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.     

A steady-state and flow-through cell for screen-printed eight-electrode arrays

An electrochemical cell has been developed enabling amperometric steady-state- and flow-injection measurements with screen-printed arrays consisting of eight working electrodes (Ø = 1 mm) arranged radially around a printed Ag/AgCl reference electrode in the centre. The cell contained a rotator, providing similar hydrodynamics over all the working electrodes in the array, which was manually centered under the rotator. The reproducibility of steady-state measurements with eight-electrode platinum or gold arrays in this cell was studied by measuring and comparing currents from ferricyanide reduction at each electrode in the array. It was found that the relative standard deviation (R.S.D.) for the currents at different electrodes on one array was below 5%. Similar R.S.D. was found if measurements were compared between several arrays. This indicates that manual insertion/positioning of the eight-electrode array in the cell and hydrodynamics at the electrodes provided measurement reproducibility similar to the reproducibility of manufacturing eight-electrode platinum or gold arrays by screen-printing. A comparative study was performed between screen-printed and through mask sprayed carbon arrays. It was found that the reproducibility of the sprayed arrays was similar to that of the platinum or gold screen-printed arrays, with R.S.D. values below 6% regarding the variation between electrodes within the same array and the variation between different arrays. To enable flow-injection measurements, a tube (0.4 mm inner diameter) was inserted into a hole drilled through the centre of the steady-state cell rotator. This construction made it possible to inject the solution into the cell through the tube (not rotating), while the rotator was spinning over the eight-electrode array. It was found that this combination of flow-injection and mixing by a rotator provided a uniform current response over the array electrodes and that, at optimum conditions, the R.S.D. values between the eight electrodes in the array were nearly the same as in case of the steady-state measurements, i.e., below 5%.     

Sensitive electrochemical immunosensor array for the simultaneous detection of multiple tumor markers

A novel electrochemical immunosensor array for the simultaneous detection of multiple tumor markers was developed by incorporating electrochemically addressing immobilization and one signal antibody strategy. As a proof-of-principle, an eight-electrode array including six carbon screen-printed working electrodes was used as a base array for the analysis of two important tumor markers, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) and a horseradish peroxidase-labeled antibody was employed as a signal antibody. The immunosensor in the array was fabricated in sequence by covalently coupling the capture antibody onto the surface of the desired working electrode, which was firstly electrochemically addressably grafted with an aminophenyl group by reduction of in situ generated aminophenyl diazonium cation generated from p-phenylenediamine, using glutaraldehyde as cross-linker. This allowed the selective immobilization of the capture antibody at the desired position on a single array via an electrochemical operation. The immunoassay in sandwich mode was performed by specifically binding the targets, second antibodies and one signal antibody to the immunosensor array. The result showed that the steady current density was directly proportional to the concentration of target CEA/AFP in the range from 0.10 to 50 ng mL(-1) with a detection limit of 0.03 ng mL(-1) for CEA and 0.05 ng mL(-1) for AFP (S/N = 3), respectively. This work demonstrates that the employment of an electrochemically addressing method for the fabrication of an immunosensor array and one signal antibody is a promising approach for the determination of multiple tumor markers in clinical samples.     

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.     

Determination of Carcinoembryonic Antigen by Surface-Enhanced Raman Spectroscopy Using Gold Nanobowl Arrays

Surface-enhanced Raman scattering (SERS) based on the double-antibody sandwich format is reported for the determination of carcinoembryonic antigen. Ordered gold nanobowl arrays were fabricated and conjugated with anticarcinoembryonic as capturing substrates, and gold nanoshells, adsorbed with 4-mercaptobenzonic acid, were modified with anticarcinoembryonic antigen as labeling tags. After the carcinoembryonic antigen was captured on ordered gold nanobowl arrays, the labeling tags were bonded to the captured carcinoembryonic antigen. The interaction of SERS substrates (ordered gold nanobowl arrays) and SERS labels (gold nanoshells) showed high sensitivity and a low detection limit for carcinoembryonic antigen. The linear dynamic range of SERS for carcinoembryonic antigen was from 5?pg/mL to 100?ng/mL with a linear relationship between carcinoembryonic antigen concentration and SERS intensity. The detection limit was 1.73?pg/mL. SERS detection may be used for other cancer biomarkers and provides potential for the clinical diagnosis of cancer biomarkers.     

Dual amplification strategy for the fabrication of highly sensitive interleukin-6 amperometric immunosensor based on poly-dopamine

An electrochemical immunosensor was studied for sensitive detection of Interleukin-6 (IL-6) based on a dual amplification mechanism resulting from Au nanoparticles (AuNP)-Poly-dopamine (PDOP) as the sensor platform and multienzyme-antibody functionalized AuNP-PDOP@carbon nanotubes (CNT). The stable and robust film, PDOP, was used to immobilize biomolecules not only for the construction of the sensor platform, but also for the signal labeling. Sensitivity was greatly amplified by using the special platform of AuNP-PDOP and synthesizing horseradish peroxidase (HRP)-antibody (Ab(2)) functionalized AuNP-PDOP@carbon nanotubes (CNT). A linear response range of IL-6 from 4.0 to 8.0 × 10(2) pg mL(-1) with a low detection limit of 1.0 pg mL(-1) was obtained by the amperometry determination. Measurements of IL-6 in human serum gave excellent correlations with standard ELISA assays. Moreover, the immunosensor exhibited high selectivity, good reproducibility, and stability.     

Detection of Alzheimer's tau protein using localised surface plasmon resonance-based immunochip

In this study, we present the detection of tau protein, at room temperature, using a multi-spot localised surface plasmon resonance (LSPR)-based immunochip. To the best of our knowledge, this is the first report of an immunochip for tau protein. The detection method includes fabrication of a gold-capped nanoparticle LSPR chip, formation and functionalisation of a self-assembled monolayer (SAM), immobilisation of a suitable linker, effective blocking of non-specific adsorption, immobilisation of a monoclonal anti-tau antibody (tau-mAb), and finally, the optimum conditions for the immuno-reaction between tau-mAb and the antigen were determined. The method has a high performance, enables detection of tau at 10pg/mL, lower than the cut-off value of 195pg/mL (for AD) for tau protein in cerebral spinal fluid (CSF). Further, we demonstrated selectivity of the technique by showing that the introduction of bovine serum albumin (BSA), perhaps the most abundant protein component in serum and CSF, does not interfere with the detection of tau. This method also offers a potential platform for studying tau interactions with other proteins and/or potential drug candidates and could also be easily adapted for detecting phosphorylated tau and other AD biomarkers.     

Disposable electrochemical immunosensor by using carbon sphere/gold nanoparticle composites as labels for signal amplification

This work designed a simple, sensitive, and low-cost immunosensor for the detection of protein marker by using a carbon sphere/gold nanoparticle (CNS/AuNP) composite as an electrochemical label. The nanoscale carbon spheres, prepared with a hydrothermal method by using glucose as raw material, were used to load AuNPs for labeling antibody by electrostatic interaction, which provided a feasible pathway for electron transfer due to the remarkable conductivity. The disposable immunosensor was constructed by coating a polyethylene glycol (PEG) film on a screen-printed carbon-working electrode and then immobilizing capture antibody on the film. With a sandwich-type immunoassay format, the analyte and then the CNS/AuNP-labeled antibody were successively bound to the immunosensor. The bound AuNPs were finally electro-oxidized in 0.1 M HCl to produce AuCl(4)(-) for differential pulse voltammetric (DPV) detection. The high-loading capability of AuNPs on CNS for the sandwich-type immunorecognition led to obvious signal amplification. By using human immunoglobulin?G (IgG) as model target, the DPV signal of AuNPs after electro-oxidized at optimal potential of +1.40?V for 40?s showed a wide linear dependence on the logarithm of target concentration ranging from 10?pg mL(-1) to 10?ng mL(-1). The detection limit was around 9?pg mL(-1). The immunosensor showed excellent analytical performance with cost effectivity, good fabrication reproducibility, and acceptable precision and accuracy, providing significant potential application in clinical analysis.     

Isolation and detection of single molecules on paramagnetic beads using sequential fluid flows in microfabricated polymer array assemblies

We report a method for isolating individual paramagnetic beads in arrays of femtolitre-sized wells and detecting single enzyme-labeled proteins on these beads using sequential fluid flows in microfabricated polymer array assemblies. Arrays of femtolitre-sized wells were fabricated in cyclic olefin polymer (COP) using injection moulding based on DVD manufacturing. These arrays were bonded to a complementary fluidic structure that was also moulded in COP to create an enclosed device to allow delivery of liquids to the arrays. Enzyme-associated, paramagnetic beads suspended in aqueous solutions of enzyme substrate were delivered fluidically to the array such that one bead per well was loaded by gravity. A fluorocarbon oil was then flowed into the device to remove excess beads from the surface of the array, and to seal and isolate the femtolitre-sized wells containing beads and enzyme substrate. The device was then imaged using standard fluorescence imaging to determine which wells contained single enzyme molecules. The analytical performance of this device as the detector for digital ELISA compared favourably to the standard method, i.e., glass arrays mechanically sealed against a silicone gasket; prostate specific antigen (PSA) could be detected from 0.011 pg mL(-1) up to 100 pg mL(-1). The use of an enclosed fluidic device to isolate beads in single-molecule arrays offers a multitude of advantages for low-cost manufacturing, ease of automation, and instrument development to enable applications in biomarker validation and medical diagnosis.     

Gold nanoparticle patterning of silicon wafers using chemical e-beam lithography

This paper demonstrates a novel facile method for fabrication of patterned arrays of gold nanoparticles on Si/SiO2 by combining electron beam lithography and self-assembly techniques. Our strategy is to use direct-write electron beam patterning to convert nitro functionality in self-assembled monolayers of 3-(4-nitrophenoxy)-propyltrimethoxysilane to amino functionality, forming chemically well-defined surface architectures on the 100 nm scale. These nanopatterns are employed to guide the assembly of citrate-passivated gold nanoparticles according to their different affinities for amino and nitro groups. This kind of nanoparticle assembly offers an attractive new option for nanoparticle patterning a silicon surface, as relevant, for example, to biosensors, electronics, and optical devices.     

Thin film processing using S-layer proteins: biotemplated assembly of colloidal gold etch masks for fabrication of silicon nanopillar arrays

We explored the bionanofabrication of silicon nanopillar structures using ordered gold nanoparticle arrays generated from microbial surface layer (S-layer) protein templates. The S-layer template used for these thin film processing experiments was isolated from the Gram-positive bacterium Deinococcus radiodurans. In this preliminary work, S-layers preimmobilized onto chemically modified silicon substrates were initially used to template the fabrication of a nanolithographic hard mask pattern comprised of a hexagonally ordered array of 5-nm gold nanoparticles (lattice constant = 18 nm). Significantly, the use of the biotemplated gold nanoparticle mask patterns in an inductively coupled plasma (ICP) etching process successfully yielded silicon nanopillar structures. However, it was found that the resultant nanopillars (8–13 nm wide at the tip, 15–20 nm wide at half-height, 20–30 nm wide at the base, and 60–90 nm tall) appeared to lack any significant degree of translational ordering. The results suggest that further studies are needed in order to elucidate the optimal plasma processing parameters that will lead to the generation of long-range ordered arrays of silicon-based nanostructures using S-layer protein templates.     

Ultrasensitive Electrochemical Immunosensor for Detection of Tumor Necrosis Factor‐α Based on Functionalized MWCNT‐Gold Nanoparticle/Ionic Liquid Nanocomposite

A novel and highly sensitive electrochemical immunosensor was developed for the detection of protein biomarker tumor necrosis factor‐alpha (TNF‐α) based on immobilization of TNF‐α‐antibody (anti‐TNF‐α) onto robust nanocomposite containing gold nanoparticles (AuNP), multiwalled carbon nanotubes (MWCNTs) and ionic liquid (1‐buthyl‐3‐methylimidazolium bis (trifluoromethyl sulfonyl)imide). Functionalized MWCNT‐gold nanoparticle was produced by one‐step synthesis based on the direct redox reaction. The electrochemical properties of nanocomposite were characterized by electrochemical impedance spectroscopy and cyclic voltammetry. The anti‐TNF‐α was immobilized or entrapped in the nanocomposite and used in a sandwich type complex immunoassay with anti‐TNF‐α labeled with horseradish peroxidase as secondary antibody. Under optimum conditions, the immunosensor could detect TNF‐α in a linear range from 6.0 to 100 pg mL^?1 with a low detection limit of 2.0 pg mL^?1. The simple fabrication method, high sensitivity, good reproducibility, stability, as well as acceptable accuracy for TNF‐α detection in human serum samples are the main advantages of this immunosensor, which might have broad applications in protein diagnostics and bioassay.     

Synthesis of monofunctionalized gold nanoparticles by fmoc solid-phase reactions

In this communication, solid-phase reactions for the synthesis of Lys-monofunctionalized gold nanoparticles are described. A controlled and selective fabrication of linear nanoparticle arrays can be achieved through peptide linkage systems, and therefore it is essential to prepare Fmoc amino acid nanoparticle building blocks susceptible to Fmoc solid-phase peptide synthesis. Gold nanoparticles containing carboxylic acids (2) in the organic shell were covalently ligated to Lys on solid supports through amide bond coupling reactions. We employed Fmoc-Lys-substituted polymer resins such as Fmoc-Lys-Wang or Fmoc-Lys-HMPA-PEGA. The low density of Lys on the matrix enabled 2 nm-sized gold nanoparticles to react with Lys in a 1:1 ratio. Subsequent cleavage reactions using 60% TFA reagent resulted in Lys transfer from the solid matrix to gold nanoparticles, and the Fmoc-Lys-monofunctionalized gold nanoparticles (5) were obtained with 3-15% yield. Synthesis using HMPA-PEGA resin increased productivity due to the superior swelling properties of PEGA resin in DMF. Monofunctionalization of nanoparticles was microscopically characterized using TEM for the ethylenediamine-bridged nanoparticle dimers (6). By counting the number of 6, we found that at least 60% of cleaved nanoparticles were monofunctionalized by Lys. This method is highly selective and efficient for the preparation of monofunctionalized nanoparticles.     

Chemically bound gold nanoparticle arrays on silicon: assembly, properties and SERS study of protein interactions

A highly reproducible and facile method for formation of ordered 2 dimensional arrays of CTAB protected 50 nm gold nanoparticles bonded to silicon wafers is described. The silicon wafers have been chemically modified with long-chain silanes terminated with thiol that penetrate the CTAB bilayer and chemically bind to the underlying gold nanoparticle. The silicon wafer provides a reproducibly smooth, chemically functionalizable and non-fluorescent substrate with a silicon phonon mode which may provide a convenient internal frequency and intensity calibration for vibrational spectroscopy. The CTAB bilayer provides a potentially biomimetic environment for analyte, yet allows a sufficiently small nanoparticle separation to achieve a significant electric field enhancement. The arrays have been characterized using SEM and Raman spectroscopy. These studies reveal that the reproducibility of the arrays is excellent both between batches (<10% RSD) and across a single batch (<5% RSD). The arrays also exhibit good stability, and the effect of temperature on the arrays was also investigated. The interaction of protein and amino acid with the nanoparticle arrays was investigated using Raman microscopy to investigate their potential in bio-SERS spectroscopy. Raman of phenylalanine and the protein bovine pancreatic trypsin inhibitor, BPTI were studied using 785 nm excitation, coincident with the surface plasmon absorbance of the array. The arrays exhibit SERS enhancements of the order of 2.6 x 10(4) for phenylalanine, the standard deviation on the relative intensity of the 1555 cm(-1) mode of phenylalanine is less than 10% for 100 randomly distributed locations across a single substrate and less than 20% between different substrates. Significantly, comparisons of the Raman spectra of the protein and phenylalanine in solution and immobilized on the nanoparticle arrays indicates that the protein is non-randomly orientated on the arrays. Selective SERS enhancements suggest that aromatic residues penetrate through the bilayer inducing conformational changes in the protein.     

Large-area and high-density gold nanoparticle arrays with sub-10 nm gaps

Electrochemical techniques are widely used for the fabrication of nanostructured materials, yet a desired high-density nanoparticle arrays remains a challenge. Here large-area and high-density gold nanoparticle arrays with sub-10 nm gaps have been, for the first time, synthesized on Si(1 0 0) substrate within an electrochemical deposition system via the application of an unusually high over-potential. The extremely high over-potential contributes to the relatively small critical island size and high nucleation rate. It is believed that this method can be extended to the electrochemical fabrication nanoparticle arrays of other materials.     

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.     

金纳米微粒作探针共振瑞利散射光谱法测定亚甲蓝

在pH为6.5~9.5的中性或弱碱性介质中, 金纳米微粒可与亚甲蓝(MB)阳离子靠静电引力及疏水作用力结合, 形成粒径较大的聚集体(平均粒径从12 nm增至20 nm), 这种聚集体的形成导致共振瑞利散射(RRS)强度显著增强, 最大散射峰位于371 nm. 在适当条件下, 散射强度(ΔI)与亚甲蓝浓度成正比. 该法具有高灵敏度, 将金纳米微粒作为测定亚甲蓝的高灵敏RRS探针, 对亚甲蓝的检出限为21.17 ng/mL, 该法简便, 快速, 且有较好的选择性, 可用于血液中亚甲蓝的测定.     

A supersandwich multienzyme-DNA label based electrochemical immunosensor

An immunosensor based on the supersandwich multienzyme-DNA label realizing the electron transfer between the enzyme's redox site and the electrode is proposed for the ultrasensitive detection of Interleukin-6 (IL-6) with a low detection limit of 0.05 pg mL(-1).     

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.     

Fabrication of an ammonia gas sensor using inkjet-printed polyaniline nanoparticles

This work details the fabrication and performance of a sensor for ammonia gas analysis which has been constructed via the inkjet-printed deposition of polyaniline nanoparticle films. The conducting films were assembled on interdigitated electrode arrays and characterised with respect to their layer thickness and thermal properties. The sensor was further combined with heater foils for operation at a range of temperatures. When operated in a conductimetric mode, the sensor was shown to exhibit temperature-dependent analytical performance to ammonia detection. At room temperature, the sensor responded rapidly to ammonia (t₅₀ = 15 s). Sensor recovery time, response linearity and sensitivity were all significantly improved by operating the sensor at temperatures up to 80 °C. The sensor was found to have a stable logarithmic response to ammonia in the range of interest (1-100 ppm). The sensor was also insensitive to moisture in the range from 35 to 98% relative humidity. The response of the sensor to a range of common potential interferents was also studied.