SERS signal showed a linear relationship to the target RNA in the range of 0-60 attomoles and the limit of detect is 2.67 attomoles. The non-complementary RNA sequences control was also detected and no spectral response was observed. The sensing process only required a single hybridization step and post-hybridization washing could also be omitted. Given that this ultrasensitive biosensor assay is free of polymerase chain reaction (PCR) amplification, it would be a potential diagnostic tool for point-of-care HPAI virus detection.

Base pairing at the abasic site in DNA duplexes and its application in adenosine aptasensors.

The binding of nucleosides to abasic site (AP site)-containing DNA duplexes (AP-DNAs) carrying complementary nucleosides opposite the AP site was investigated by thermal denaturation and isothermal titration calorimetric (ITC) experiments. Purine nucleosides show high affinities (K(d) =14.1 µM for adenosine and 41.8 µM for guanosine) for binding to the AP-DNAs, and the interactions are driven primarily by the enthalpy change, similarly to the case of DNA intercalators. In contrast, pyrimidine nucleosides do not show noticeable binding to the AP-DNAs, thus suggesting that stacking interaction at the AP site plays a key role in the binding of purine nucleosides to the AP-DNAs, as revealed by ITC measurements. Next, to apply an AP-DNA as an aptasensor for adenosine, a competitive assay between adenosine and AP-site-binding fluorescent ligand was performed. The assay employs a fluorescent ligand, riboflavin, that binds to the AP site in a DNA duplex, thereby causing fluorescence quenching. By adding adenosine to the riboflavin/AP-DNA complex, the binding of adenosine to the AP site causes release of riboflavin from the AP site, thereby resulting in restoration of riboflavin fluorescence. AP-DNAs can serve as a new class of aptasensors-a limit of detection of 0.7 µM was obtained for adenosine. In contrast to conventional aptasensors for adenosine, the present method shows high selectivity for adenosine over the other nucleotides (AMP, ADP and ATP). The method does not require covalent labelling of fluorophores, and thus it is cost-effective; finally, the method was successfully demonstrated to be applicable for the detection of adenosine in horse serum.