Magnetic particle plug-based assays for biomarker analysis

Conventional immunoassays offer selective and quantitative detection of a number of biomarkers, but are laborious and time-consuming. Magnetic particle-based assays allow easy and rapid selection of analytes, but still suffer from the requirement of tedious multiple reaction and washing steps. Here, we demonstrate the trapping of functionalised magnetic particles within a microchannel for performing rapid immunoassays by flushing consecutive reagent and washing solutions over the trapped particle plug. Three main studies were performed to investigate the potential of the platform for quantitative analysis of biomarkers: (i) a streptavidin-biotin binding assay; (ii) a sandwich assay of the inflammation biomarker, C-reactive protein (CRP); and (iii) detection of the steroid hormone, progesterone (P4), towards a competitive assay. Quantitative analysis with low limits of detection was demonstrated with streptavidin-biotin, while the CRP and P4 assays exhibited the ability to detect clinically relevant analytes, and all assays were completed in only 15 min. These preliminary results show the great potential of the platform for performing rapid, low volume magnetic particle plug-based assays of a range of clinical biomarkers via an exceedingly simple technique

Diamagnetic repulsion of particles for multilaminar flow assays

© The Royal Society of Chemistry. We demonstrate diamagnetic repulsion forces for performing continuous multilaminar flow assays on particles based on their intrinsic properties and with a simple setup. The platform could be applied to sandwich assays on polystyrene particles, and to cell-based assays via their suspension in biologically benign magnetic media

On-chip determination of C-reactive protein using magnetic particles in continuous flow

We demonstrate the application of a multilaminar flow platform, in which functionalized magnetic particles are deflected through alternating laminar flow streams of reagents and washing solutions via an external magnet, for the rapid detection of the inflammatory biomarker, C-reactive protein (CRP). The two-step sandwich immunoassay was accomplished in less than 60 s, a vast improvement on the 80−300 min time frame required for enzyme-linked immunosorbent assays (ELISA) and the 50 min necessary for off-chip magnetic particle-based assays. The combination of continuous flow and a stationary magnet enables a degree of autonomy in the system, while a detection limit of 0.87 μg mL−1 makes it suitable for the determination of CRP concentrations in clinical diagnostics. Its applicability was further proven by assaying real human serum samples and comparing those results to values obtained using standard ELISA tests

Immobilised-enzyme microreactors for the identification and synthesis of conjugated drug metabolites

The study of naturally circulating drug metabolites has been a focus of interest, since these metabolites may have different therapeutic and toxicological effects compared to the parent drug. The synthesis of metabolites outside of the human body is vital in order to conduct studies into the pharmacological activities of drugs and bioactive compounds. Current synthesis methods require significant purification and separation efforts or do not provide sufficient quantities for use in pharmacology experiments. Thus, there is a need for simple methods yielding high conversions whilst bypassing the requirement for a separation. Here we have developed and optimised flow chemistry methods in glass microfluidic reactors utilising surface-immobilised enzymes for sulfonation (SULT1a1) and glucuronidation (UGT1a1). Conversion occurs in flow, the precursor and co-factor are pumped through the device, react with the immobilised enzymes and the product is then simply collected at the outlet with no separation from a complex biological matrix required. Conversion only occurred when both the correct co-factor and enzyme were present within the microfluidic system. Yields of 0.97 ± 0.26 mg were obtained from the conversion of resorufin into resorufin sulfate over 2 h with the SULT1a1 enzyme and 0.47 mg of resorufin glucuronide over 4 h for UGT1a1. This was demonstrated to be significantly more than static test tube reactions at 0.22 mg (SULT1a1) and 0.19 mg (UGT1a1) over 4 h. With scaling out and parallelising, useable quantities of hundreds of micrograms for use in pharmacology studies can be synthesised simply

Phaseguide assisted liquid lamination for magnetic particle-based assays

We have developed a magnetic particle-based assay platform in which functionalised magnetic particles are transferred sequentially through laminated volumes of reagents and washing buffers. Lamination of aqueous liquids is achieved via the use of phaseguide technology; microstructures that control the advancing air–liquid interface of solutions as they enter a microfluidic chamber. This allows manual filling of the device, eliminating the need for external pumping systems, and preparation of the system requires only a few minutes. Here, we apply the platform to two on-chip strategies: (i) a one-step streptavidin–biotin binding assay, and (ii) a two-step C-reactive protein immunoassay. With these, we demonstrate how condensing multiple reaction and washing processes into a single step significantly reduces procedural times, with both assay procedures requiring less than 8 seconds

Artificial leaf device for hydrogen generation from immobilised C. reinhardtii microalgae

We developed a fully biomimetic leaf-like device for hydrogen production which allows incorporated fabric-immobilised microalgae culture to be simultaneously hydrated with media and harvested from the produced hydrogen in a continuous flow regime without the need to replace the algal culture. Our leaf device produces hydrogen by direct photolysis of water resulting from redirecting the photosynthetic pathways in immobilised microalgae due to the lack of oxygen. In contrast to the many other reports in the literature on batch photobioreactors producing hydrogen from suspension culture of microalgae, we present the first report where this is done in a continuous manner from a fabric-immobilised microalgae culture. The reported artificial leaf device maximises the sunlight energy utilisation per gram of algae and can be upscaled cheaply and easily to cover large areas. We compared the production of hydrogen from both immobilised and suspended cultures of C. reinhardtii microalgae under sulphur, phosphorus and oxygen deprived conditions. The viability and potential of this approach is clearly demonstrated. Even though this is a first prototype, the hydrogen yield of our artificial leaf device is twenty times higher per gram of algae than in previously the reported batch reactors. Such leaf-like devices could potentially be made from flexible plastic sheets and installed on roofs and other sun-exposed surfaces that are inaccessible by photovoltaic cells. The ability to continuously produce inexpensive hydrogen by positioning inexpensive sheets onto any surface could have an enormous importance in the field of biofuels. The proposed new concept can provide a cleaner and very inexpensive way of bio-hydrogen generation by flexible sheet-like devices

FISH and chips: a review of microfluidic platforms for FISH analysis

Fluorescence in situ hybridization (FISH) allows visualization of specific nucleic acid sequences within an intact cell or a tissue section. It is based on molecular recognition between a fluorescently labeled probe that penetrates the cell membrane of a fixed but intact sample and hybridizes to a nucleic acid sequence of interest within the cell, rendering a measurable signal. FISH has been applied to, for example, gene mapping, diagnosis of chromosomal aberrations and identification of pathogens in complex samples as well as detailed studies of cellular structure and function. However, FISH protocols are complex, they comprise of many fixation, incubation and washing steps involving a range of solvents and temperatures and are, thus, generally time consuming and labor intensive. The complexity of the process, the relatively high-priced fluorescent probes and the fairly high-end microscopy needed for readout render the whole process costly and have limited wider uptake of this powerful technique. In recent years, there have been attempts to transfer FISH assay protocols onto microfluidic lab-on-a-chip platforms, which reduces the required amount of sample and reagents, shortens incubation times and, thus, time to complete the protocol, and finally has the potential for automating the process. Here, we review the wide variety of approaches for lab-on-chip-based FISH that have been demonstrated at proof-of-concept stage, ranging from FISH analysis of immobilized cell layers, and cells trapped in arrays, to FISH on tissue slices. Some researchers have aimed to develop simple devices that interface with existing equipment and workflows, whilst others have aimed to integrate the entire FISH protocol into a fully autonomous FISH on-chip system. Whilst the technical possibilities for FISH on-chip are clearly demonstrated, only a small number of approaches have so far been converted into off-the-shelf products for wider use beyond the research laboratory

Detection of doxycycline hyclate and oxymetazoline hydrochloride in pharmaceutical preparations via spectrophotometry and microfluidic paper-based analytical device (μPADs)

There is growing demand for simple to operate, sensitive, on-site quantitative assays to investigate concentrations of drug molecules in pharmaceutical preparations for quality assurance. Here, we report on the development of two colorimetric analysis methods for the study the antibiotic doxycycline hyclate (DOX) and the nasal decongestant oxymetazoline hydrochloride (OXY), in solution as well as in their respective formulations. We compare a UV/vis spectrophotometry method with a color change recorded on a microfluidic paper-based analytical device (μPAD). Detection is based on the pharmaceutical compounds coupling with diazotized 4-aminoacetophenone (DAAP) under alkaline conditions to produce colored azo-dye products. These azo-compounds were monitored by absorbance at 425 nm for DOX and 521 nm for OXY, with linear calibration graphs in the concentration range of 0.5–35 mg L−1 (DOX) and 1.0–40 mg L−1 (OXY) and limits of detection of 0.24 mg L−1 (DOX) and 0.32 mg L−1 (OXY). For the μPAD method, color intensity was measured from photographs and a linear increase was observed at concentrations from above approximately 15 mg L−1 for both compounds and up to 35 mg L−1 for DOX and 40 mg L−1 for OXY. The developed methods were also applied to the formulated pharmaceuticals and no interference was found from the excipient. Thus, the paper-based device provides an inexpensive, simple alternative approach for use outside centralized laboratories with semi-quantitative capability

Magnetic particles-based chemiluminescence immunoassay for progesterone determination

© 2017, Chiang Mai University. All rights reserved. A magnetic particles-based chemiluminescence immunoassay was investigated for progesterone detection by using luminometer. In this work, progesterone was determined based on the competitive binding between progesterone in the sample and progesterone-horseradish peroxidase (HRP) conjugate for a constant amount of rabbit anti-progesterone. Initially, anti-rabbit IgG coated magnetic particles conjugated with primary progesterone antibody were bound to progesterone in the samples. Then, the amount of proge sterone was quantified by reacting with the residual unoccupied antibody sites with HRP-progesterone, followed by HRP substrate (luminol, H 2 O 2 , and p-iodophenol (PIP)) and finally detection of the generated chemiluminescence by a luminometer. The intensity of the emitting light was proportional to the amount of enzyme present (HRP-progesterone) and was inversely related to the amount of unlabeled progesterone in the sample. The optimum conditions for determination of progesterone were obtained at 0.15 μg L -1 magnetic particles, 5.0x10-4 mol L -1 luminol, 5.0 × 10 -3 mol L -1 H 2 O 2 , 1.0 × 10 -3 mol L -1 PIP, and phosphate buffer saline buffer pH 9. The optimal dilutions of both anti-progesterone antibody and HRP-progesterone conjugate were 1:1000. The linear relationship between chemiluminescence intensity (RLU) and various concentrations of progesterone was over the concentration range of 0.5-50.0 μg L -1 . This proposed method had been successfully applied to the evaluation of progesterone in human sera

Multiplex sorting of foodborne pathogens by on-chip free-flow magnetophoresis

This study reports multiplex sorting of Salmonella typhimurium and Escherichia coli 0157, from broth cultures and from pathogen-spiked skinned chicken breast enrichment broths by employing microfluidic free-flow magnetophoresis. Magnetic beads of different sizes and magnetite content, namely Dynabeads anti-salmonella and Hyglos-Streptavidin beads together with the corresponding pathogen-specific biotinylated recombinant phages, were utilised as affinity solid phases for the capture and concentration of viable S. typhimurium and E. coli 0157. Following optimisation, the protocol was used to demonstrate continuous magnetophoretic sorting of the two pathogen-bound magnetic bead populations from mixed cultures and from pathogen-spiked chicken pre-enrichment broths under the influence of a Halbach magnet array. For example, in the la tter case, a pure population of S. typhimurium-bound Dynabeads (72% recovery) was sorted from a 100 μL mixture containing E. coli 0157-bound Hyglos beads (67% recovery) within 1.2 min in the presence of 0.1% Tween 20. This proof-of-principle study demonstrates how more than one pathogen type can be simultaneously isolated/enriched from a single food pre-enrichment broth (e.g. Universal food enrichment broth)