Development and validation of spectrophotometric method and paper-based microfluidic devices for the quantitative determination of Amoxicillin in pure form and pharmaceutical formulations.

There is a growing need for easy-to-use, low cost and portable quantitative assays to determine active pharmaceutical ingredients in the pharmaceutical industry. Here, we developed a batch spectrophotometric method and a method employing a paper-based microfluidic device for the estimation of Amoxicillin (AMX) in pure solution and pharmaceutical preparations. The detection depends on the coupling reaction of Amoxicillin with diazotized sulfadimidine (DSDM) in an alkaline medium. The yellow azo dye reaction product was measured at λmax 425 nm and linearity was observed from 2 to 30 mg L-1 with a detection limit of 0.32 mg L-1 and a quantification limit of 1.2 mg L-1 was found. The reaction was then transferred onto the paper-based microfluidic device and a plateau change in color intensity was found above 10 mg L-1. Thus, the paper-based microfluidic device can be applied for the semi-quantitative determination of Amoxicillin in pure solution and commercial pharmaceutical products for rapid screening.

Integration of IFAST-based nucleic acid extraction and LAMP for on-chip rapid detection of Agroathelia rolfsii in soil.

Agroathelia rolfsii (A. rolfsii) is a fungal infection and poses a significant threat to over 500 plant species worldwide. It can reduce crop yields drastically resulting in substantial economic losses. While conventional detection methods like PCR offer high sensitivity and specificity, they require specialized and expensive equipment, limiting their applicability in resource-limited settings and in the field. Herein, we present an integrated workflow with nucleic acid extraction and isothermal amplification in a lab-on-a-chip cartridge based on immiscible filtration assisted by surface tension (IFAST) to detect A. rolfsii fungi in soil for point-of-need application. Our approach enabled both DNA extraction of A. rolfsii from soil and subsequent colorimetric loop-mediated isothermal amplification (LAMP) to be completed on a single chip, termed IFAST-LAMP. LAMP primers targeting ITS region of A. rolfsii were newly designed and tested. Two DNA extraction methods based on silica paramagnetic particles (PMPs) and three LAMP assays were compared. The best-performing assay was selected for on-chip extraction and detection of A. rolfsii from soil samples inoculated with concentrations of 3.75, 0.375 and 0.0375 mg fresh weight per 100-g soil (%FW). The full on-chip workflow was achieved within a 1-h turnaround time. The platform was capable of detecting as low as 3.75 %FW at 2 days after inoculation and down to 0.0375 %FW at 3 days after inoculation. The IFAST-LAMP could be suitable for field-applicability for A. rolfsii detection in low-resource settings.

Development of a dual-flow tissue perfusion device for modeling the gastrointestinal tract-brain axis.

Despite the large number of microfluidic devices that have been described over the past decade for the study of tissues and organs, few have become widely adopted. There are many reasons for this lack of adoption, primarily that devices are constructed for a single purpose or because they are highly complex and require relatively expensive investment in facilities and training. Here, we describe a microphysiological system (MPS) that is simple to use and provides fluid channels above and below cells, or tissue biopsies, maintained on a disposable, poly(methyl methacrylate), carrier held between polycarbonate outer plates. All other fittings are standard Luer sizes for ease of adoption. The carrier can be coated with cells on both sides to generate membrane barriers, and the devices can be established in series to allow medium to flow from one cell layer to another. Furthermore, the carrier containing cells can be easily removed after treatment on the device and the cells can be visualized or recovered for additional off-chip analysis. A 0.4 µm membrane with cell monolayers proved most effective in maintaining separate fluid flows, allowing apical and basal surfaces to be perfused independently. A panel of different cell lines (Caco-2, HT29-MTX-E12, SH-SY5Y, and HUVEC) were successfully maintained in the MPS for up to 7 days, either alone or on devices connected in series. The presence of tight junctions and mucin was expressed as expected by Caco-2 and HT-29-MTX-E12, with Concanavalin A showing uniform staining. Addition of Annexin V and PI showed viability of these cells to be >80% at 7 days. Bacterial extracellular vesicles (BEVs) produced by Bacteroides thetaiotaomicron and labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbo-cyanine perchlorate (DiD) were used as a model component of the human colonic microbiota and were visualized translocating from an apical surface containing Caco-2 cells to differentiated SH-SY5Y neuronal cells cultured on the basal surface of connected devices. The newly described MPS can be easily adapted, by changing the carrier to maintain spheroids, pieces, or slices of biopsy tissue and joined in series to study a variety of cell and tissue processes. The cell layers can be made more complex through the addition of multiple cell types and/or different patterning of extracellular matrix and the ability to culture cells adjacent to one another to allow study of cell:cell transfer, e.g., passive or active drug transfer, virus or bacterial entry or BEV uptake and transfer.

Liquid Crystal-Templated Porous Microparticles via Photopolymerization of Temperature-Induced Droplets in a Binary Liquid Mixture.

Porous polymeric microspheres are an emerging class of materials, offering stimuli-responsive cargo uptake and release. Herein, we describe a new approach to fabricate porous microspheres based on temperature-induced droplet formation and light-induced polymerization. Microparticles were prepared by exploiting the partial miscibility of a thermotropic liquid crystal (LC) mixture composed of 4-cyano-4'-pentylbiphenyl (5CB, unreactive mesogens) with 2-methyl-1,4-phenylene bis4-[3-(acryloyloxy)propoxy] benzoate (RM257, reactive mesogens) in methanol (MeOH). Isotropic 5CB/RM257-rich droplets were generated by cooling below the binodal curve (20 °C), and the isotropic-to-nematic transition occurred after cooling below 0 °C. The resulting 5CB/RM257-rich droplets with radial configuration were subsequently polymerized under UV light, resulting in nematic microparticles. Upon heating the mixture, the 5CB mesogens underwent a nematic-isotropic transition and eventually became homogeneous with MeOH, while the polymerized RM257 preserved its radial configuration. Repeated cycles of cooling and heating resulted in swelling and shrinking of the porous microparticles. The use of a reversible materials templating approach to obtain porous microparticles provides new insights into binary liquid manipulation and potential for microparticle production.

Integrated microscale immiscible phase extraction and isothermal amplification for colorimetric detection of Neisseria gonorrhoeae.

Gonorrhea is the second most common sexually transmitted infection (STI) with around 87 million cases worldwide estimated in 2016 by the World Health Organization. With over half of the cases being asymptomatic, potential life-threatening complications and increasing numbers of drug-resistant strains, routine monitoring of prevalence and incidence of infections are key preventive measures. Whilst gold standard qPCR tests have excellent accuracy, they are neither affordable nor accessible in low-resource settings. In this study, we developed a lab-on-a-chip platform based on microscale immiscible filtration to extract, concentrate and purify Neisseria gonorrhoeae DNA with an integrated detection assay based on colorimetric isothermal amplification. The platform was capable of detecting as low as 500 copies/mL from spiked synthetic urine and showed no cross-reactivity when challenged with DNAs from other common STIs. The credit card-size device allows DNA extraction and purification without power or centrifuges, and the detection reaction only needs a low-tech block heater, providing a straightforward and visual positive/negative result within 1 h. These advantages offer great potential for accurate, affordable and accessible monitoring of gonorrhea infection in resource-poor settings.

Investigating the effects of arginine methylation inhibitors on microdissected brain tumour biopsies maintained in a miniaturised perfusion system.

Arginine methylation is a post-translational modification that consists of the transfer of one or two methyl (CH3) groups to arginine residues in proteins. Several types of arginine methylation occur, namely monomethylation, symmetric dimethylation and asymmetric dimethylation, which are catalysed by different protein arginine methyltransferases (PRMTs). Inhibitors of PRMTs have recently entered clinical trials to target several types of cancer, including gliomas (NCT04089449). People with glioblastoma (GBM), the most aggressive form of brain tumour, are among those with the poorest quality of life and likelihood of survival of anyone diagnosed with cancer. There is currently a lack of (pre)clinical research on the possible application of PRMT inhibitors to target brain tumours. Here, we set out to investigate the effects of clinically-relevant PRMT inhibitors on GBM biopsies. We present a new, low-cost, easy to fabricate perfusion device that can maintain GBM tissue in a viable condition for at least eight days post-surgical resection. The miniaturised perfusion device enables the treatment of GBM tissue with PRMT inhibitors ex vivo, and we observed a two-fold increase in apoptosis in treated samples compared to parallel control experiments. Mechanistically, we show thousands of differentially expressed genes after treatment, and changes in the type of arginine methylation of the RNA binding protein FUS that are consistent with hundreds of differential gene splicing events. This is the first time that cross-talk between different types of arginine methylation has been observed in clinical samples after treatment with PRMT inhibitors.

A sample-to-answer COVID-19 diagnostic device based on immiscible filtration and CRISPR-Cas12a-assisted detection.

In response to the ongoing coronavirus disease 2019 (COVID-19) pandemic and disparities of vaccination coverage in low-and middle-income countries, it is vital to adopt a widespread testing and screening programme, combined with contact tracing, to monitor and effectively control the infection dispersion in areas where medical resources are limited. This work presents a lab-on-a-chip device, namely 'IFAST-LAMP-CRISPR', as an affordable, rapid and high-precision molecular diagnostic means for detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The herein proposed 'sample-to-answer' platform integrates RNA extraction, amplification and molecular detection with lateral flow readout in one device. The microscale dimensions of the device containing immiscible liquids, coupled with the use of silica paramagnetic beads and guanidine hydrochloride, streamline sample preparation (including RNA extraction, concentration and purification) in 15 min with minimal hands-on steps. The pre-amplification in combination with CRISPR-Cas12a detection assays targeting the nucleoprotein (N) gene achieved visual identification of ≥ 470 copies mL-1 genomic SARS-CoV-2 samples in 45 min. On-chip assays showed the ability to isolate and detect SARS-CoV-2 RNA from 100 genome copies mL-1 of replication-deficient viral particles in 1 h. This simple, affordable and integrated platform demonstrated a visual, faster, and yet specificity- and sensitivity-comparable alternative to the costly gold-standard reverse transcription-polymerase chain reaction (RT-PCR) assay, requiring only a simple heating source. Initial testing illustrates the platform viability both on nasopharyngeal swab and saliva samples collected using the easily accessible Swan-brand cigarette filter, providing a complete workflow for COVID-19 diagnostics in low-resource settings.

Citizen-led sampling to monitor phosphate levels in freshwater environments using a simple paper microfluidic device.

Contamination of waterways is of increasing concern, with recent studies demonstrating elevated levels of antibiotics, antidepressants, household, agricultural and industrial chemicals in freshwater systems. Thus, there is a growing demand for methods to rapidly and conveniently monitor contaminants in waterways. Here we demonstrate how a combination of paper microfluidic devices and handheld mobile technology can be used by citizen scientists to carry out a sustained water monitoring campaign. We have developed a paper-based analytical device and a 3 minute sampling workflow that requires no more than a container, a test device and a smartphone app. The contaminant measured in these pilots are phosphates, detectable down to 3 mg L-1. Together these allow volunteers to successfully carry out cost-effective, high frequency, phosphate monitoring over an extended geographies and periods.

Spheroid-on-chip microfluidic technology for the evaluation of the impact of continuous flow on metastatic potential in cancer models in vitro.

The majority of cancer deaths are linked to tumor spread, or metastasis, but 3D in vitro metastasis models relevant to the tumor microenvironment (including interstitial fluid flow) remain an area of unmet need. Microfluidics allows us to introduce controlled flow to an in vitro cancer model to better understand the relationship between flow and metastasis. Here, we report new hybrid spheroid-on-chip in vitro models for the impact of interstitial fluid flow on cancer spread. We designed a series of reusable glass microfluidic devices to contain one spheroid in a microwell under continuous perfusion culture. Spheroids derived from established cancer cell lines were perfused with complete media at a flow rate relevant to tumor interstitial fluid flow. Spheroid viability and migratory/invasive capabilities were maintained on-chip when compared to off-chip static conditions. Importantly, using flow conditions modeled in vitro, we are the first to report flow-induced secretion of pro-metastatic factors, in this case cytokines vascular endothelial growth factor and interleukin 6. In summary, we have developed a new, streamlined spheroid-on-chip in vitro model that represents a feasible in vitro alternative to conventional murine in vivo metastasis assays, including complex tumor environmental factors, such as interstitial fluid flow, extracellular matrices, and using 3D models to model nutrient and oxygen gradients. Our device, therefore, constitutes a robust alternative to in vivo early-metastasis models for determination of novel metastasis biomarkers as well as evaluation of therapeutically relevant molecular targets not possible in in vivo murine models.

A lab-on-a-chip platform for integrated extraction and detection of SARS-CoV-2 RNA in resource-limited settings.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the unprecedented global pandemic of coronavirus disease-2019 (COVID-19). Efforts are needed to develop rapid and accurate diagnostic tools for extensive testing, allowing for effective containment of the infection via timely identification and isolation of SARS-CoV-2 carriers. Current gold standard nucleic acid tests require many separate steps that need trained personnel to operate specialist instrumentation in laboratory environments, hampering turnaround time and test accessibility, especially in low-resource settings. We devised an integrated on-chip platform coupling RNA extraction based on immiscible filtration assisted by surface tension (IFAST), with RNA amplification and detection via colorimetric reverse-transcription loop mediated isothermal amplification (RT-LAMP), using two sets of primers targeting open reading frame 1a (ORF1a) and nucleoprotein (N) genes of SARS-CoV-2. Results were identified visually, with a colour change from pink to yellow indicating positive amplification, and further confirmed by DNA gel electrophoresis. The specificity of the assay was tested against HCoV-OC43 and H1N1 RNAs. The assay based on use of gene N primers was 100% specific to SARS-CoV-2 with no cross-reactivity to HCoV-OC43 nor H1N1. Proof-of-concept studies on water and artificial sputum containing genomic SARS-CoV-2 RNA showed our IFAST RT-LAMP device to be capable of extracting and detecting 470 SARS-CoV-2 copies mL-1 within 1 h (from sample-in to answer-out). IFAST RT-LAMP is a simple-to-use, integrated, rapid and accurate COVID-19 diagnostic platform, which could provide an attractive means for extensive screening of SARS-CoV-2 infections at point-of-care, especially in resource-constrained settings.

Inertial focusing of microparticles, bacteria, and blood in serpentine glass channels.

Early detection of pathogenic microorganisms is pivotal to diagnosis and prevention of health and safety crises. Standard methods for pathogen detection often rely on lengthy culturing procedures, confirmed by biochemical assays, leading to >24 h for a diagnosis. The main challenge for pathogen detection is their low concentration within complex matrices. Detection of blood-borne pathogens via techniques such as PCR requires an initial positive blood culture and removal of inhibitory blood components, reducing its potential as a diagnostic tool. Among different label-free microfluidic techniques, inertial focusing on microscale channels holds great promise for automation, parallelization, and passive continuous separation of particles and cells. This work presents inertial microfluidic manipulation of small particles and cells (1-10 µm) in curved serpentine glass channels etched at different depths (deep and shallow designs) that can be exploited for (1) bacteria preconcentration from biological samples and (2) bacteria-blood cell separation. In our shallow device, the ability to focus Escherichia coli into the channel side streams with high recovery (89% at 2.2× preconcentration factor) could be applied for bacteria preconcentration in urine for diagnosis of urinary tract infections. Relying on differential equilibrium positions of red blood cells and E. coli inside the deep device, 97% red blood cells were depleted from 1:50 diluted blood with 54% E. coli recovered at a throughput of 0.7 mL/min. Parallelization of such devices could process relevant volumes of 7 mL whole blood in 10 min, allowing faster sample preparation for downstream molecular diagnostics of bacteria present in bloodstream.

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.

Microfluidic-Based Electrochemical Immunosensing of Ferritin.

Ferritin is a clinically important biomarker which reflects the state of iron in the body and is directly involved with anemia. Current methods available for ferritin estimation are generally not portable or they do not provide a fast response. To combat these issues, an attempt was made for lab-on-a-chip-based electrochemical detection of ferritin, developed with an integrated electrochemically active screen-printed electrode (SPE), combining nanotechnology, microfluidics, and electrochemistry. The SPE surface was modified with amine-functionalized graphene oxide to facilitate the binding of ferritin antibodies on the electrode surface. The functionalized SPE was embedded in the microfluidic flow cell with a simple magnetic clamping mechanism to allow continuous electrochemical detection of ferritin. Ferritin detection was accomplished via cyclic voltammetry with a dynamic linear range from 7.81 to 500 ng·mL-1 and an LOD of 0.413 ng·mL-1. The sensor performance was verified with spiked human serum samples. Furthermore, the sensor was validated by comparing its response with the response of the conventional ELISA method. The current method of microfluidic flow cell-based electrochemical ferritin detection demonstrated promising sensitivity and selectivity. This confirmed the plausibility of using the reported technique in point-of-care testing applications at a much faster rate than conventional techniques.

Rapid detection of Group B Streptococcus (GBS) from artificial urine samples based on IFAST and ATP bioluminescence assay: from development to practical challenges during protocol testing in Kenya.

We report the rapid detection (20 min) of Streptococcus agalactiae, Group B Streptococcus (GBS) employing on-chip magnetic isolation of GBS based on immiscible filtration assisted by surface tension (IFAST), followed by detection of the isolated GBS using an adenosine triphosphate (ATP) bioluminescence assay. Up to 80% GBS cells were isolated from spiked artificial urine samples with linear responses of bioluminescence signals from isolated cells at 2.3 × 102-9.1 × 105 CFU mL-1, demonstrating great promise for point-of-care detection of pathogenic bacteria in screening urine samples from pregnant women. Practical challenges during initial testing of the developed protocol with urine samples in Kenya are also described.

Computational modeling and fluorescence microscopy characterization of a two-phase magnetophoretic microsystem for continuous-flow blood detoxification.

Magnetic beads can be functionalized to capture and separate target pathogens from blood for extracorporeal detoxification. The beads can be magnetically separated from a blood stream and collected into a coflowing buffer solution using a two-phase liquid-liquid continuous-flow microfluidic device in the presence of an external field. However, device design and process optimization, i.e. high bead recovery with minimum blood loss or dilution remain a substantial technological challenge. We introduce a CFD-based Eulerian-Lagrangian computational model that enables the rational design and optimization of such systems. The model takes into account dominant magnetic and hydrodynamic forces on the beads as well as coupled bead-fluid interactions. Fluid flow (Navier-Stokes equations) and mass transfer (Fick's law) between the coflowing fluids are solved numerically, while the magnetic force on the beads is predicted using analytical methods. The model is demonstrated via application to a prototype device and used to predict key performance metrics; degree of bead separation, flow patterns, and mass transfer, i.e. blood diffusion to the buffer phase. The impact of different process variables and parameters - flow rates, bead and magnet dimensions and fluid viscosities - on both bead recovery and blood loss or dilution is quantified for the first time. The performance of the prototype device is characterized using fluorescence microscopy and the experimental results are found to match theoretical predictions within an absolute error of 15%. While the model is demonstrated here for analysis of a detoxification device, it can be readily adapted to a broad range of magnetically-enabled microfluidic applications, e.g. bioseparation, sorting and sensing.

Microfluidic Technique for the Simultaneous Quantification of Emulsion Instabilities and Lipid Digestion Kinetics.

Quantifying the impact of environmental physicochemical changes on the microstructure of lipid delivery systems is challenging. Therefore, we have developed a methodology to quantify the coalescence of oil-in-water emulsion droplets during lipid digestion in situ on a single droplet level. This technique involves a custom-made glass microfluidic platform, in which oil droplets can be trapped as single droplets, or several droplets per trap. The physicochemical environment can be controlled, and droplet digestion, as well as coalescence, can be visualized. We show that the exchange of the physicochemical conditions in the entire reaction chamber can be reached in under 30 s. Microparticle image velocimetry allowed mapping of the flow profile and demonstrated the tuneability of the shear profile in the device. The extraction of quantitative information regarding the physical characteristics of the droplets during digestion was performed using an automated image analysis throughout the digestion process. Therefore, we were able to show that oil-in-water emulsions stabilized by proteins coalesced under human gastric conditions. This coalescence delayed the overall lipid digestion kinetics. The droplets that coalesced during digestion were hydrolyzed 1.4 times slower than individually trapped droplets. Thus, the microstructural evolution of lipid delivery systems is a crucial factor in lipid digestion kinetics. This novel technique allows the simultaneous quantification of the impact that the physicochemical environment has on both the lipid droplet microstructure and the lipid release patterns.

A Microfluidic Device for Rapid Screening of E.†coli O157:H7 Based on IFAST and ATP Bioluminescence Assay for Water Analysis.

We present a simple microfluidic system for rapid screening of Escherichia coli (E. coli) O157:H7 employing the specificity of immunomagnetic separation (IMS) via immiscible filtration assisted by surface tension (IFAST), and the sensitivity of the subsequent adenosine triphosphate (ATP) assay by the bioluminescence luciferin/luciferase reaction. The developed device was capable of detecting E. coli O157:H7 from just 6 colony forming units (CFU) in 1 mL spiked buffer within 20 min. When tested with wastewater discharged effluent samples, without pre-concentration, the device demonstrated the ability to detect 104  CFU per mL seeded; suggesting great potential for point-of-need microbiological water quality monitoring.

Cell sorting by endocytotic capacity in a microfluidic magnetophoresis device.

Magnetically labelled cells are finding a wealth of applications for in vitro analysis as well as in vivo treatments. Sorting of cells into subpopulations based on their magnetite loading is an important step in such procedures. Here, we study the sorting of monocytes and macrophages which internalise nanoparticles to different extents based on their endocytotic capacity. Macrophages featured a high endocytotic activity and were found to internalise between 4 and 60 pg of iron per cell. They were successfully sorted into five subpopulations of narrow iron loading distributions via on-chip free-flow magnetophoresis, thus demonstrating the potential of sorting of relatively similarly loaded cells. Monocytes featured a low endocytotic capacity and took on 1 to 4 pg of iron per cell. Mixtures of monocytes and macrophages were successfully sorted within the free-flow magnetophoresis chip and good purity (>88%), efficacy (>60%) and throughput (from 10 to 100 cells s(-1)) could be achieved. The introduced method constitutes a viable tool for studies of endocytotic capacity and sorting/selection of cells based on this functionality.

Rapid, multistep on-chip DNA hybridisation in continuous flow on magnetic particles.

DNA hybridisation is an important tool for bioanalytical research and clinical diagnostics; conventional methods, however, require long incubation times and numerous washing steps, rendering the procedure time consuming and labour intensive. In this paper, we report on a rapid method for DNA hybridisation and isolation within a microfluidic device, where all reaction and washing steps are performed in continuous flow in an automated fashion within less than two minutes. Magnetic particles were used as a solid support and manipulated through laminar flow streams containing reagents and buffers by means of an external magnet. Thus, hybridisation, washing, intercalation, fluorescence detection and isolation were performed in continuous flow on the surface of the particles. Initially, the sensitivity of the system was investigated for a one-step DNA hybridisation of Alexa Fluor 555 labelled target DNA to a capture probe immobilised on the particle surface. Hybridisation and washing steps were performed in half a minute and target DNA was readily detected down to 20 nmol L(-1). Then a two-step assay, label-free DNA hybridisation followed by intercalation with PicoGreen was performed. All reaction and washing steps were carried out in continuous flow with a total assay time of about 1 min. This is a significant reduction in procedural time compared to conventional methods and opens the door for developing fully automated continuous flow integrated DNA analysis platforms.

Mobile magnetic particles as solid-supports for rapid surface-based bioanalysis in continuous flow.

An extremely versatile microfluidic device is demonstrated in which multi-step (bio)chemical procedures can be performed in continuous flow. The system operates by generating several co-laminar flow streams, which contain reagents for specific (bio)reactions across a rectangular reaction chamber. Functionalized magnetic microparticles are employed as mobile solid-supports and are pulled from one side of the reaction chamber to the other by use of an external magnetic field. As the particles traverse the co-laminar reagent streams, binding and washing steps are performed on their surface in one operation in continuous flow. The applicability of the platform was first demonstrated by performing a proof-of-principle binding assay between streptavidin coated magnetic particles and biotin in free solution with a limit of detection of 20 ng mL(-1) of free biotin. The system was then applied to a mouse IgG sandwich immunoassay as a first example of a process involving two binding steps and two washing steps, all performed within 60 s, a fraction of the time required for conventional testing.