A renewable electrochemical magnetic immunosensor based on gold nanoparticle labels

A particle-based renewable electrochemical magnetic immunosensor was developed by using magnetic beads and gold nanoparticle labels. Anti-IgG antibody-modified magnetic beads were attached to a renewable carbon paste transducer surface by magnet that was fixed inside the sensor. Gold nanoparticle labels were capsulated to the surface of magnetic beads by sandwich immunoassay. Highly sensitive electrochemical stripping analysis offers a simple and fast method to quantify the capatured gold nanoparticle tracers and avoid the use of an enzyme label and substrate. The stripping signal of gold nanoparticles is related to the concentration of target IgG in the sample solution. A transmission electron microscopy image shows that the gold nanoparticles were successfully capsulated to the surface of magnetic beads through sandwich immunoreaction events. The parameters of immunoassay, including the loading of magnetic beads, the amount of gold nanoparticle conjugate, and the immunoreaction time, were optimized. The detection limit of 0.02 microg ml(-1) of IgG was obtained under optimum experimental conditions. Such particle-based electrochemical magnetic immunosensors could be readily used for simultaneous parallel detection of multiple proteins by using multiple inorganic metal nanoparticle tracers and are expected to open new opportunities for disease diagnostics and biosecurity.     

Magneto immunoassays for Plasmodium falciparum histidine-rich protein 2 related to malaria based on magnetic nanoparticles

Magneto immunoassay-based strategies for the detection of Plasmodium falciparum histidine-rich protein 2 (HRP2) related to malaria are described for the first time by using magnetic micro- and nanoparticles. The covalent immobilization of a commercial monoclonal antibody toward the HRP2 protein in magnetic beads and nanoparticles was evaluated and compared. The immunological reaction for the protein HRP2 was successfully performed in a sandwich assay on magnetic micro- and nanoparticles by using a second monoclonal antibody labeled with the enzyme, horseradish peroxidase (HRP). Then, the modified magnetic particles were easily captured by a magneto sensor made of graphite-epoxy composite (m-GEC) which was also used as the transducer for the electrochemical detection. The performance of the immunoassay-based strategy with the electrochemical magneto immunosensors was successfully evaluated and compared with a novel magneto-ELISA based on optical detection using spiked serum samples. Improved sensitivity was obtained when using 300 nm magnetic nanoparticles in both cases. The electrochemical magneto immunosensor coupled with magnetic nanoparticles have shown better analytical performance in terms of limit of detection (0.36 ng mL(-1)), which is much lower than the LOD reported by other methods. Moreover, at a low level of HRP2 concentration of 31.0 ng mL(-1), a signal of 15.30 μA was reached with a cutoff value of 0.34 μA, giving a clear positive result with a non-specific adsorption ratio of 51. Due to the high sensitivity, this novel strategy offers great promise for rapid, simple, cost-effective, and on-site detection of falciparum malaria disease in patients, but also to screen out at-risk blood samples for prevention of transfusion-transmitted malaria.     

Aptamer-based detection of plasma proteins by an electrochemical assay coupled to magnetic beads

The DNA thrombin aptamer has been extensively investigated, and the coupling of this aptamer to different transduction principles has demonstrated the wide applicability of aptamers as bioreceptors in bioanalytical assays. The goal of this work was to design an aptamer-based sandwich assay with electrochemical detection for thrombin analysis in complex matrixes, using a simple target capturing step by aptamer-functionalized magnetic beads. The conditions for the aptamer immobilization and for the protein binding have been first optimized by surface plasmon resonance, and then transferred to the electrochemical-based assay performed onto screen-printed electrodes. The assay was then applied to the analysis of thrombin in buffer, spiked serum, and plasma and high sensitivity and specificity were found. Moreover, thrombin was generated in situ in plasma by the conversion of its precursor prothrombin, and the formation of thrombin was followed at different times. The concentrations detected by the electrochemical assay were in agreement with a simulation software that mimics the formation of thrombin over time (thrombogram). The proposed work demonstrates that the high specificity of aptamers together with the use of magnetic beads are the key features for aptamer-based analysis in complex matrixes, opening the possibility of a real application to diagnostics or medical investigation.     

Label-free cell separation using a tunable magnetophoretic repulsion force

This paper describes a new label-free cell separation method using a magnetic repulsion force resulting from the magnetic susceptibility difference between cells and a paramagnetic buffer solution in a microchannel. The difference in the magnetic forces acting on different-sized cells is enhanced by adjusting the magnetic susceptibility of the surrounding medium, which depends on the concentration of paramagnetic salts, such as biocompatible gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA), dissolved therein. As a proof-of-concept demonstration, Gd-DTPA solutions at concentrations of 0-80 mM were applied to separate U937 cells from red blood cells (RBCs) and to distinguish two different-sized polystyrene (PS) beads (8 and 10 μm in diameter). By increasing the Gd-DTPA concentration from 0 to 40 mM, the separation resolution of PS beads was increased from 0.08 to 0.91. Additionally, we successfully achieved label-free separation of U937 cells from RBCs with >90% purity and 1 × 10(5) cells/h throughput using a 40 mM Gd-DTPA solution.     

Integrated inductive sensors for the detection of magnetic microparticles

In this paper, we deal with novel inductive microsensors, realized by using standard CMOS microelectronic technologies, for the detection of small amounts of magnetic beads that are placed in selected regions over the surface of the microsensor. The sensor proposed here represents a step toward the development of integrated inductive biosensors for application in the area of magnetic immunoassay where magnetic markers, carrying specific antibodies that selectively bind to the cells or molecules to be detected, are used; the measurement of the analyte concentration is therefore accomplished by determining the concentration of magnetic particles tied to it. A planar differential transformer structure is proposed here as part of the measurement strategy. The analysis, simulation, and design of the proposed device are reported, and its sensitivity to the quantity of micromagnetic beads deposited over its surface has been also demonstrated through experiments.     

Quantum dot biolabeling coupled with immunomagnetic separation for detection of Escherichia coli O157:H7

A sensitive, specific, and rapid method for the detection of E. coli O157:H7 was demonstrated using quantum dots (QDs) as a fluorescence marker coupled with immunomagnetic separation. Magnetic beads coated with anti-E. coli O157 antibodies were employed to selectively capture the target bacteria, and biotin-conjugated anti-E. coli antibodies were added to form sandwich immuno complexes. After magnetic separation, the immuno complexes were labeled with QDs via biotin-streptavidin conjugation. This was followed by a fluorescence measurement using a laptop-controlled portable device, which consisted of a blue LED and a CCD-array spectrometer. The peak intensity of the fluorescence emission was proportional to the initial cell concentration of E. coli O157:H7 in the range of 10(3)-10(7) CFU/mL with a detection limit at least 100 times lower than that of the FITC-based method. The total detection time was less than 2 h. Neither E. coli K12 nor Salmonella typhimurium interfered with the detection of E. coli O157:H7.     

Microelectrode control of surface-bound enzymatic activity

Microelectrodes have been used to control the microscopically local reaction environment of surface-bound alcohol dehydrogenase (ADH). A surface comprised of agarose beads coated with immobilized ADH was prepared on a microscope slide and exhibited maximum activity toward the oxidation of ethanol, in the presence of nicotinamide adenine dinucleotide (NAD(+)), at a pH of 9.0. Microelectrode control of activity was achieved by submerging the slide in a solution of pH 6.0, well below the optimum value, and generating hydroxide at the microelectrode tip through the reduction of oxygen or water. An alkaline "sphere of influence" was set up around the microelectrode tip that, when positioned in close proximity to the enzyme surface, created a favorable reaction environment. The increased enzymatic activity was monitored by observing fluorescence of the reduced cofactor, NADH, using a fluorescence microscope equipped with an imaging camera. The fluorescent sphere diameter was characterized as a function of time, potential, and solution buffer strength. Optimum spatial resolution for enzymatic control was 7-12 μm.     

Multiplexed, waveguide approach to magnetically assisted transport evanescent field fluoroassays

This paper, expanding upon the recently developed magnetically assisted transport evanescent field fluoroassays (MATEFFs), takes advantage of several innovations in order to successfully integrate a microfluidic platform and planar waveguide technology for exploitation of multiplexing advantages. In the current adaptation of MATEFFs, a multiple internal reflection element (waveguide) is created using a simple microscope slide and PDMS microfluidic architecture, allowing simultaneous detection of multiple samples. Furthermore, the magnetic beads are manipulated using a passive pumping technique and a simple external permanent magnet, thereby circumventing the need for electromagnetic fabrication or complicated architectures and equipment. Initial testing, optimization, and calibration were performed using a model sandwich immunoassay system for the detection of rabbit IgG, with which we demonstrate a linear dynamic range of 3 orders of magnitude and physiologically relevant detection limits of nanograms per milliliter. Further work employed a sandwich immunoassay for the detection of interleukin-4, a cytokine that promotes proliferation and differentiation of B cells, to demonstrate technique reproducibility with RSD values of 5% and reported LOD of 10 ng/mL. The use of harvesting magnetic beads resulted in assays with mass-sensing behavior. Using IgG as a model cross-reactant with the interleukin-4 system, we additionally illustrate technique selectivity and multiplexing capability. A DNA hybridization assay is carried out using magnetic bead-immobilized single-stranded DNA with hybridization detected via ethidium bromide intercalation, further establishing technique versatility.     

Electrochemical magnetoimmunosensing strategy for the detection of pesticides residues

A novel electrochemical immunosensing strategy for the detection of atrazine based on magnetic beads is presented. Different coupling strategies for the modification of the magnetic beads with the specific anti-atrazine antibody have been developed. The immunological reaction for the detection of atrazine performed on the magnetic bead is based on a direct competitive assay using a peroxidase (HRP) tracer as the enzymatic label. After the immunochemical reactions, the modified magnetic beads can be easily captured by a magnetosensor made of graphite-epoxy composite, which is also used as the transducer for the electrochemical immunosensing. The electrochemical detection is thus achieved through a suitable substrate and mediator for the enzyme HRP. The electrochemical approach is also compared with a novel magneto-ELISA based on optical detection. The performance of the electrochemical immunosensing strategy based on magnetic beads was successfully evaluated using spiked real orange juice samples. The detection limit for atrazine using the competitive electrochemical magnetoimmunosensing strategy with anti-atrazine-specific antibody covalent coupled with tosyl-activated magnetic beads was found to be 6 x 10(-3) microg L(-1) (0.027 nmol L(-1)). This strategy offers great promise for rapid, simple, cost-effective, and on-site analysis of biological, food, and environmental samples.     

Simultaneous detection of single molecules and singulated ensembles of molecules enables immunoassays with broad dynamic range

We report a method for combining the detection of single molecules (digital) and an ensemble of molecules (analog) that is capable of detecting enzyme label from 10(-19) M to 10(-13) M, for use in high sensitivity enzyme-linked immunosorbent assays (ELISA). The approach works by capturing proteins on microscopic beads, labeling the proteins with enzymes using a conventional multistep immunosandwich approach, isolating the beads in an array of 50-femtoliter wells (Single Molecule Array, SiMoA), and detecting bead-associated enzymatic activity using fluorescence imaging. At low concentrations of proteins, when the ratio of enzyme labels to beads is less than ～1.2, beads carry either zero or low numbers of enzymes, and protein concentration is quantified by counting the presence of "on" or "off" beads (digital regime). (1) At higher protein concentrations, each bead typically carries multiple enzyme labels, and the average number of enzyme labels present on each bead is quantified from a measure of the average fluorescence intensity (analog regime). Both the digital and analog concentration ranges are quantified by a common unit, namely, average number of enzyme labels per bead (AEB). By combining digital and analog detection of singulated beads, a linear dynamic range of over 6 orders of magnitude to enzyme label was achieved. Using this approach, an immunoassay for prostate specific antigen (PSA) was developed. The combined digital and analog PSA assay provided linear response over approximately four logs of concentration ([PSA] from 8 fg/mL to 100 pg/mL or 250 aM to 3.3 pM). This approach extends the dynamic range of ELISA from picomolar levels down to subfemtomolar levels in a single measurement.     

Highly sensitive detection of proteins and bacteria in aqueous solution using surface-enhanced Raman scattering and optical fibers

We report the detection of the proteins lysozyme and cytochrome c as well as the live bacterial cells of Shewanella oneidensis MR-1 in aqueous solutions with sensitivities order(s) of magnitude higher than those previously reported. Two highly sensitive surface-enhanced Raman scattering (SERS)-based biosensors using optical fibers have been employed for such label-free macromolecule detections. The first sensor is based on a tip-coated multimode fiber (TCMMF) with a double-substrate "sandwich" structure, and a detection limit of 0.2 μg/mL is achieved in protein detections. The second sensor is based on a liquid core photonic crystal fiber (LCPCF) with a better confinement of light inside the fiber core, and a detection limit of 10(6) cells/mL is achieved for the bacteria detection. Both SERS biosensors show great potential for highly sensitive and molecule-specific detection and identification of biomolecules.     

DNA-wrapped carbon nanotubes as sensitive electrochemical labels in controlled-assembly-mediated signal transduction for the detection of sequence-specific DNA

A novel electrochemical strategy that uses DNA-wrapped carbon nanotubes (CNTs) as electrochemical labels is developed for sensitive and selective detection of sequence-specific DNA. The presence of target DNA mediates the formation of a sandwiched complex between the DNA-wrapped CNT and a hairpin DNA capture probe immobilized on magnetic beads. This allows target-selective collection of the CNT labels by magnetic separation and transfer on the electrode surface modified with an insulating self-assembled monolayer (SAM). After treatment with N,N-dimethylformamide, the collected sandwiched complex releases the bare CNTs and facilitates the removal of magnetic beads from the electrode surface. The bare CNTs can then assemble on the SAM-modified electrode surface and mediate efficient electron transfer between the electrode and the electroactive species in the solution with a strong current signal generated. The results indicate that the developed strategy shows a sensitive response to target DNA with a desirable signal gain and a low detection limit of 0.9 pM. This strategy is also demonstrated to provide excellent differentiation of single-base mismatch in target DNA. It is expected that this electrochemical strategy may hold great potential as a novel platform for clinical diagnostics and genetic analysis.     

基于小波变换的低碳钢连铸坯超声探伤

利用离散小波变换对粗晶钢材超声检测信号进行处理,比较了使用不同的小波基,并对不同尺度中突出体现的信息分别进行处理后重构信号的去噪效果。结果表明,对不同尺度系数作不同的阈值处理有利于缺陷的识别,这种方法对粗晶钢材料的检测有积极的意义。     

Aptameric peptide for one-step detection of protein kinase

Protein kinases are significant regulators in the cell signal pathway, and it is difficult to achieve quick kinase detection because traditional kinase assays normally rely on a time-consuming kinase phosphorylation process. Herein, we present a novel one-step strategy to detect protein kinase by using a kinase-specific aptameric peptide-functionalized quartz crystal microbalance (QCM) electrode, in which the detection can be finished in less than 10 min. A peptide kinase inhibitor (IP(20)) was used as the aptameric peptide because of its selective and strong interaction with the target protein kinase (cyclic adenosine monophosphate-dependent protein kinase A, PKA), high stability, and ease of inexpensive synthesis, presenting a new direct recognition element for kinase. The aptameric peptide was immobilized on the Au-coated quartz electrode through dual-thiol anchoring and the binding of His-tagged peptide with a nitrilotriacetic acid/Ni(II) complex, fabricating a highly specific and stable detection platform. The interaction of aptameric peptide with kinase was monitored with the QCM in real time, and the concentration of protein kinase was sensitively measured by the frequency response of the QCM with the low detection limit for PKA at 0.061 mU μL(-1) and a linear range from 0.64 to 22.33 mU μL(-1). This method is rapid and reagentless and does not require a phosphorylation process. The versatility of our aptameric peptide-based strategy has also been demonstrated by the application in kinase assay using electrochemical impedance spectroscopy. Moreover, this method was successfully applied to detect the forskolin/3-isobutyl-1-methylxanthine-stimulated activation of PKA in cell lysate.     

PCR-free DNA detection using a magnetic bead-supported polymeric transducer and microelectromagnetic traps

A fluorescent polymeric hybridization transducer supported on magnetic microbeads was investigated for the rapid, ultrasensitive, and sequence-specific detection of DNA. We show that the polymer derivative can be used to detect target DNA directly on magnetic particles by preparing "target-ready" microbeads grafted with the polymer and suitable DNA probes. A detection limit of approximately 200 target copies in a probed volume of 150 muL (1.4 copies/muL) was obtained for a DNA sequence specific to Candida albicans This detection scheme does not require the release of the hybridized target DNA prior to its detection or the labeling or amplification of the nucleic acids. Furthermore, we show that the fluorescence from these biosensing magnetic beads can be read while magnetically confined in a small volume by a microelectromagnetic trap, which offers the possibility of performing both the preconcentration and detection steps simultaneously on the same support. The combination of the fluorescent polymer biosensor with magnetic particle-assisted DNA preconcentration extends the application of this ultrasensitive biosensor to biological samples with complex matrixes and to integrated lab-on-a-chip platforms, where it could be used for fast multitarget DNA detection in point-of-care diagnostics and field analysis.     

Rapid rotation of micron and submicron dielectric particles measured using optical tweezers

Abstract

We demonstrate the use of a laser trap (‘optical tweezers’) and back-focal-plane position detector to measure rapid rotation in aqueous solution of single particles with sizes in the vicinity of 1 μm. Two types of rotation were measured: electrorotation of polystyrene microspheres and rotation of the flagellar motor of the bacterium Vibrio alginolyticus. In both cases, speeds in excess of 1000 Hz (rev s^?1) were measured. Polystyrene beads of diameter about 1 μm labelled with smaller beads were held at the centre of a microelectrode array by the optical tweezers. Electrorotation of the labelled beads was induced by applying a rotating electric field to the solution using microelectrodes. Electrorotation spectra were obtained by varying the frequency of the applied field and analysed to obtain the surface conductance of the beads. Single cells of V. alginolyticus were trapped and rotation of the polar sodium-driven flagellar motor was measured. Cells rotated more rapidly in media containing higher concentrations of Na⁺, and photodamage caused by the trap was considerably less when the suspending medium did not contain oxygen. The technique allows single-speed measurements to be made in less than a second and separate particles can be measured at a rate of several per minute.     

Aptamer based electrochemical sensor system for protein using the generation/collection mode of scanning electrochemical microscope (SECM)

To detect the target molecules, aptamers are currently focused on and the use of aptamers for biosensing is particularly interesting, as aptamers could substitute antibodies in bioanalytical sensing. So this paper describes the novel electrochemical system for protein in sandwich manner by using the aptamers and the scanning electrochemical microscope (SECM). For protein detection, sandwich system is ideal since labeling of the target protein is not necessary. To develop the electrochemical protein sensor system, thrombin was chosen as a target protein since many aptamers for it were already reported and two different aptamers, which recognize different positions of thrombin, were chosen to construct sandwich type sensing system. In order to obtain the electrochemical signal, the glucose oxidase (GOD) used for labeling the detection aptamers since it has large amount of stability in aqueous solution. One aptamer was immobilized onto the gold electrode and the other aptamer for detection was labeled with GOD for generation of the electric signal. Thrombin was detected in sandwich manner with aptamer immobilized onto the gold electrode and the GOD labeled aptamer. The enzymatic signal, generated from glucose addition after the formation of the complex of thrombin, was measured. The generation-collection mode of SECM was used for amperometric H2O2 detection.     

Fast colorimetric detection of copper ions using L-cysteine functionalized gold nanoparticles

This communication reports an efficient visual detection method of Cu2+ by L-cysteine functionalized gold nanoparticles in aqueous solution. Upon exposure to Cu2+, the gold nanoparticle solution changed from red to blue, in response to surface plasmon absorption of dispersed and aggregated nanoparticles. This colorimetric sensor allows a rapid quantitative assay of Cu2+ down to the concentration range of 10(-5) M. Recognition of Cu2+ and formation of the aggregates are proposed to occur via a 2 : 1 sandwich complex between L-cysteine and Cu2+.     

Synthesis and applications of magnetic nanoparticles for biorecognition and point of care medical diagnostics

Functionalized magnetic nanoparticles are important components in biorecognition and medical diagnostics. Here, we present a review of our contribution to this interdisciplinary research field. We start by describing a simple one-step process for the synthesis of highly uniform ferrite nanoparticles (d = 20-200 nm) and their functionalization with amino acids via carboxyl groups. For real-world applications, we used admicellar polymerization to produce 200 nm diameter 'FG beads', consisting of several 40 nm diameter ferrite nanoparticles encapsulated in a co-polymer of styrene and glycidyl methacrylate for high throughput molecular screening. The highly dispersive FG beads were functionalized with an ethylene glycol diglycidyl ether spacer and used for affinity purification of methotrexate-an anti-cancer agent. We synthesized sub-100 nm diameter magnetic nanocapsules by exploiting the self-assembly of viral capsid protein pentamers, where single 8, 20, and 27 nm nanoparticles were encapsulated with VP1 pentamers for applications including MRI contrast agents. The FG beads are now commercially available for use in fully automated bio-screening systems. We also incorporated europium complexes inside a polymer matrix to produce 140 nm diameter fluorescent-ferrite beads (FF beads), which emit at 618 nm. These FF beads were used for immunofluorescent staining for diagnosis of cancer metastases to lymph nodes during cancer resection surgery by labeling tumor cell epidermal growth factor receptor (EGFRs), and for the detection of brain natriuretic peptide (BNP)-a hormone secreted in excess amounts by the heart when stressed-to a level of 2.0 pg ml(-1). We also describe our work on Hall biosensors made using InSb and GaAs/InGaAs/AlGaAs 2DEG heterostructures integrated with gold current strips to reduce measurement times. Our approach for the detection of sub-200 nm magnetic bead is also described: we exploit the magnetically induced capture of micrometer sized 'probe beads' by nanometer sized 'target beads', enabling the detection of small concentrations of beads as small as 8 nm in 'pumpless' microcapillary systems. Finally, we describe a 'label-less homogeneous' procedure referred to as 'magneto-optical transmission (MT) sensing', where the optical transmission of a solution containing rotating linear chains of magnetic nanobeads was used to detect biomolecules with pM-level sensitivity with a dynamic range of more than four orders of magnitude. Our research on the synthesis and applications of nanoparticles is particularly suitable for point of care diagnostics.     

Biosensing based on magnetically induced self-assembly of particles in magnetic colloids

Superparamagnetic beads and nonmagnetic beads of different sizes were assembled to form a "ring-structure" in a magnetorheological (MR) fluid solution by the application of external magnetic fields. For superparamagnetic beads and non-magnetic beads functionalized with probe and target molecules, respectively, the ring-structure was maintained even after removing the external magnetic field due to biomolecular bonding. Several experiments are described, including the formation process of ring-structures with and without molecular interactions, the accelerating effect of external magnetic fields, and the effect of biotin concentration on the structures of the rings. We define the small nonmagnetic particles as "petals" because the whole structure looks like a flower. The number of remnant ring petals was a function of the concentration of target molecules in the concentration range of 0.0768 ng/ml-3.8419 ng/ml which makes this protocol a promising method for biosensing. Not only was the formation process rapid, but the resulting two-dimensional colloidal system also offers a simple method for reducing reagent consumption and waste generation.