A microfluidic surface-enhanced Raman scattering (SERS) sensor for microRNA in extracellular vesicles with nucleic acid-tyramine cascade amplification

Exosomal microRNA (miRNA) is an ideal candidate of noninvasive biomarker for the early diagnosis of cancer. Sensitive and accurate sensing of abnormal exosomal miRNA plays essential role for clinical promotion due to its close correlation with tumor proliferation and progression. Herein, a microfluidic surface-enhanced Raman scattering (SERS) sensor was proposed for an on-line detection of exosomal miRNA based on rolling circle amplification (RCA) and tyramine signal amplification (TSA) strategy. The microfluidic chip consists of a magnetic enrichment chamber, a serpentine fluidic mixer and a plasmonic SERS substrate functionalized with capture probes. The released miRNA activates the capture probe, triggers RCA reaction, and generates a large number of single-stranded DNA products to drive the catalysis of nanotags deposition via TSA, producing numerous “hot spots” to enhance the SERS signals. In merit of the microfluidics chip and nucleic acid-tyramine cascade amplification, the developed SERS sensor significantly improves the sensitivity for the exosomal miRNA assay, resulting in a limit of detection (LOD) as low as 1 pmol/L and can be successfully applied in the analysis of exosomes secreted from breast tumor cells, which demonstrates the potential utility in practical applications.     

纸质空心金纳米笼SERS传感器的制备及对非小细胞肺癌患者痰液中miRNAs的快速高灵敏检测

利用油水界面自组装法获得了单层空心金纳米笼(HGNCs)阵列基底. 通过时域有限差分方法, 证明HGNCs间隙可提供大量“热点”, 从而使基底表现出优异的表面增强拉曼散射(SERS)性能. 同时, 将拉曼信号分子标记的发夹结构DNA与基底链接, 在与目标miRNAs互补杂交后进行SERS信号检测. 结果表明, 基于单层HGNCs阵列基底的SERS传感器具有优良的灵敏度、 可重复性和特异性, 对痰液中miR-196a和miR-21的检出限分别为10.00和36.15 amol/L. 为了验证该SERS传感器在临床检测中的准确性, 利用其对非小细胞肺癌(NSCLC)患者痰液中miR-196a和miR-21进行检测, 并将结果与实时定量多聚核苷酸链式反应技术(qRT-PCR)的检测结果进行了比较. 2种检测方法均显示NSCLC患者痰液中miR-196a和miR-21的表达高于健康人, 检测结果间没有统计学差异, 且相对标准偏差均低于10%. 因此, 纸质空心金纳米笼SERS传感器在NSCLC诊断中具有应用价值, 可能成为生物医学诊断领域miRNAs研究的一个替代工具.     

Biocompatible core-shell nanoparticle-based surface-enhanced Raman scattering probes for detection of DNA related to HIV gene using silica-coated magnetic nanoparticles as separation tools

A novel, highly selective DNA hybridization assay has been developed based on surface-enhanced Raman scattering (SERS) for DNA sequences related to HIV. This strategy employs the Ag/SiO₂ core-shell nanoparticle-based Raman tags and the amino group modified silica-coated magnetic nanoparticles as immobilization matrix and separation tool. The hybridization reaction was performed between Raman tags functionalized with 3′-amino-labeled oligonucleotides as detection probes and the amino group modified silica-coated magnetic nanoparticles functionalized with 5′-amino-labeled oligonucleotides as capture probes. The Raman spectra of Raman tags can be used to monitor the presence of target oligonucleotides. The utilization of silica-coated magnetic nanoparticles not only avoided time-consuming washing, but also amplified the signal of hybridization assay. Additionally, the results of control experiments show that no or very low signal would be obtained if the hybridization assay is conducted in the presence of DNA sequences other than complementary oligonucleotides related to HIV gene such as non-complementary oligonucleotides, four bases mismatch oligonucleotides, two bases mismatch oligonucleotides and even single base mismatch oligonucleotides. It was demonstrated that the method developed in this work has high selectivity and sensitivity for DNA detection related to HIV gene.     

Surface-enhanced Raman scattering detection of DNAs derived from virus genomes using Au-coated paramagnetic nanoparticles

A magnetic capture-based, surface-enhanced Raman scattering (SERS) assay for DNA detection has been developed which utilizes Au-coated paramagnetic nanoparticles (Au@PMPs) as both a SERS substrate and effective bioseparation reagent for the selective removal of target DNAs from solution. Hybridization reactions contained two target DNAs, sequence complementary reporter probes conjugated with spectrally distinct Raman dyes distinct for each target, and Au@PMPs conjugated with sequence complementary capture probes. In this case, target DNAs were derived from the RNA genomes of the Rift Valley Fever virus (RVFV) or West Nile virus (WNV). The hybridization reactions were incubated for a short period and then concentrated within the focus beam of an interrogating laser by magnetic pull-down. The attendant SERS response of each individually captured DNA provided a limit of detection sensitivity in the range 20-100 nM. X-ray diffraction and UV-vis analysis validated both the desired surface plasmon resonance properties and bimetallic composition of synthesized Au@PMPs, and UV-vis spectroscopy confirmed conjugation of the Raman dye compounds malachite green (MG) and erythrosin B (EB) with the RVFV and WNV reporter probes, respectively. Finally, hybridization reactions assembled for multiplexed detection of both targets yielded mixed MG/EB spectra and clearly differentiated peaks which facilitate the quantitative detection of each DNA target. On the basis of the simple design of a single-particle DNA detection assay, the opportunity is provided to develop magnetic capture-based SERS assays that are easily assembled and adapted for high-level multiplex detection using low-cost Raman instrumentation.     

Separation and detection of multiple pathogens in a food matrix by magnetic SERS nanoprobes

A rapid and sensitive method was developed here for separation and detection of multiple pathogens in food matrix by magnetic surface-enhanced Raman scattering (SERS) nanoprobes. Silica-coated magnetic probes (MNPs@SiO₂) of ∼100 nm in diameter were first prepared via the reverse microemulsion method using cetyltrimethylammonium bromide as a surfactant and tetraethyl orthosilicate as the silica precursor. The as-prepared MNPs@SiO₂ were functionalized with specific pathogen antibodies to first capture threat agents directly from a food matrix followed by detection using an optical approach enabled by SERS. In this scheme, pathogens were first immuno-magnetically captured with MNPs@SiO₂, and pathogen-specific SERS probes (gold nanoparticles integrated with a Raman reporter) were functionalized with corresponding antibodies to allow the formation of a sandwich assay to complete the sensor module for the detection of multiple pathogens in selected food matrices, just changing the kinds of Raman reporters on SERS probes. Here, up to two key pathogens, Salmonella enterica serovar Typhimurium and Staphylococcus aureus, were selected as a model to illustrate the probability of this scheme for multiple pathogens detection. The lowest cell concentration detected in spinach solution was 10³ CFU/mL. A blind test conducted in peanut butter validated the limit of detection as 10³ CFU/mL with high specificity, demonstrating the potential of this approach in complex matrices.     

Surface-enhanced Raman scattering molecular sentinel nanoprobes for viral infection diagnostics

In this paper, we describe a surface-enhanced Raman scattering (SERS)-based detection approach, referred to as “molecular sentinel” (MS) plasmonic nanoprobes, to detect an RNA target related to viral infection. The MS method is essentially a label-free technique incorporating the SERS effect modulation scheme associated with silver nanoparticles and Raman dye-labeled DNA hairpin probes. Hybridization with target sequences opens the hairpin and spatially separates the Raman label from the silver surface thus reducing the SERS signal of the label. Herein, we have developed a MS nanoprobe to detect the human radical S-adenosyl methionine domain containing 2 (RSAD2) RNA target as a model system for method demonstration. The human RSAD2 gene has recently emerged as a novel host-response biomarker for diagnosis of respiratory infections. Our results showed that the RSAD2 MS nanoprobes exhibits high specificity and can detect as low as 1 nM target sequences. With the use of a portable Raman spectrometer and total RNA samples, we have also demonstrated for the first time the potential of the MS nanoprobe technology for detection of host-response RNA biomarkers for infectious disease diagnostics.     

空间稳定的SERS标记金纳米棒探针用于免疫检测

报道了空间稳定的表面增强拉曼散射(SERS)标记的金纳米棒探针在免疫检测方面的应用.该探针是将拉曼活性分子4-巯基苯甲酸和生物亲和性高分子α-巯基-ω-羧基聚乙二醇共吸附于金纳米棒表面而制得.其中,聚乙二醇高分子链为探针提供保护作用和空间稳定,使之可以耐受较苛性的条件;其端位的羧基与抗体等靶向实体结合,从而赋予探针检测识别功能.当探针检测待测抗原时(通过固体基底上的捕获抗体、待测抗原和探针上的抗体之间的特异性结合,形成经典“三明治”夹心结构),探针上4-巯基苯甲酸的SERS信号就能示踪出这种识别.该探针对单组分抗原的检出浓度能低至1×10^-9mg·mL^-1.     

Fast and sensitive trace analysis of malachite green using a surface-enhanced Raman microfluidic sensor

A rapid and highly sensitive trace analysis technique for determining malachite green (MG) in a polydimethylsiloxane (PDMS) microfluidic sensor was investigated using surface-enhanced Raman spectroscopy (SERS). A zigzag-shaped PDMS microfluidic channel was fabricated for efficient mixing between MG analytes and aggregated silver colloids. Under the optimal condition of flow velocity, MG molecules were effectively adsorbed onto silver nanoparticles while flowing along the upper and lower zigzag-shaped PDMS channel. A quantitative analysis of MG was performed based on the measured peak height at 1615 cm⁻¹ in its SERS spectrum. The limit of detection, using the SERS microfluidic sensor, was found to be below the 1–2 ppb level and this low detection limit is comparable to the result of the LC-Mass detection method. In the present study, we introduce a new conceptual detection technology, using a SERS microfluidic sensor, for the highly sensitive trace analysis of MG in water.     

An electrochemical biosensor based on DNA “nano-bridge” for amplified detection of exosomal microRNAs

Exosomal miRNAs, as potential biomarkers in liquid biopsy for cancer early diagnosis, have aroused widespread concern. Herein, an electrochemical biosensor based on DNA “nano-bridge” was designed and applied to detect exosomal microRNA-21 (miR-21) derived from breast cancer cells. In brief, the target miR-21 can specifically open the hairpin probe 1(HP1) labeled on the gold electrode (GE) surface through strand displacement reaction. Thus the exposed loop region of HP1 can act as an initiator sequence to activate the hybridization chain reaction (HCR) between two kinetically trapped hairpin probes: HP2 immobilized on the GE surface and biotin labeled HP3 in solution. Cascade HCR leads to the formation of DNA “nano-bridge” tethered to the GE surface with a great deal of “piers”. Upon addition of avidin-modified horseradish peroxidase (HRP), numerous HRP were bound to the formed “nano-bridge” through biotin-avidin interaction to arouse tremendous current signal. In theory, only a single miR-21 is able to trigger the continuous HCR between HP2 and HP3 until all of the HP2 are exhausted. Therefore the proposed biosensor achieved ultrahigh sensitivity toward miR-21 with the detection limit down to 168 amol/L, as well as little cross-hybridization even at the single-base-mismatched level. Successful attempts were also made in the detection of exosomal miR-21 obtained from the MCF-7 of breast cancer cell line. To our knowledge, this is the first attempt to built horizontal DNA nano-structure on the electrode surface for exosomal miRNAs detection. In a word, the high sensitivity, selectivity, low cost make the proposed method hold great potential application for early point-of-care (POC) diagnostics of cancer.     

Detection of single nucleotide polymorphisms by a gold nanowire-on-film SERS sensor coupled with S1 nuclease treatment

Single nucleotide polymorphisms (SNPs) can serve as important biomarkers for genetic diseases, for which accurate detection of SNPs is essential for early diagnosis. We have developed a novel SNP sensor by combining a Au nanowire-on-film surface-enhanced Raman scattering (SERS) platform with S1 nuclease reaction. The combined sensor system provides reproducible SERS signals only in the presence of perfectly matched target DNAs, to probe DNAs as a result of single-stranded DNA-specific degradation by S1 nuclease. Furthermore, point mutations in DNA causing Wilson disease and Avellino corneal dystrophy were successfully identified by this sensor, thereby indicating its practical ability to diagnose genetic diseases.     

A new aptameric biosensor for cocaine based on surface-enhanced Raman scattering spectroscopy

The present study reports the proof of principle of a reagentless aptameric sensor based on surface-enhanced Raman scattering (SERS) spectroscopy with "signal-on" architecture using a model target of cocaine. This new aptameric sensor is based on the conformational change of the surface-tethered aptamer on a binding target that draws a certain Raman reporter in close proximity to the SERS substrate, thereby increasing the Raman scattering signal due to the local enhancement effect of SERS. To improve the response performance, the sensor is fabricated from a cocaine-templated mixed self-assembly of a 3'-terminal tetramethylrhodamine (TMR)-labeled DNA aptamer on a silver colloid film by means of an alkanethiol moiety at the 5' end. This immobilization strategy optimizes the orientation of the aptamer on the surface and facilitates the folding on the binding target. Under optimized assay conditions, one can determine cocaine at a concentration of 1 muM, which compares favorably with analogous aptameric sensors based on electrochemical and fluorescence techniques. The sensor can be readily regenerated by being washed with a buffer. These results suggest that the SERS-based transducer might create a new dimension for future development of aptameric sensors for sensitive determination in biochemical and biomedical studies.     

Silver nanoparticle aggregates on copper foil for reliable quantitative SERS analysis of polycyclic aromatic hydrocarbons with a portable Raman spectrometer

Silver nanoparticle aggregates were synthesized on copper foil, which was used for the surface-enhanced Raman spectroscopy (SERS) detection of polycyclic aromatic hydrocarbons (PAHs) with a portable Raman spectrometer. Silver nanoparticle aggregates were prepared by immersing copper foil in the solution of Sn(2+) and AgNO(3) in a cyclic fashion. A four-cycle process was selected for the following experiments due to its high enhancement and relatively convenient experimental procedure. The substrate has greater temporal stability under continuous laser radiation, good uniformity and reproducibility, which indicated that the substrate could provide reliable measurements. The relationship between SERS intensity and concentrations of PAHs was studied. Quantitative analysis of PAHs in aqueous solution was further performed based on the prepared substrate. The log-log plot of normalized SERS intensity to PAHs concentration exhibited a good linear relationship, with the detection limits in the range of 5-500 μg L(-1). Thus, due to the stability, reproducibility and quantitative results, the prepared substrate could be used as a potential SERS sensor for the analysis of environmental pollutants.     

Plasmonics nanoprobes: detection of single-nucleotide polymorphisms in the breast cancer BRCA1 gene

This paper describes the application of plasmonics-based nanoprobes that combine the modulation of the plasmonics effect to change the surface-enhanced Raman scattering (SERS) of a Raman label and the specificity of a DNA hairpin loop sequence to recognize and discriminate a variety of molecular target sequences. Hybridization with target DNA opens the hairpin and physically separates the Raman label from the metal nanoparticle thus reducing the plasmonics effect and quenching the SERS signal of the label. We have successfully demonstrated the specificity and selectivity of the nanoprobes in the detection of a single-nucleotide polymorphism (SNP) in the breast cancer BRCA1 gene in a homogenous solution at room temperature. In addition, the potential application of plasmonics nanoprobes for quantitative DNA diagnostic testing is discussed.     

树枝状银微纳结构的表面增强拉曼散射效应研究

利用两电极电化学沉积法制备出一种树枝状银微纳结构基体.扫描电子显微镜(SEM)的表征结果证实所制备的银基体呈现出完整的树枝状结构,具有对称性的树枝和树干,且树叶清晰可见.实验结果表明,树枝状银微纳结构的表面增强拉曼散射(Surface-enhanced Raman scattering,SERS)可以检测到超低浓度的罗丹明6G(Rhodamine 6G,R6G,10-10 mol/L)光谱信号,即树枝状银微纳结构作为SERS基体表现出较好的灵敏性;当R6G的浓度在10-5~10-10 mol/L范围依次降低一个数量级时,谱带610 cm-1处的拉曼散射强度的相对标准偏差分别为12.1%,12.0%,11.7%,10.9%,13.2%和14.3%,表明所制备银基体的SERS"热点"(Hot spots)分布较均一,树枝状银微纳结构作为SERS基体具有较好的重现性;当低SERS活性的3-巯基丙酸(3-Mercaptopropionic acid,3MPA)的检测浓度为10-5 mol/L时,利用树枝状银基体能检测到3MPA的SERS光谱,说明所制备的银基体对低活性物质也具有较好的SERS灵敏性.     

Single-step multiplex detection of toxic metal ions by Au nanowires-on-chip sensor using reporter elimination

We have developed a Au nanowires (NWs)-on-chip surface-enhanced Raman scattering (SERS) multiplex sensor that can sensitively detect multiple toxic metal ions. Most importantly, the reporter elimination method simplified the detection procedure to a single step, which has been much desired for remote environmental monitoring. This sensor has several notable features. First, it shows high reproducibility based on well-defined single-crystalline Au NWs. Second, single-NW-sensors that can detect a specific metal ion are combined for multiplex sensing of metal ions. Third, when a sample solution is put onto the NWs-on-chip sensor, a decrease in the SERS signal of a specific NW-sensor identifies the target metal ion. Simple, rapid, sensitive and quantitative detection of metal ions becomes possible through the measurement of the SERS signals. We successfully detected ions of mercury (Hg(2+)), silver (Ag(+)), and lead (Pb(2+)) coexisting in the same solution by using this sensor.     

SERS标记的金纳米棒探针用于免疫检测

报道了基于金纳米棒表面增强拉曼散射(SERS)的免疫检测. 将拉曼活性分子对巯基苯甲酸吸附于金纳米棒表面, 制备出SERS标记的金纳米棒探针. 该探针和蛋白抗体结合形成SERS标记抗体. 通过SERS标记抗体、待测抗原和俘获抗体(固体基底上修饰的抗体, 即俘获抗体)之间的免疫应答反应, 将金纳米棒探针组装到固体基底上, 形成SERS标记抗体-抗原-俘获抗体 “三明治”夹心复合体. 待测抗原浓度越大, 固体基底上俘获的金纳米棒探针的数目越多, 从而可通过SERS信号的强弱来检测待测抗原的浓度. 由于金纳米棒的表面等离子体共振(SPR)峰位置可以在较宽的范围内调控, 可通过激发光和SPR的耦合来提高SERS信号, 从而提高免疫检测的灵敏度. 单组分抗原可检出的浓度范围高于1×10－8 mg/mL.     

复合纳米材料Cu2O@Au对水体中罗丹明B的检测应用研究

制备了多面体Cu2 O纳米粒子,利用Cu2 O的还原性,在其表面原位生成了不同密度的Au纳米粒子,制备了Au、Cu共同增强拉曼信号的复合纳米粒子Cu2 O@Au.利用透射电镜(TEM)、扫描电镜(SEM)、X射线衍射(XRD)等对制备的Cu2 O和Cu2 O@Au的形貌、粒径、表面性能等进行了表征.研究了Cu2 O表面金纳米粒子的分布密度对水样中目标检测物罗丹明B的拉曼增强效果.结果表明,氯金酸浓度在1 mmol/L时制备的Cu2 O@Au表面均匀覆盖一层金纳米粒子,其表面增强拉曼效果最为显著,对水样中罗丹明B检测范围为1×10-2~5×10-6 mol/L.研究了此探针在PBS(1×)和酸性水溶液(0.01 mol/L HCl)中的稳定性,并将其用于沂河水样中靶标的检测实验,结果表明,其稳定性较好.     

Detection of human serum albumin through surface-enhanced Raman scattering using gold "pearl necklace" nanomaterials as substrates

We describe a surface-enhanced Raman scattering (SERS)-based sensor for the detection of human serum albumin (HSA) using gold "pearl necklace" nanomaterials (Au PNNs) as the substrate and AB 580 as the reporter.     

Surface-enhanced Raman spectroscopy of half-mustard agent

The detection and identification of chemical warfare agents is an important analytical goal. Herein, it is demonstrated that 2-chloroethyl ethyl sulfide (half-mustard, CEES) can be successfully analysed using surface-enhanced Raman spectroscopy (SERS). A critical component in this detection system is the fabrication of a robust, yet highly enhancing, sensor surface. Recent advances in substrate fabrication and in the fundamental understanding of the SERS phenomenon enable the development of improved substrates for practical SERS applications.