Aptamers-based sandwich assay for silver-enhanced fluorescence multiplex detection

In this work, aptamers-modified silver nanoparticles (AgNPs) were prepared as capture substrate, and fluorescent dyes-modified aptamers were synthesized as detection probes. The sandwich assay was based on dual aptamers, which was aimed to accomplish the highly sensitive detection of single protein and multiplex detection of proteins on one-spot. We found that aptamers-modified AgNPs based microarray was much superior to the aptamer based microarray in fluorescence detection of proteins. The result shows that the detection limit of the sandwich assay using AgNPs probes for thrombin or platelet-derived growth factor-BB (PDGF-BB) is 80 or 8 times lower than that of aptamers used directly. For multiplex detection of proteins, the detection limit was 625 pM for PDGF-BB and 21 pM for thrombin respectively. The sandwich assay based on dual aptamers and AgNPs was sensitive and specific.     

A photopatterned SERS substrate with a sandwich structure for multiplex detection

Substrate photopatterning has provided versatile applications in biomedical fields. Herein, an universal and efficient photoligation reaction has been used to prepare a patterned capture substrate for a sandwich SERS immunoassay. Photoirradiation induces mild and efficient immobilization of antibodies at the desired region of a gold surface, and the antibody-antigen interaction helps the substrate to capture the antigens in solution specifically. After exposing to SERS probes, i.e., the gold nan...     

A photopatterned SERS substrate with a sandwich structure for multiplex detection

Substrate photopatterning has provided versatile applications in biomedical fields. Herein, an universal and efficient photoligation reaction has been used to prepare a patterned capture substrate for a sandwich SERS immunoassay. Photoirradiation induces mild and efficient immobilization of antibodies at the desired region of a gold surface, and the antibody-antigen interaction helps the substrate to capture the antigens in solution specifically. After exposing to SERS probes, i.e., the gold nan...     

Duplexed sandwich immunoassays on a fiber-optic microarray

In this paper, we describe a duplexed imaging optical fiber array-based immunoassay for immunoglobulin A (IgA) and lactoferrin. To fabricate the individually addressable array, microspheres were functionalized with highly specific monoclonal antibodies. The microspheres were loaded in microwells etched into the distal face of an imaging optical fiber bundle. Two microsphere-based sandwich immunoassays were developed to simultaneously detect IgA and lactoferrin, two innate immune system proteins found in human saliva. Individual microspheres could be interrogated for the simultaneous measurement of both proteins. The working concentration range for IgA detection was between 700 pM and 100 nM, while the working concentration range for lactoferrin was between 385 pM and 10 nM. The cross-reactivity between detection antibodies and their non-specific targets was relatively low in comparison to the signal generated by the specific binding with their targets. These results suggest that the degree of multiplexing on this fiber-optic array platform can be increased beyond a duplex.     

A flow-through microarray cell for the online SERS detection of antibody-captured E. coli bacteria

We present an immunoassay microarray flow-through system for the surface-enhanced Raman scattering (SERS) analysis of bacteria. The system has been constructed to support and automatize the nondestructive in situ analysis of different microorganisms in aqueous environment. After the immobilization of the desired antibodies to an activated PEG-coated surface, the chip is placed into the flow cell which is then flushed with the contaminated sample. Finally, colloidal metal nanoparticles are added and the cells are detected label-free by SERS. Here, we introduce the successful imaging of single microorganisms in the flow cell as well as the quantification of microorganisms in water by SERS mapping with a linear range between 4.3 × 10³ to 4.3 × 10⁵ cells/mL. The method has potential for routine application, e.g. for drinking water control.     

SERS opens a new way in aptasensor for protein recognition with high sensitivity and selectivity

SERS aptasensors for protein recognition based on Au nanoparticles labeled with aptamers and Raman reporters have been developed, which opens a new way for protein recognition of high sensitivity and selectivity.     

Application of external micro-spectrophotometric detection to improve sensitivity on microchips

The goal of this work was to increase the sensitivity of a UV–Vis spectrophotometer by decreasing the background noise and lengthening the optical path. A microphotometer has been modified to precisely select very small parts of a microfluidic channel pattern of a chip and to measure light absorbance on a magnified area of the selected part of the channel. The viability of combining a projection microscope and a spectrophotometer for external absorbance measurements on disposable PDMS chips was studied. Besides the external direct detection above a microfluidic channel, the optical pathlength was lengthened by detecting in the region of the perpendicular exit port. Increasing the cross-sectional area of the zone of irradiation improved the signal-to-noise ratio and the limits of detection (LOD).     

Efforts to improve detection sensitivity for capillary electrophoresis coupled to atmospheric pressure photoionization mass spectrometry

Electrospray ionization performs best with volatile buffers. However, generally the best separation performance for capillary electrophoresis (CE) is achieved with non‐volatile buffers. Hyphenation of CE with mass spectrometry (MS) utilizing atmospheric pressure photoionization (APPI) enables use of a wider range of separation buffers without compromising detection sensitivity. As APPI is considered to be mass flow sensitive, the use of a larger inner diameter separation capillary (75 µm) allows larger volumes to be injected, without decreased separation performance, thus providing improved sensitivity (approx. a factor of 10), compared to the use of a 25 µm capillary. However, nebulizing gas flow and position of capillary tip in the sprayer have to be carefully optimized to prevent excessive band broadening. Further improvement in sensitivity (approx. a factor of 2) was obtained by decreasing the distance between the sprayer and ionization region, indicating that a specially designed CE/APPI‐MS interface for low flow rates will be favourable. Copyright © 2010 John Wiley & Sons, Ltd.     

Silica encapsulated SERS nanoprobe conjugated to the bacteriophage tailspike protein for targeted detection of Salmonella

Silica-encapsulated Raman-reporter embedded SERS nanoprobes, named nanoaggregate embedded beads (NAEBs), were conjugated to the Salmonella specific tailspike protein (TSP) isolated from the P22 bacteriophage to enable a highly specific and ultrasensitive optical transduction platform. We demonstrate three successful surface conjugation strategies and highlight the detection of a single bacterium using SERS.     

Dye-doped silica nanoparticle labels/protein microarray for detection of protein biomarkers

We report a dye-encapsulated silica nanoparticle as a label, with the advantages of high fluorescence intensity, photostability, and biocompatibility, in conjunction with microarray technology for sensitive immunoassay of a biomarker, interleukin-6 (IL-6), on a microarray format. The tris(2,2'-bipyridyl)ruthenium(ii) chloride hexahydrate (Rubpy) dye was incorporated into silica nanoparticles using a simple one-step microemulsion synthesis. In this synthesis process, Igepal CA520 was used as the surfactant, therefore, no requirement of cosolvent during the synthesis and the particle size was reduced comparing to the commonly used Triton surfactant system. The nanoparticles are uniform in size with a diameter of 50 nm. The microarray fluorescent immunoassay approach based on dye-doped silica nanoparticle labels has high sensitivity for practical applications with a limit of detection for IL-6 down to 0.1 ng mL(-1). The calibration curve is linear over the range from 0.1 ng mL(-1) to 10 ng mL(-1). Furthermore, results illustrated that the assay is highly specific for IL-6 in the presence of range of cytokines or proteins. The RuDS dye-labeled nanoparticles in connection with protein microarrays show the promise for clinical diagnosis of biomarkers.     

Rhodamine B doped silica nanoparticle labels for protein microarray detection

A core-shell Rhodamine B-doped SiO2 nanoparticle was synthesized and its fluorescent intensity was found to be 1000 times higher than that of individual Rhodamine B molecule. The doped nanoparticles were further conjugated with streptavidin and the resulting nanoparticles were used in the detection of reverse-phase protein microarrays, in which human IgG of various concentrations was first immobilized on aldehyde-modified glass slides and then biotinlyated goat anti human IgG as well as the labeled nanopart...     

SERS detection of protein biochip fabricated by etching polystyrene template

In this study, a nanoscale protein chip is prepared by using an etched polystyrene (PS) template. This protein chip can be directly used for immunoassay, with the help of Surface Enhanced Raman Scattering (SERS) spectra. Some glass slides submerged in aldehyde is initially prepared, modified with antibodies, human immunoglobulin G (IgG). Then PS arrays are self-assembled on these slides with the Langmuir–Blodgett method. The PS template pattern is transferred to the human IgG substrate using an etching process—slides are exposed to O₂ plasma for 90 s. The PS nanoparticles are then washed away using phosphate buffered saline solution. Next, the slides are dipped into bovine serum albumin solution to ensure that the anti IgG would bond only to the human IgG. At this moment, a patterned protein chip is obtained. When used for protein detection, the protein chip could be immersed into labeled specificity antigen solution. Here we chose fluorescein isothiocyanate anti-human IgG. After washing, only bonded antigens remain. Fluorescence microscopy and SERS is used to characterize the samples. The SERS spectra intensity shows liner correlation with the concentration of anti-human IgG. All the experiments are conducted in a phosphate buffered saline solution at 37 °C for 2 h.     

Quantum dot self-assembly for protein detection with sub-picomolar sensitivity

A novel approach to sensitive and rapid antigen detection is described. In the presence of a specific antigen, quantum dot-antibody conjugates rapidly self-assemble into agglomerates that are typically more than 1 order of magnitude larger than their individual components. The size distribution of the agglomerated colloids depends on, among other things, the relative concentration of quantum dot conjugates and antigen molecules. Quantum dot agglomerates mediated by antigen recognition were characterized by measuring their light scattering and fluorescence characteristics in an unmodified flow cytometer. Protein antigens angiopoietin-2 and mouse IgG were detected to sub-picomolar concentrations using this method. This simple technique enables the potential simultaneous detection of multiple antigenic biomarkers directly from physiological media and could be used for early detection and frequent screening of cancers and other diseases.     

Traps and cages for universal SERS detection

In this tutorial review, we provide an overview of the recent research toward surface functionalization of plasmonic nanoparticles for the generation of advanced optical sensors that make possible the analysis of various moieties by means of surface enhanced Raman scattering (SERS). Such moieties include atomic ions, low affinity target molecules, inorganic anions, biometabolites, pathogen markers and/or other analytes of interest even under very demanding circumstances such as those related to real life samples. We expect this review to be of interest to researchers in a broad diversity of fields that can take advantage of the unprecedented sensitivity of this type of molecular spectroscopy, in a wide variety of analytical and bioanalytical problems.     

A simple double-bead sandwich assay for protein detection in serum using UV-vis spectroscopy

In this study a double-bead sandwich assay, employing magnetic nanoparticles and gold nanoparticles is proposed. The magnetic nanoparticles allow specific capturing of the analyte in biological samples, while the optical properties of the gold nanoparticles provide the signal transduction. We demonstrated that a major improvement in the assay sensitivity was obtained by selecting an optimal gold nanoparticle size (60 nm). A detection limit of 5-8 ng/mL, a sensitivity of 0.6-0.8 (pg/mL)⁻¹ and a dynamic range of 3 orders of magnitude were achieved without any further amplification using the detection of prostate specific antigen in serum as a model system. The proposed assay has the ability to be easily implemented within a microfluidic device for point-of-care applications whereby the readout can be executed by a fast and cheap optical measurement.     

A SERS DNAzyme biosensor for lead ion detection

SERS biosensor for sensitive and selective detection of lead ions (Pb(2+)) based on DNAzyme was developed by taking advantage of the specific catalytic reaction of DNAzyme upon binding to Pb(2+) ions. Detection was accomplished by SERS nanoprobe labeled with DNA and Raman reporters for signal amplification.     

分子印迹膜协同银增强电化学信号高灵敏特异性检测啶虫脒

以啶虫脒为模板分子,壳聚糖为功能单体,戊二醛为交联剂,协同Ag颗粒增强导电性,在丝网印刷电极表面合成分子印迹膜,研制了测定啶虫脒的分子印迹传感器。通过循环伏安法(CV)对传感器的性能进行研究,在扫描速率100 mV/s的最优条件下,CV测试表明,啶虫脒浓度在0.01~1.0μmol/L的浓度范围内呈现良好的线性关系(线性相关系数R^2=0.9342)。分子印迹传感器具有良好的印迹效果,相较于其他农药如吡虫啉、水胺硫磷、乙草胺,对啶虫脒有较高的特异性识别能力。构建了啶虫脒分子印迹传感器的动力学吸附模型,测得传感器的印迹因子(β)为3.61,结合速率(k)为19.46 s。传感器用于水样中啶虫脒的测定,加标回收率在92.9%~100.4%。     

DNA microarray: a high throughput approach for methylation detection

We described a DNA microarray-based method combined with bisulphite treatment of DNA and regular PCR to examine hyper-methylation in promoter 1A of APC gene. A set of oligonucleotide probes were designed and immobilized on the aldehyde-coated glass slides for detecting the methylation pattern of 15 selected CpG sites in the region. The methylation status of 30 colorectal tumor samples have been examined by both of methylation-specific PCR (MS-PCR) and the present microarray method. The methylation pattern of the 15 CpG sites for the samples have been obtained with the microarray. A total of 19 samples out of 30 were methylated by microarray, in which five samples cannot be detected by MS-PCR due to the methylated CpG patterns not accordant to the MS-PCR primers. The detecting ratio for methylation of APC gene of colorectal tumor samples increased from 46.7% with MS-PCR to 63.3% with the microarray, which successfully demonstrated that DNA microarray-based method not only can obtained the methylation patterns for the related genes, but also decrease the false-negative results of methylation status by the conventional MS-PCR for the investigated genes.