Novel and simple route to fabricate 2D ordered gold nanobowl arrays based on 3D colloidal crystals

In this study, we present a new method to fabricate large-area two-dimensionally (2D) ordered gold nanobowl arrays based on 3D colloidal crystals by wet chemosynthesis, which combines the advantages of a very simple preparation and an applicability to "real" nanomaterials. By combination of in situ growth of gold nanoshell (GNSs) arrays based on three-dimensional (3D) colloidal silica crystals, a monolayer ordered reversed GNS array (2D ordered GNS array) was conveniently manufactured by an acrylic ester modified biaxial oriented polypropylene (BOPP). 2D ordered gold nanobowl array with adjustable periodic holes, good stability, reproducibility, and repeatability could be obtained when the silica core was etched by HF solution. The surface-enhanced Raman scattering (SERS) enhancement factor (EF) of this 2D ordered gold nanobowl array could reach 1.27 × 10(7), which shows high SERS enhancing activity and can be used as a universal SERS substrate.     

Controlled fabrication of nanopillar arrays as active substrates for surface-enhanced Raman spectroscopy

Highly ordered gold nanopillar arrays were fabricated using anodized aluminum oxide (AAO) templates. Nanopillars with a dimension of 110 +/- 15 nm in vertical height and 75 +/- 10 nm in base diameter were formed with a density of 150 microm(-2). The ordered nanopillar arrays give reproducible surface-enhanced Raman scattering (SERS) at a detection limit of 10(-8) M using thionine as probing molecules. The enhancement by the Au nanopillar arrays was comparable with or better than that of dispersed gold nanoparticle SERS substrates. This work demonstrates a new technique for producing highly ordered and reproducible SERS substrates potentially applicable for chemical and biological assay.     

有序Au/Ag纳米碗阵列的简易制备及其表面增强拉曼散射性能

通过湿法化学合成基于SiO₂胶体晶体的大面积有序Au/Ag纳米碗(Au/AgNB)阵列。首先,在玻璃基板上以3D SiO₂胶体晶体作为模板。然后,在Au纳米颗粒(AuNP)种子的帮助下,通过原位生长方法在模板上沉积一层Au纳米壳(AuNS)。再通过HCHO还原Ag⁺使AuNS表面进一步沉积Ag纳米壳,形成Ag/Au双纳米壳(Ag/AuNS)阵列。通过丙烯酸酯改性双向取向聚丙烯(BOPP)方便地获得了单层有序反转Ag/AuNB阵列。这种有序Au/AgNB阵列具有更佳的表面增强拉曼散射(SERS)活性,其SERS分析增强因子(AEF)可达2.23×10⁷。     

有序Au/Ag纳米碗阵列的简易制备及其表面增强拉曼散射性能

通过湿法化学合成基于SiO₂胶体晶体的大面积有序Au/Ag纳米碗(Au/AgNB)阵列。首先,在玻璃基板上组装3D SiO₂胶体晶体作为模板。然后,以Au纳米颗粒(AuNP)为种子,通过原位生长法在SiO₂模板上沉积一层Au纳米壳(AuNS)。再通过HCHO还原Ag⁺成Ag⁰,进一步在AuNS表面沉积Ag纳米壳,形成Ag/Au双纳米壳(Ag/AuNS)阵列。最后通过丙烯酸酯改性双向取向聚丙烯(BOPP)膜方便地获得了单层有序反转Ag/AuNB阵列。这种有序Au/AgNB阵列具有更佳的表面增强拉曼散射(SERS)活性,其SERS分析增强因子(AEF)可达2.23×10⁷。     

A Novel Electrochemical Aptasensor for Carcinoembryonic Antigen Detection Based on Target‐induced Bridge Assembly

The present study describes a novel electrochemical aptasensor for detection of carcinoembryonic antigen (CEA), a key cancer biomarker. The sensing strategy relied on the CEA‐induced bridge assembly, as a physical barrier, on the surface of gold electrode, resulting in a significant increase of the sensor sensitivity. Under optimal conditions, the aptasensing platform showed a wide linear range (3 pg/mL to 40 ng/mL) and a low detection limit (0.9 pg/mL). Some possible interfering materials were also assessed and the results indicated that the designed aptasensor had good specificity toward CEA. The quantitation of CEA in the spiked human serum samples confirmed the reliability and applicability of the electrochemical aptasensor. So, the developed sensing method has a potential application in the clinical diagnosis.     

Polymerization-assisted signal amplification for electrochemical detection of biomarkers

We present a novel immunosensor by using polymerization-assisted signal amplification strategy coupled with electrochemical detection. A sandwich immunoassay process was used to immobilize a polymerization reaction center, the initiator-conjugated polyclonal prostate specific antigen (PSA) or polyclonal carcinoembryonic antigen (CEA) antibodies on the surface of the electrode. Activator generated electron transfer for atom transfer radical polymerization (AGET ATRP) subsequently triggered the local accumulation of glycidyl methacrylate (GMA) monomers. Growth of long chain polymers provided excess epoxy groups for electrochemical tags aminoferrocene (FcNH(2)) coupling, which in turn significantly increased the loading of the signal molecules and enhanced the electrochemical readouts. The detection limit was ～0.14 pg mL(-1) for PSA and ～0.10 pg mL(-1) for CEA in PBS buffers. The proposed immunosensor was highly sensitive, selective and has a good match to the clinical electrochemiluminescent method. This suggested that the polymerization-assisted immunosensing strategy could be used as an effective method to significantly enhance signal output of the sandwich immunoassays and acted as a promising platform for the clinical screening of cancer biomarkers.     

基于杂交链式反应的可视化免疫分析检测癌胚抗原

构建了基于杂交链反应的比色免疫分析方法,实现了对肿瘤标志物癌胚抗原的检测。在抗原-抗体的特异性结合作用下,在磁珠表面构建夹心式免疫复合物,进一步结合杂交链式反应(HCR)作为信号放大策略,将染料曙红Y嵌入至DNA长链中。在可见光的照射下,能使反应底液中的四甲基联苯胺(TMB)氧化,发生明显的颜色变化,由无色变为蓝色,且与癌胚抗原的浓度呈正相关性。在最优实验条件下,癌胚抗原的浓度在1 pg/mL^5 ng/mL范围内呈线性变化,检出限为1 pg/mL。     

Streptococcus suis II immunoassay based on thorny gold nanoparticles and surface enhanced Raman scattering

An immunoassay based on surface enhanced Raman scattering (SERS) spectroscopy was developed to detect muramidase released protein (MRP) antibody against Streptococcus suis II (SS2) utilizing thorny gold nanoparticles (tAuNPs) as SERS substrates. Initially, tAuNPs with multi-branches were prepared by the seed-mediated growth method in the absence of templates and surfactants, facilitating p-mercaptobenzoic acid (pMBA) conjugation covalently onto the tAuNPs through S-Au bonds. The obtained immuno-SERS tag affording strong Raman signals made it possible to establish an application of indirect detection of the MRP antibody against SS2 with a sandwich assay at a highly sensitive level. The Raman intensity at 1588 cm(-1) was proportional to the logarithm of the concentration of MRP antibody in the range of 10 pg mL(-1) to 0.1 μg mL(-1). The detection sensitivity was significantly improved to 0.1 pg mL(-1) by using the immuno-SERS tags. Furthermore, the proposed SERS approach was applied to detect MRP antibody in pig serum samples, and the results agreed well with those of ELISA, indicating great potential for clinical application in diagnostic immunoassays.     

Synthesis and Characterization of Silver Nanoparticle Modified 3-Aminophenol Resin Microspheres with Application for Determination of Carcinoembryonic Antigens by Surface-Enhanced Raman Scattering

Uniform phenolic resin microspheres were prepared by the polycondensation of 3-aminophenol and formaldehyde. On the surface of the 3-aminophenol resin microspheres, silver nanoparticles were synthesized in situ and immobilized by simple heating. The composite was employed as a substrate for surface-enhanced Raman scattering (SERS). The SERS enhancement factor was evaluated using 4-mercaptobenzoic acid and Nile blue A as signal molecules. A highly sensitive SERS immunoassay that combined labeled antibody conjugated silver nanoparticle modified 3-aminophenol resin microspheres and coating antibody conjugated magnetic nanoparticles was fabricated to determine carcinoembryonic antigen. A linear relationship was obtained between the Raman intensity and the concentration of carcinoembryonic antigen. The limit of detection was 1.2 picograms per milliliter at a signal-to-noise ratio of three. This is believed to be the first report of a SERS immunoassay using silver nanoparticle modified 3-aminophenol resin microspheres as substrates.     

Inkjet-printed gold nanoparticle electrochemical arrays on plastic. Application to immunodetection of a cancer biomarker protein

Electrochemical detection combined with nanostructured sensor surfaces offers potentially low-cost, high-throughput solutions for detection of clinically significant proteins. Inkjet printing offers an inexpensive non-contact fabrication method for microelectronics that is easily adapted for incorporating into protein immunosensor devices. Herein we report the first direct fabrication of inkjet-printed gold nanoparticle arrays, and apply them to electrochemical detection of the cancer biomarker interleukin-6 (IL-6) in serum. The gold nanoparticle ink was printed on a flexible, heat resistant polyimide Kapton substrate and subsequently sintered to create eight-electrode arrays costing <0.2 euro per array. The inkjet-printed working electrodes had reproducible surface areas with RSD <3%. Capture antibodies for IL-6 were linked onto the eight-electrode array, and used in sandwich immunoassays. A biotinylated secondary antibody with 16-18 horseradish peroxidase labels was used, and detection was achieved by hydroquinone-mediated amperometry. The arrays provided a clinically relevant detection limit of 20 pg mL(-1) in calf serum, sensitivity of 11.4 nA pg(-1) cm(-2), and a linear dynamic range of 20-400 pg mL(-1).     

Liposome-mediated enhancement of the sensitivity in immunoassay based on surface-enhanced Raman scattering at gold nanosphere array substrate

A novel immunoassay based on surface-enhanced Raman scattering (SERS) has been developed. The method exploits the SERS-derived signal from reporter molecules (crystal violet, CV) encapsulated in antibody-modified liposome particles. The antigen is firstly captured by the primary antibody immobilized in microwell plates and then sandwiched by secondary antibody-modified liposome. The CV molecules are released from the liposome and transferred to specially designed substrate of gold nanosphere arrays with sub-10-nm gaps. The concentration of the antigen is indirectly read out by the SERS intensity of the CVs. The substrate used could substantially improve the sensitivity and reproducibility of SERS measurement. The SERS intensity responses are linearly correlated to logarithm of antigen concentration in the range of 1.0 x 10(-8) to 1.0 x 10(-4) gm L(-1) with a detection limit of 8 ng mL(-1). To our knowledge, this is the first report describing liposome-mediated enhancement of the sensitivity in immunoassay based on surface-enhanced Raman scattering. Experimental results show that the proposed method illustrates a potential prospect of applications in immunoassay.     

Ultrasensitive electrochemiluminescence immunoassay for tumor marker detection using functionalized Ru-silica@nanoporous gold composite as labels

In this work, we reported a simple and sensitive sandwich-type electrochemiluminescence (ECL) immunosensor for carcinoembryonic antigen (CEA) on a gold nanoparticles (AuNPs) modified glassy carbon electrode (GCE). The Ru-silica (Ru(bpy)(3)(2+)-doped silica) capped nanoporous gold (NPG) (Ru-silica@NPG) composite was used as an excellent label with amplification techniques. The NPG was prepared with a simple dealloying strategy, by which silver was dissolved from silver/gold alloys in nitric acid. The primary antibody was immobilized on the AuNPs modified electrode through l-cysteine and glutaraldehyde, and then the antigen and the functionalized Ru-silica@NPG composite labeled secondary antibody were conjugated successively to form a sandwich-type immunocomplex through the specific interaction. The concentrations of CEA were obtained in the range from 1 pg mL(-1) to 10 ng mL(-1) with a detection limit of 0.8 pg mL(-1). The as-proposed ECL immunosensor has the advantages of high sensitivity, specificity and stability and could become a promising technique for tumor marker detection.     

Study on urinary metabolic profile of phenylketonuria by micellar electrokinetic capillary chromatography with dual electrochemical detection--potential clinical application in fast diagnosis of phenylketonuria

In this report, we present a novel approach to detect clenbuterol based on competitive surface-enhanced Raman scattering (SERS) immunoassay. Herein, a SERS nanoprobe that relies on gold nanoparticle (GNP) is labeled by 4,4'-dipyridyl (DP) and clenbuterol antibody, respectively. The detection of clenbuterol is carried out by competitive binding between free clenbuterol and clenbuterol-BSA fastened on the substrate with their antibody labeled on SERS nanoprobes. The present method allows us to detect clenbuterol over a much wider concentration range (0.1-100 pg mL(-1)) with a lower limit of detection (ca. 0.1 pg mL(-1)) than the conventional methods. Furthermore, by the use of this new competitive SERS immunoassay, the clenbuterol-BSA (antigen) is chosen to fasten on the substrate instead of the clenbuterol antibody, which could reduce the cost of the assay. Results demonstrate that the proposed method has the wide potential applications in food safety and agonist control.     

Fabrication of immunosensor microwell arrays from gold compact discs for detection of cancer biomarker proteins

A simple method is reported to fabricate gold arrays featuring microwells surrounding 8-electrodes from gold compact discs (CDs) for less than $0.2 per chip. Integration of these disposable gold CD array chips with microfluidics provided inexpensive immunoarrays that were used to measure a cancer biomarker protein quickly at high sensitivity. The gold CD sensor arrays were fabricated using thermal transfer of laserjet toner from a computer-printed pattern followed by selective chemical etching. Sensor elements had an electrochemically addressable surface area of 0.42 mm(2) with RSD <2%. For a proof-of-concept application, the arrays were integrated into a simple microfluidic device for electrochemical detection of cancer biomarker interleukin-6 (IL-6) in diluted serum. Capture antibodies of IL-6 were chemically linked onto the electrode arrays and a sandwich immunoassay protocol was developed. A biotinylated detection antibody with polymerized horseradish peroxidase labels was used for signal amplification. The detection limit of IL-6 in diluted serum was remarkably low at 10 fg mL(-1) (385 aM) with a linear response with log of IL-6 concentration from 10 to 1300 fg mL(-1). These easily fabricated, ultrasensitive, microfluidic immunosensors should be readily adapted for sensitive detection of multiple biomarkers for cancer diagnostics.     

Solution-based direct readout surface enhanced Raman spectroscopic (SERS) detection of ultra-low levels of thiram with dogbone shaped gold nanoparticles

We report the use of two different sizes of dogbone shaped gold nanoparticles as colloidal substrates for surface enhanced Raman spectroscopy (SERS) based detection of ultra-low levels of thiram, a dithiocarbamate fungicide. We demonstrate the ability to use a solution based, direct readout SERS method as a quantitative tool for the detection of ultra-low levels of thiram. The two different sizes of dogbone shaped gold nanoparticles are synthesized by using the seed-mediated growth method and characterized by using UV-visible spectroscopy and transmission electron microscopy (TEM). The smaller dogbone shaped nanoparticles have an average size of 43 ± 13 nm. The larger dogbone shaped gold nanoparticles have an average size of 65 ± 15 nm. The nanoparticle concentration is 1.25 × 10(11) nanoparticles per mL for the smaller dogbone shaped gold nanoparticles and is 1.13 × 10(11) nanoparticles per mL for the larger dogbone shaped gold nanoparticles. Different concentrations of thiram are allowed to bind to the two different sizes of dogbone shaped gold nanoparticles and the SERS spectra are obtained. From the calibration curve, the limit of detection for thiram is 43.9 ± 6.2 nM when the smaller dogbone shaped gold nanoparticles are used as colloidal SERS substrates In the case of the larger dogbone shaped gold nanoparticles, the limit of detection for thiram is 11.8 ± 3.2 nM. The lower limit of detection obtained by using the larger dogbone shaped gold nanoparticles as colloidal substrates is due to the lightning rod effect, higher contributions from the electromagnetic enhancement effect, and larger number of surface sites for thiram to bind.     

Capacitance Polypyrrole‐based Impedimetric Immunosensor for Interleukin‐10 Cytokine Detection

In the present study, we developed a novel label‐free capacitance impedimetric immunosensor based on the immobilization of the human monoclonal antibody anti‐interleukin‐10 (anti‐IL‐10 mAb) onto polypyrrole (PPy)‐modified silicon nitride (Si₃N₄) substrates. The immunosensor was used for the detection of the recombinant interleukin‐10 antigen (rh IL‐10) that may be secreted in patients at the early stage of inflammation. The immunosensor was created by chemical deposition of PPy conducting layer on pyrrole?silane (SPy)‐treated Si/SiO₂/Si₃N₄ substrates (Si/SiO₂/Si₃N₄?SPy), followed by anti‐IL‐10 mAb immobilization through carboxyl‐functionalized diazonium (CMA) protocol and carbodiimide chemistry. The surface characterization and the biofunctionalization steps were characterized by SEM, FTIR and cyclic voltammetry (CV) while the detection process was carried out by using electrochemical impedance spectroscopy (EIS) analyses. The created immunosensor showed two linear fittings (R²=0.999) for the detection of rh IL‐10 within the concentration range from 1–50 pg/mL. It exhibited high sensitivity (0.1128 (pg/mL)^?1) with a very low limit of detection (LOD)=0.347 pg/mL, more particularly, at the low concentration range (1–10 pg/mL). Thus, this developed polypyrrole‐based immunosensor represents a promising strategy for creation of miniaturized label‐free, fast and highly sensitive biosensors for diagnosis of inflammation biomarkers at very low concentrations with reduced cost.     

Microchip-based multiplex electro-immunosensing system for the detection of cancer biomarkers

Microfluidic-based microchips have become the focus of research interest for immunoassays and biomarker diagnostics. This is due to their aptitude for high-throughput processing, small sample volume, and short analysis times. In this paper, we describe the development of a microchip-based multiplex electro-immunosensing system for simultaneous detection of cancer biomarkers using gold nanoparticles and silver enhancer. Our microchip is composed of biocompatible poly(PDMS) and glass substrates. To fix the antibody-immobilized microbeads, we used pillar-type microfilters within a reaction chamber. An immunogold silver staining (IGSS) method was used to amplify the electrical signal that corresponded to the immune complex. To demonstrate this approach, we simultaneously assayed three cancer biomarkers, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and prostate-specific antigen (PSA) on the microchip. The electrical signal generated from the result of the immunoreaction was measured and monitored by a PC-based system. The overall assay time was reduced from 3-8 h to about 55 min when compared to conventional immunoassays. The working range of the proposed microchip was from 10(-3) to 10(-1) microg/mL of the target antigen.     

Nanoparticle-based substrates for surface-enhanced Raman scattering detection of bacterial spores

The development of ultrasensitive and rapid methods for the detection of bacterial spores is important for medical diagnostics of infectious diseases. While Surface-Enhanced Raman Spectroscopic (SERS) techniques have been increasingly demonstrated for achieving this goal, a key challenge is the development of sensitive and stable SERS substrates or probes. This Minireview highlights recent progress in exploring metal nanoparticle-based substrates, especially gold nanoparticle-based substrates, for the detection of biomarkers released from bacterial spores. One recent example involves assemblies of gold nanoparticles on a gold substrate for the highly sensitive detection of dipicolinic acid (DPA), a biomarker for bacterial spores such as Bacillus anthracis. This type of substrate exploits a strong SERS effect produced by the particle-particle and particle-substrate plasmonic coupling. It is capable of accurate speciation of the biomarker but also selective detection under various reactive or non-reactive conditions. In the case of detecting Bacillus subtilis spores, the limit of detection is quite comparable (0.1 ppb for DPA, and 1.5 × 10(9) spores per L (or 2.5 × 10(-14) M)) with those obtained using silver nanoparticle-based substrates. Implications of the recent findings for improving the gold nanoparticle-based SERS substrates with ultrahigh sensitivity for the detection of bacterial spores are also discussed.     

Adaptive silver films for detection of antibody-antigen binding

Antibody-antigen binding events at a monolayer protein concentration have been demonstrated on nanostructured adaptive silver films (ASFs) using surface-enhanced Raman scattering (SERS) and luminescence-based assays. It is shown that proteins stabilize and restructure the ASF to increase the SERS signal while preserving antigen-binding activity. Evidence for antibody-antigen binding on the ASF substrates is the distinct SERS spectral changes of the surface-bound antibody or antigen without special tags. The activity of the surface-bound proteins and their practical application are validated by independent immunochemical assays. Results are presented to demonstrate that these surfaces can be extended to protein arrays with detection applications distinct from current SERS, fluorescence, or luminescence methods.     

Multiplexed detection of serological cancer markers with plasmon-enhanced Raman spectro-immunoassay

Circulating biomarkers have emerged as promising non-invasive, real-time surrogates for cancer diagnosis, prognostication and monitoring of therapeutic response. Emerging data, however, suggest that single markers are inadequate in describing complex pathologic transformations. Architecting assays capable of parallel measurements of multiple biomarkers can help achieve the desired clinical sensitivity and specificity while conserving patient specimen and reducing turn-around time. Here we describe a plasmon-enhanced Raman spectroscopic assay featuring nanostructured biomolecular probes and spectroscopic imaging for multiplexed detection of disseminated breast cancer markers cancer antigen (CA) 15-3, CA 27-29 and cancer embryonic antigen (CEA). In the developed SERS assay, both the assay chip and surface-enhanced Raman spectroscopy (SERS) tags are functionalized with monoclonal antibodies against CA15-3, CA27-29 and CEA, respectively. Sequential addition of biomarkers and functionalized SERS tags onto the functionalized assay chip enable the specific recognition of these biomarkers through the antibody-antigen interactions, leading to a sandwich spectro-immunoassay. In addition to offering extensive multiplexing capability, our method provides higher sensitivity than conventional immunoassays and demonstrates exquisite specificity owing to selective formation of conjugated complexes and fingerprint spectra of the Raman reporter. We envision that clinical translation of this assay may further enable asymptomatic surveillance of cancer survivors and speedy assessment of treatment benefit through a simple blood test.