An integrated microfluidic biochemical detection system for protein analysis with magnetic bead-based sampling capabilities

This paper presents the development and characterization of an integrated microfluidic biochemical detection system for fast and low-volume immunoassays using magnetic beads, which are used as both immobilization surfaces and bio-molecule carriers. Microfluidic components have been developed and integrated to construct a microfluidic biochemical detection system. Magnetic bead-based immunoassay, as a typical example of biochemical detection and analysis, has been successfully performed on the integrated microfluidic biochemical analysis system that includes a surface-mounted biofilter and electrochemical sensor on a glass microfluidic motherboard. Total time required for an immunoassay was less than 20 min including sample incubation time, and sample volume wasted was less than 50 microl during five repeated assays. Fast and low-volume biochemical analysis has been successfully achieved with the developed biofilter and immunosensor, which is integrated to the microfluidic system. Such a magnetic bead-based biochemical detection system, described in this paper, can be applied to protein analysis systems.     

Electrochemical behavior of kryptand 222 on a mercury electrode

The electrochemical behavior of kryptand 222 at the interface between a mercury electrode and an aqueous solution is studied by the impedance spectroscopy, polarography, and cyclic voltammetry methods. It is established that kryptand 222 possesses high surface activity at this interface. Adsorption parameters of kryptand 222 are found on the basis of the model of two parallel capacitors supplemented by the Frumkin isotherm by the regression analysis method. The potential dependences of the differential capacitance (C,E curves), calculated using these parameters satisfactorily agree with experiment. It is shown that the maximum in the C,E curves in the region of negative potentials is caused by catalytic process of hydrogen evolution.     

Magnetic bead-based label-free chemiluminescence detection of telomeres

For the first time we report on the detection of telomeres by coupling of the label-free guanine CL detection route with an efficient magnetic isolation of the hybrid.     

A magnetic bead-based assay for the rapid detection of methicillin-resistant Staphylococcus aureus by using a microfluidic system with integrated loop-mediated isothermal amplification

This study reports a new diagnostic assay for the rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) by combing nucleic acid extraction and isothermal amplification of target nucleic acids in a magnetic bead-based microfluidic system. By using specific probe-conjugated magnetic beads, the target deoxyribonucleic acid (DNA) of the MRSA can be specifically recognized and hybridized onto the surface of the magnetic beads which are then mixed with clinical sample lysates. This is followed by purifying and concentrating the target DNA from the clinical sample lysates by applying a magnetic field. Nucleic acid amplification of the target genes can then be performed by the use of a loop-mediated isothermal amplification (LAMP) process via the incorporation of a built-in micro temperature control module, followed by analyzing the optical density (OD) of the LAMP amplicons using a spectrophotometer. Significantly, experimental results show that the limit of detection (LOD) for MRSA in the clinical samples is approximately 10 fg μL(-1) by performing this diagnostic assay in the magnetic bead-based microfluidic system. In addition, the entire diagnostic protocol, from bio-sample pre-treatment to optical detection, can be automatically completed within 60 min. Consequently, this miniature diagnostic assay may become a powerful tool for the rapid purification and detection of MRSA and a potential point-of-care platform for detection of other types of infections.     

Magnetic bead-based hybridization assay for electrochemical detection of microRNA

Aberrant expression of microRNAs (miRNAs), short non-coding RNA molecules regulating gene expression, is often found in tumor cells, making the miRNAs suitable candidates as cancer biomarkers. Electrochemistry is an interesting alternative to current standard methods of miRNA detection by offering cheaper instrumentation and faster assays times. In this paper, we labeled miRNA in a quick, simple, two-step procedure with electroactive complex of osmium(VI) and 2,2′-bipyridine, Os(VI)bipy, which specifically binds to the ribose at the 3′-end of the miRNA, and hybridized such labeled miRNA with biotinylated capture probe attached to the streptavidin magnetic beads. Labeled miRNA was then detected at hanging mercury drop electrode at femtomole level due to an electrocatalytic nature of the peak from the Os(VI)bipy label. We obtained good selectivity of the assay using elevated hybridization temperatures for better discrimination of perfect duplex from single and double mismatches. After optimization of the protocol, we demonstrated feasibility of our assay by detecting target miRNA in real total RNA samples isolated from human cancer cells.     

Magnetic bead-based label-free electrochemical detection of DNA hybridization.

Magnetic bead capture has been used for eliminating non-specific adsorption effects hampering label-free detection of DNA hybridization based on stripping potentiometric measurements of the target guanine at graphite electrodes. In particular, the efficient magnetic separation has been extremely useful for discriminating against unwanted constituents, including a large excess of co-existing mismatched and non-complementary oligomers, chromosomal DNA, RNA and proteins. The new protocol involves the attachment of biotinylated oligonucleotide probes onto streptavidin-coated magnetic beads, followed by the hybridization event, dissociation of the DNA hybrid from the beads, and potentiometric stripping measurements at a renewable graphite pencil electrode. Such coupling of magnetic hybridization surfaces with renewable graphite electrode transducers and label-free electrical detection results in a greatly simplified protocol and offers great promise for centralized and decentralized genetic testing. A new magnetic carbon-paste transducer, combining the solution-phase magnetic separation with an instantaneous magnetic collection of the bead-captured hybrid, is also described. The characterization, optimization and advantages of the genomagnetic label-free electrical protocol are illustrated below for assays of DNA sequences related to the breast-cancer BRCA1 gene.     

Spatially addressable bead-based biosensor for rapid detection of beta-thalassemia mutations

A simple glass-polymer bead-based biosensor was validated for the detection of beta-thalassemia mutations. Different bead types, each carrying allele-specific probes targeting a particular mutation on the beta-globin gene, were immobilized and distinguished on the chip based on their spatial addresses. Genomic DNA samples carrying various single nucleotide transitions and transversions in the beta-globin gene were subjected to polymerase chain reaction and asymmetric amplification in the presence of Cy3-labeled primers, followed by hybridization onto the chip and detection under an epifluorescent microscope. Mutations that were heterozygous or homozygous were easily detected on the device based on the signal intensity difference (or similarity) between the wildtype and mutant probes. This device successfully detected all six common beta-globin gene mutations within 30 min. The number of targeted mutations on this chip can be easily expandable through the introduction of additional probe sets.     

硼掺杂金刚石纳米棒电极对比阿培南的电化学检测研究

在自制的硅纳米线上采用热丝化学气相沉积方法制备了硼掺杂金刚石纳米棒电极.采用循环伏安及计时电流方法测定了在磷酸缓冲溶液中的药物比阿培南的浓度,灵敏度达到0.038μA μM-1较相同条件下制备得到的普通硼掺杂金刚石电极(0.028μA μM-1)相比有所提高.该纳米棒电极由于特殊的表面形貌,较普通硼掺杂金刚石电极表现出...     

Aptamer-functionalized magnetic nanoparticle-based bioassay for the detection of ochratoxin A using upconversion nanoparticles as labels

A sensitive luminescent bioassay for the detection of ochratoxin A (OTA), a small molecular mycotoxin, was developed using aptamer-conjugated magnetic nanoparticles (MNPs) as the recognition and concentration element and upconversion nanoparticles (UCNPs) as highly sensitive labels. The bioassay system was fabricated by immobilizing aptamer DNA 1 sequence onto the surface of Fe(3)O(4) MNPs, which were implemented to capture and concentrate OTA from bulk samples. The aptamer DNA 1 sequence then hybridized with UCNPs modified with DNA 2 sequence, which could dissociate from DNA 1 and result in a decreased luminescent signal when aptamer DNA 1 recognized and bound to target OTA. Under the optimal conditions, the decreased luminescent intensity (ΔI) is proportional to the concentration of OTA in the range of 1 × 10(-13) to 1 × 10(-9) g mL(-1) with a detection limit of 1 × 10(-13) g mL(-1). The proposed method then was successfully applied to measure OTA in naturally contaminated maize samples and validated by a commercially available enzyme-linked immunosorbent assay (ELISA) method. Benefiting from the magnetic separation and concentration effect of MNPs, the high sensitivity of UCNPs, as well as the selectivity and stability of the aptamer, the present upconversion luminescent bioassay offers a promising approach for the screening of small molecular mycotoxins because it is simple, rapid, highly sensitive, specific, does not require sample pre-concentration and lacks interference from autofluorescence of other biomolecules.     

Magnetic actuator for the control and mixing of magnetic bead-based reactions on-chip

While magnetic bead (MB)-based bioassays have been implemented in integrated devices, their handling on-chip is normally either not optimal—i.e. only trapping is achieved, with aggregation of the beads—or requires complex actuator systems. Herein, we describe a simple and low-cost magnetic actuator to trap and move MBs within a microfluidic chamber in order to enhance the mixing of a MB-based reaction. The magnetic actuator consists of a CD-shaped plastic unit with an arrangement of embedded magnets which, when rotating, generate the mixing. The magnetic actuator has been used to enhance the amplification reaction of an enzyme-linked fluorescence immunoassay to detect Escherichia coli O157:H7 whole cells, an enterohemorrhagic strain, which have caused several outbreaks in food and water samples. A 2.7-fold sensitivity enhancement was attained with a detection limit of 603?colony-forming units (CFU) /mL, when employing the magnetic actuator. Graphical Abstract

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Microdrop analysis of a bead-based immunoassay

The progress to electrochemical detection of a microbead-based immunoassay in small volumes has led to a reduced assay time and lower detection limits. Three electrochemical techniques are described for an immunoassay with detection in a microdrop. The techniques are amperometric detection with a rotating disk electrode (RDE), a microelectrode, and an interdigitated array (IDA) electrode. An enzyme-labeled sandwich immunoassay with mouse IgG as the model analyte is used to demonstrate the three techniques. The microbead assay is carried out in a test tube using a magnet to control bead collection. Once the immunocomplex is formed on the microbead, the beads are transferred to a microdrop where the enzyme, either alkaline phosphatase or β-galactosidase, generates 4-aminophenol (PAP). PAP is oxidized at the electrode with an applied potential of +290 mV vs. Ag/AgCl. For all three techniques, the upper limit of the dynamic range was 1000 ng/ml mouse IgG, and the detection limits were: 50 ng/ml for the RDE, 40 ng/ml for the microelectrode, and 26 ng/ml for the IDA electrode.     

Impedimetric genosensor for miRNA-34a detection in cell lysates using polypyrrole

Journal of Solid State Electrochemistry - This work reports the development of simple, practical, cost effective and label free genosensor prepared by electropolymerization of polypyrrole on pencil...     

Electrochemical antioxidant detection technique based on guanine-bonded graphene and magnetic nanoparticles composite materials

An antioxidant (AO) amperometric technique based on guanine attached to graphene and Fe(3)O(4) nanoparticles (NPs) magnetic materials was developed. Guanine molecules acted as an antioxidant competitor were bonded with graphene nanosheets, onto which magnetic Fe(3)O(4) NPs were attached and the as-prepared magnetic composite can be attracted to the electrode surface by an external magnetic field. When applied with negative potentials, the dissolved oxygen was reduced to H(2)O(2) at the electrode surface, and then reacted with the EDTA-Fe(ii) complex via a Fenton-like reaction to produce OH radicals. After oxidation damage by OH radicals, the electrochemical oxidation of guanine gave a decreased current. In the presence of AOs, the reactive oxygen species (ROS, e.g. OH radicals and H(2)O(2)) were scavenged by AOs and fewer guanine probe molecules were oxidized, thus inducing a higher electrochemical oxidation current of guanine. So AOs competed with the guanine probe molecules toward oxidation by ROS. The current signals of the guanine probe molecules were proportional to the concentrations of AOs. A kinetic model was proposed to quantify the ROS scavenging capacities of the AOs. Using guanine as an oxidizable probe and OH radicals and H(2)O(2) as endogenous ROS, this kind of AO detection technique mimicks the antioxidant protection mechanism by small AO molecules in the human body.     

Electrochemical detection for trace analysis by capillary HPLC

An amperometric-type electrochemical detector is proposed for use in capillary HPLC. Its cell (volume 60 nl) contains a cylindrical working electrode for detecting the presence of electroactive compounds. The noise level is 3–5 pA depending on the potential. The minimum detectable concentrations of the o-, m-, and p- isomers of nitrophenol (after separation of a 20 μl sample on a fused silica column) lies between 4 and 18 ng/l. The relative standard deviation of the height of the chromatographic peaks of phenols present at concentrations of 10 ppb does not exceed 0. 08 (n = 5).     

Electrochemical detection in a microfluidic device of oxidative stress generated by macrophage cells

The release of reactive oxygen species (ROS) or reactive nitrogen species (RNS), i.e., the initial phase of oxidative stress, by macrophage cells has been studied by electrochemistry within a microfluidic device. Macrophages were first cultured into a detection chamber containing the three electrodes system and were subsequently stimulated by the microinjection of a calcium ionophore (A23187). Their production of ROS and RNS was then measured by amperometry at the surface of a platinized microelectrode. The fabricated microfluidic device provides an accurate measurement of oxidative release kinetics with an excellent reproducibility. We believe that such a method is simple and versatile for a number of advanced applications based on the detection of biological processes of secretion by a few or even a single living cell.     

Electrochemical detection of receptor-mediated endocytosis by scanning electrochemical microscopy

We report a scanning electrochemical microscopy (SECM)-based receptor-mediated endocytosis detection method. Epidermal growth factor receptor (EGFR), which is one of the key membrane proteins associated with cancer, was used as a model for receptor-mediated endocytosis. EGFR molecules on the outer cell membrane were detected by SECM by using alkaline phosphatase (ALP) as a labeling enzyme. Since SECM detected the ALP activity on the outer membrane, the procedure helped discriminate the EGFR on the outer membrane from the intracellular EGFR involved in endocytosis. SECM showed a marked decrease in the current responses generated due to ALP activity by 93% on addition of the epidermal growth factor, indicating clearly that EGF triggered the endocytosis, which led to the withdrawal of most EGFRs from the outer membrane.     

Applicability of a yeast bioassay in the detection of steroid esters in hair

The aim of the present study was to demonstrate the applicability of a yeast androgen and estrogen bioassay in the detection of steroid esters in hair samples of animals treated with a hormone ester cocktail. The outcome of the activity screenings was critically compared with the results previously obtained with LC-MS/MS analysis. Hair samples of one pour-on treated animal, 10 ml DMSO containing 25 mg estradiol benzoate (EB), 60 mg testosterone decanoate (TD) and 60 mg testosterone cypionate (TC), were selected and analyzed with the androgen and estrogen yeast bioassay. Results showed that by the introduction of a hydrolysis step, bioassays can be used to screen for the presence of hormone esters in hair samples. Based on the difference in fluorescence responses between the non-hydrolyzed and the hydrolyzed hair samples, it was possible to detect the presence of EB up to at least 56 days after a single pour-on treatment and to detect the presence of TC and TD up to at least 14 days after the treatment. Although the LC-MS/MS analysis could detect TC and TD up to 49 days after treatment, bioassays have the advantage that they can also detect any (un)known steroid ester.     

Electrochemical detection of arsenic on a gold nanoparticle array

The detection of As(III) was investigated on a gold nanoparticle array. At the first stage, gold nanoparticles were synthesized on glassy carbon microspheres. The resulting hybrid material was characterized by SEM and the sizes of the nanoparticles were found to be in the range 20–200 nm. At the second stage, glassy carbon microspheres decorated with Au nanoparticles were abrasively attached to the surface of a basal-plane pyrolytic electrode. The resulting gold nanoarray was characterized by the reduction of surface gold oxides. Furthermore, it was found to have good characteristics for the sensing of arsenic via anodic stripping voltammetry with a limit of detection of 0.8 μM and a sensitivity of 0.91 C M⁻¹. The text was submitted by the authors in English.