The generation of multi-laminar reagent streams for rapid, sequential (bio)chemical reactions on magnetic particles in a continuous flow microreactor

This paper was presented at the 2nd Micro and Nano Flows Conference (MNF2009), which was held at Brunel University, West London, UK. The conference was organised by Brunel University and supported by the Institution of Mechanical Engineers, IPEM, the Italian Union of Thermofluid dynamics, the Process Intensification Network, HEXAG - the Heat Exchange Action Group and the Institute of Mathematics and its Applications.We demonstrate a versatile microfluidic system for performing rapid, consecutive (bio)chemical reactions in continuous flow. Surface-functionalised magnetic microparticles are introduced into a chamber and pulled, via a magnet, across a series of laminar flow streams containing different reagents, thus performing multiple sequential reactions on the particles’ surface. Such a continuous flow method eliminates many of the inefficiencies associated with batch techniques, such as the time-consuming, laborious sequential reaction and washing steps, to yield a system that can perform analyses far more rapidly and with less reagent volume than conventional methods. This innovative device has been applied to a two-reaction step mouse IgG sandwich immunoassay and one- and two-reaction step DNA hybridisation assays, all of which were completed within one minute. These results pave the way for a multi-purpose microreactor that can perform a variety of analytical and synthetic processes.This study is funded by the Engineering and Physical Sciences Research Council (EPSRC)

Miniaturized Protein Microarray with Internal Calibration as Point-of-Care Device for Diagnosis of Neonatal Sepsis

Neonatal sepsis is still a leading cause of death among newborns. Therefore a protein-microarray for point-of-care testing that simultaneously quantifies the sepsis associated serum proteins IL-6, IL-8, IL-10, TNF alpha, S-100, PCT, E-Selectin, CRP and Neopterin has been developed. The chip works with only a 4 μL patient serum sample and hence minimizes excessive blood withdrawal from newborns. The 4 μL patient samples are diluted with 36 μL assay buffer and distributed to four slides for repetitive measurements. Streptavidin coated magnetic particles that act as distinct stirring detection components are added, not only to stir the sample, but also to detect antibody antigen binding events. We demonstrate that the test is complete within 2.5 h using a single step assay. S-100 conjugated to BSA is spotted in increasing concentrations to create an internal calibration. The presented low volume protein-chip fulfills the requirements of point-of-care testing for accurate and repeatable (CV < 14%) quantification of serum proteins for the diagnosis of neonatal sepsis

A Renewable and Ultrasensitive Electrochemiluminescence Immunosenor Based on Magnetic RuL@SiO2-Au∼RuL-Ab2 Sandwich-Type Nano-Immunocomplexes

An ultrasensitive and renewable electrochemiluminescence (ECL) immunosensor was developed for the detection of tumor markers by combining a newly designed trace tag and streptavidin-coated magnetic particles (SCMPs). The trace tag (RuL@SiO2-Au∼RuL-Ab2) was prepared by loading Ru(bpy)32+(RuL)-conjuged secondary antibodies (RuL-Ab2) on RuL@SiO2 (RuL-doped SiO2) doped Au (RuL@SiO2-Au). To fabricate the immunosensor, SCMPs were mixed with biotinylated AFP primary antibody (Biotin-Ab1), AFP, and RuL@SiO2-Au∼RuL-Ab2 complexes, then the resulting SCMP/Biotin-Ab1/AFP/RuL@SiO2-Au∼RuL-Ab2 (SBAR) sandwich-type immunocomplexes were absorbed on screen printed carbon electrode (SPCE) for detection. The immunocomplexes can be easily washed away from the surface of the SPCE when the magnetic field was removed, which made the immunosensor reusable. The present immunosensor showed a wide linear range of 0.05–100 ng mL−1 for detecting AFP, with a low detection limit of 0.02 ng mL−1 (defined as S/N = 3). The method takes advantage of three properties of the immunosensor: firstly, the RuL@SiO2-Au∼RuL-Ab2 composite exhibited dual amplification since SiO2 could load large amount of reporter molecules (RuL) for signal amplification. Gold particles could provide a large active surface to load more reporter molecules (RuL-Ab2). Accordingly, through the ECL response of RuL and tripropylamine (TPA), a strong ECL signal was obtained and an amplification analysis of protein interaction was achieved. Secondly, the sensor is renewable because the sandwich-type immunocomplexes can be readily absorbed or removed on the SPCE’s surface in a magnetic field. Thirdly, the SCMP modified probes can perform the rapid separation and purification of signal antibodies in a magnetic field. Thus, the present immunosensor can simultaneously realize separation, enrichment and determination. It showed potential application for the detection of AFP in human sera

Magnetic Scanometric DNA Microarray Detection of Methyl Tertiary Butyl Ether Degrading Bacteria for Environmental Monitoring

A magnetoresistive biosensing platform based on a single magnetic tunnel junction (MTJ) scanning probe and DNA microarrays labeled with magnetic particles has been developed to provide an inexpensive, sensitive and reliable detection of DNA. The biosensing platform was demonstrated on a DNA microarray assay for quantifying bacteria capable of degrading methyl tertiary butyl ether (MTBE), where concentrations as low as 10 pM were detectable. Synthetic probe bacterial DNA was immobilized on a microarray glass slide surface, hybridized with the 48 base pair long biotinylated target DNA and subsequently incubated with streptavidin-coated 2.8 μm diameter magnetic particles. The biosensing platform then makes use of a micron-sized MTJ sensor that was raster scanned across a 3 mm by 5 mm glass slide area to capture the stray magnetic field from the tagged DNA and extract two dimensional magnetic field images of the microarray. The magnetic field output is then averaged over each 100 μm diameter DNA array spot to extract the magnetic spot intensity, analogous to the fluorescence spot intensity used in conventional optical scanners. The magnetic scanning result is compared with results from a commercial laser scanner and particle coverage optical counting to demonstrate the dynamic range and linear sensitivity of the biosensing platform as a potentially inexpensive, sensitive and portable alternative for DNA microarray detection for field applications

Development and characterization of microsatellite loci for Tabebuia cassinoides (Bignoniaceae)

Tabebuia cassinoides (Lam.) DC., popularly known as caxeta, is a tree species that belongs to the plant family Bignoniaceae. This species is endemic to the Brazilian Atlantic Forest and is widely exploited commercially. To date, little is known about its genetic structure, preventing the establishment of adequate management plans for this taxon. The objective of this study was to construct a microsatellite-enriched genomic library for T. cassinoides to select polymorphic loci, and standardize polymerase chain reaction amplification conditions. Of the 15 loci examined, 5 were polymorphic. The number of alleles per locus ranged from 2 to 8, with a mean of 4.4. The microsatellite loci described here represent the basis for detailed population genetic studies of this species, which will greatly contribute for the development of better conservation strategies for this taxon. Tabebuia cassinoides (Lam.) DC., popularly known as caxeta, is a tree species that belongs to the plant family Bignoniaceae. This species is endemic to the Brazilian Atlantic Forest and is widely exploited commercially. To date, little is known about its genetic structure, preventing the establishment of adequate management plans for this taxon. The objective of this study was to construct a microsatellite-enriched genomic library for T. cassinoides to select polymorphic loci, and standardize polymerase chain reaction amplification conditions. Of the 15 loci examined, 5 were polymorphic. The number of alleles per locus ranged from 2 to 8, with a mean of 4.4. The microsatellite loci described here represent the basis for detailed population genetic studies of this species, which will greatly contribute for the development of better conservation strategies for this taxon13356015605CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP142040/2009-6; 305102/2010-9sem informação2010/20548-

Development And Characterization Of Microsatellite Loci For Tabebuia Cassinoides (bignoniaceae).

Tabebuia cassinoides (Lam.) DC., popularly known as caxeta, is a tree species that belongs to the plant family Bignoniaceae. This species is endemic to the Brazilian Atlantic Forest and is widely exploited commercially. To date, little is known about its genetic structure, preventing the establishment of adequate management plans for this taxon. The objective of this study was to construct a microsatellite-enriched genomic library for T. cassinoides to select polymorphic loci, and standardize polymerase chain reaction amplification conditions. Of the 15 loci examined, 5 were polymorphic. The number of alleles per locus ranged from 2 to 8, with a mean of 4.4. The microsatellite loci described here represent the basis for detailed population genetic studies of this species, which will greatly contribute for the development of better conservation strategies for this taxon.135601-

Development of Multiple Polymorphic Microsatellite Markers for Ceratina calcarata (Hymenoptera: Apidae) Using Genome-Wide Analysis

The small carpenter bee, Ceratina calcarata (Robertson), is a widespread native pollinator across eastern North America. The behavioral ecology and nesting biology of C. calcarata has been relatively well-studied and the species is emerging as a model organism for both native pollinator and social evolution research. C. calcarata is subsocial: reproductively mature females provide extended maternal care to their brood. As such, studies of C. calcarata may also reveal patterns of relatedness and demography unique to primitively social Hymenoptera. Here, we present 21 microsatellite loci, isolated from the recently completed C. calcarata genome. Screening in 39 individuals across their distribution revealed that no loci were in linkage disequilibrium, nor did any deviate significantly from Hardy-Weinberg following sequential Bonferroni correction. Allele count ranged from 2 to 14, and observed and expected heterozygosities ranged from 0.08 to 0.82 (mean 0.47) and 0.26 to 0.88 (mean 0.56), respectively. These markers will enable studies of population-wide genetic structuring across C. calcarata’s distribution. Such tools will also allow for exploration of between and within-colony relatedness in this subsocial native pollinator

Giant magnetoresistive sensors and magnetic labels for chip-scale detection of immunosorbent assays

Giant magnetoresistive sensors utilized for the detection of bioanalytes modified with magnetic labels is a field of study in its beginning stages. Giant magnetoresistors (GMRs) are the magnetic sensors that are used in the read-heads of computer hard drives, and are small, microfabricated devices that display a measurable change in resistance in response to an external magnetic field;The research described in this dissertation focuses on the creation of a small, low-cost, sensitive, and high-speed readout method for the detection of magnetically-labeled bioanalytes. Several methods were examined, including the direct modification of the GMR sensor surface to be a bioactive address for the capture of magnetically-labeled alpha-mouse IgG through an immunosorbent assay. The GMR sensor was coated with gold, which was further modified with mouse IgG through a succinimidyl-terminated thiolate monolayer. The GMR sensors were found to respond linearly to the presence of the magnetically-labeled alpha-mouse IgG;Further explorations of the use of the GMR sensor moved the assay from directly on the sensor surface to a sample stick consisting of 200 x 200 microm alternating nickel reference and gold addresses. The sample stick was then scanned over the GMR sensor in a credit-card reader fashion. The nickel reference addresses served as a reference-normalizing tool, and compensated for any variation in sensor to sample separation distance. This detection methodology was verified with the detection of streptavidin-coated magnetic particles (MPs) captured on biotinylated gold addressees. The reference-normalized GMR response varied linearly with MP surface concentration;The utilization of the sample stick for the detection of rabbit IgG in a sandwich assay format was also described. Goat alpha-rabbit IgG capture antibody was bound to the gold addresses through reaction with a succinimidyl-terminated monolayer. After capture of the rabbit IgG and further labeling with biotinylated goat alpha-rabbit IgG, the sample sticks were exposed to streptavidin-coated MPs. Issues related to the small size of the gold addresses, assessments of GMR detection capabilities, and potential applications are described. Finally, the simultaneous detection of three IgG proteins was demonstrated by using the same sandwich assay tactics and modifying each gold address with an individual capture antibody