Time- and distance-resolved robotic imaging of fluid flow in vertical microfluidic strips: a new technique for quantitative, multiparameter measurement of global haemostasis.

Measuring the complex processes of blood coagulation, haemostasis and thrombosis that are central to cardiovascular health and disease typically requires a choice between high-resolution low-throughput laboratory assays, or simpler less quantitative tests. We propose combining mass-produced microfluidic devices with open-source robotic instrumentation to enable rapid development of affordable and portable, yet high-throughput and performance haematological testing. A time- and distance-resolved fluid flow analysis by Raspberry Pi imaging integrated with controlled sample addition and illumination, enabled simultaneous tracking of capillary rise in 120 individual capillaries (∼160, 200 or 270 µm internal diameter), in 12 parallel disposable devices. We found time-resolved tracking of capillary rise in each individual microcapillary provides quantitative information about fluid properties and most importantly enables quantitation of dynamic changes in these properties following stimulation. Fluid properties were derived from flow kinetics using a pressure balance model validated with glycerol-water mixtures and blood components. Time-resolved imaging revealed fluid properties that were harder to determine from a single endpoint image or equilibrium analysis alone. Surprisingly, instantaneous superficial fluid velocity during capillary rise was found to be largely independent of capillary diameter at initial time points. We tested if blood function could be measured dynamically by stimulating blood with thrombin to trigger activation of global haemostasis. Thrombin stimulation slowed vertical fluid velocity consistent with a dynamic increase in viscosity. The dynamics were concentration-dependent, with highest doses reducing flow velocity faster (within 10 s) than lower doses (10-30 s). This open-source imaging instrumentation expands the capability of affordable microfluidic devices for haematological testing, towards high-throughput multi-parameter blood analysis needed to understand and improve cardiovascular health.

Moving microcapillary antibiotic susceptibility testing (mcAST) towards the clinic: unravelling kinetics of detection of uropathogenic E. coli, mass-manufacturing and usability for detection of urinary tract infections in human urine.

Innovation in infection based point-of-care (PoC) diagnostics is vital to avoid unnecessary use of antibiotics and the development of antimicrobial resistance. Several groups including our research team have in recent years successfully miniaturised phenotypic antibiotic susceptibility tests (AST) of isolated bacterial strains, providing validation that miniaturised AST can match conventional microbiological methods. Some studies have also shown the feasibility of direct testing (without isolation or purification), specifically for urinary tract infections, paving the way for direct microfluidic AST systems at PoC. As rate of bacteria growth is intrinsically linked to the temperature of incubation, transferring miniaturised AST nearer the patient requires building new capabilities in terms of temperature control at PoC, furthermore widespread clinical use will require mass-manufacturing of microfluidic test strips and direct testing of urine samples. This study shows for the first-time application of microcapillary antibiotic susceptibility testing (mcAST) directly from clinical samples, using minimal equipment and simple liquid handling, and with kinetics of growth recorded using a smartphone camera. A complete PoC-mcAST system was presented and tested using 12 clinical samples sent to a clinical laboratory for microbiological analysis. The test showed 100% accuracy for determining bacteria in urine above the clinical threshold (5 out of 12 positive) and achieved 95% categorical agreement for 5 positive urines tested with 4 antibiotics (nitrofurantoin, ciprofloxacin, trimethoprim and cephalexin) within 6 h compared to the reference standard overnight AST method. A kinetic model is presented for metabolization of resazurin, demonstrating kinetics of degradation of resazurin in microcapillaries follow those observed for a microtiter plate, with time for AST dependent on the initial CFU ml-1 of uropathogenic bacteria in the urine sample. In addition, we show for the first time that use of air-drying for mass-manufacturing and deposition of AST reagents within the inner surface of mcAST strips matches results obtained with standard AST methods. These results take mcAST a step closer to clinical application, for example as PoC support for antibiotic prescription decisions within a day.

Rapid Bacterial Motility Monitoring Using Inexpensive 3D-Printed OpenFlexure Microscopy Allows Microfluidic Antibiotic Susceptibility Testing.

Antibiotic susceptibility testing is vital to tackle the emergence and spread of antimicrobial resistance. Inexpensive digital CMOS cameras can be converted into portable digital microscopes using 3D printed x-y-z stages. Microscopic examination of bacterial motility can rapidly detect the response of microbes to antibiotics to determine susceptibility. Here, we present a new simple microdevice-miniature microscope cell measurement system for multiplexed antibiotic susceptibility testing. The microdevice is made using melt-extruded plastic film strips containing ten parallel 0.2 mm diameter microcapillaries. Two different antibiotics, ceftazidime and gentamicin, were prepared in Mueller-Hinton agar (0.4%) to produce an antibiotic-loaded microdevice for simple sample addition. This combination was selected to closely match current standard methods for both antibiotic susceptibility testing and motility testing. Use of low agar concentration permits observation of motile bacteria responding to antibiotic exposure as they enter capillaries. This device fits onto the OpenFlexure 3D-printed digital microscope using a Raspberry Pi computer and v2 camera, avoiding need for expensive laboratory microscopes. This inexpensive and portable digital microscope platform had sufficient magnification to detect motile bacteria, yet wide enough field of view to monitor bacteria behavior as they entered antibiotic-loaded microcapillaries. The image quality was sufficient to detect how bacterial motility was inhibited by different concentrations of antibiotic. We conclude that a 3D-printed Raspberry Pi-based microscope combined with disposable microfluidic test strips permit rapid, easy-to-use bacterial motility detection, with potential for aiding detection of antibiotic resistance.

Miniaturised broth microdilution for simplified antibiotic susceptibility testing of Gram negative clinical isolates using microcapillary devices.

Antibiotic resistance is a major global challenge. Although microfluidic antibiotic susceptibility tests (AST) offer great potential for rapid and portable testing to inform correct antibiotic selection, the impact of miniaturisation on broth microdilution (BMD) is not fully understood. We developed a 10-plex microcapillary based broth microdilution using resazurin as a colorimetric indicator for bacterial growth. Each capillary had a 1 microlitre capillary volume, 100 times smaller than microplate broth microdilution. The microcapillary BMD was compared to an in-house standard microplate AST and commercial Vitek 2 system. When tested with 25 uropathogenic isolates (20 Escherichia coli and 5 Klebsiella pneumoniae) and 2 reference E. coli, these devices gave 96.1% (441/459 isolate/antibiotic combinations) categorical agreement, across 17 therapeutically beneficial antibiotics, compared to in-house microplate BMD with resazurin. A further 99 (50 E. coli and 49 K. pneumoniae) clinical isolates were tested against 10 antibiotics and showed 92.3% categorical agreement (914/990 isolate/antibiotic combinations) compared to the Vitek 2 measurements. These microcapillary tests showed excellent analytical agreement with existing AST methods. Furthermore, the small size and simple colour change can be recorded using a smartphone camera or it is feasible to follow growth kinetics using very simple, low-cost readers. The test strips used here are produced in large batches, allowing hundreds of multiplex tests to be made and tested rapidly. Demonstrating performance of miniaturised broth microdilution with clinical isolates paves the way for wider use of microfluidic AST.

Label-free 1D microfluidic dipstick counting of microbial colonies and bacteriophage plaques.

Counting viable bacterial cells and functional bacteriophage is fundamental to microbiology underpinning research, surveillance, biopharmaceuticals and diagnostics. Colony forming unit (CFU) and plaque forming unit (PFU) counting still requires slow and laborious solid culture on agar in Petri dishes or plates. Here, we show that dip-stick microfluidic strips can be used without growth indicator dye for rapid and simple CFU ml-1 and PFU ml-1 measurement. We demonstrate for the first time that fluoropolymer microcapillaries combined with digital imaging allow bacteriophage plaques to be counted rapidly in a dip-and-test format. The microfluidic length scales offer a linear 1-dimensional alternative to a 2D solid agar medium surface, with colonies or plaques clearly visible as "dashes" or "gaps". An inexpensive open source darkfield biosensor system using Raspberry Pi imaging permits label-free detection and counting of colonies or plaques within 4-8 hours in a linear, liquid matrix within ∼200 µm inner diameter microcapillaries. We obtained full quantitative agreement between 1D microfluidic colony counting in dipsticks versus conventional 2D solid agar Petri dish plates for S. aureus and E. coli, and for T2 phage and phage K, but up to 6 times faster. Time-lapse darkfield imaging permitted detailed kinetic analysis of colony growth in the microcapillaries, providing new insight into microfluidic microbiology and colony growth, not possible with Petri dishes. Surprisingly, whilst E. coli colonies appeared earlier, subsequent colony expansion was faster along the microcapillaries for S. aureus. This may be explained by the microenvironment offered for 1D colony growth within microcapillaries, linked to a mass balance between nutrient (glucose) diffusion and bacterial growth kinetics. Counting individual colonies in liquid medium was not possible for motile strains that spread rapidly along the capillary, however inclusion of soft agar inhibited spreading, making this new simple dip-and-test counting method applicable to both motile and non-motile bacteria. Label-free dipstick colony and plaque counting has potential for many analytical microbial tasks, and the innovation of 1D colony counting has relevance to other microfluidic microbiology.

Gravity-Driven Microfluidic Siphons: Fluidic Characterization and Application to Quantitative Immunoassays.

A range of biosensing techniques including immunoassays are routinely used for quantitation of analytes in biological samples and available in a range of formats, from centralized lab testing (e.g., microplate enzyme-linked immunosorbent assay (ELISA)) to automated point-of-care (POC) and lateral flow immunochromatographic tests. High analytical performance is intrinsically linked to the use of a sequence of reagent and washing steps, yet this is extremely challenging to deliver at the POC without a high level of fluidic control involving, e.g., automation, fluidic pumping, or manual fluid handling/pipetting. Here we introduce a microfluidic siphon concept that conceptualizes a multistep ″dipstick″ for quantitative, enzymatically amplified immunoassays using a strip of microporous or microbored material. We demonstrated that gravity-driven siphon flow can be realized in single-bore glass capillaries, a multibored microcapillary film, and a glass fiber porous membrane. In contrast to other POC devices proposed to date, the operation of the siphon is only dependent on the hydrostatic liquid pressure (gravity) and not capillary forces, and the unique stepwise approach to the delivery of the sample and immunoassay reagents results in zero dead volume in the device, no reagent overlap or carryover, and full start/stop fluid control. We demonstrated applications of a 10-bore microfluidic siphon as a portable ELISA system without compromised quantitative capabilities in two global diagnostic applications: (1) a four-plex sandwich ELISA for rapid smartphone dengue serotype identification by serotype-specific dengue virus NS1 antigen detection, relevant for acute dengue fever diagnosis, and (2) quantitation of anti-SARS-CoV-2 IgG and IgM titers in spiked serum samples. Diagnostic siphons provide the opportunity for high-performance immunoassay testing outside sophisticated laboratories, meeting the rapidly changing global clinical and public health needs.

Antibody Surface Coverage Drives Matrix Interference in Microfluidic Capillary Immunoassays.

The performance of biosensors is often optimized in buffers, which brings inconsistencies during applications with biological samples. Current strategies for minimizing sample (matrix) interference are complex to automate and miniaturize, involving, e.g., sample dilution or recovery of serum/plasma. This study shows the first systematic analysis using hundreds of actual microfluidic immunoassay fluoropolymer strips to understand matrix interference in microflow systems. As many interfering factors are assay-specific, we have explored matrix interference for a range of enzymatic immunoassays, including a direct mIgG/anti-mIgG, a sandwich cancer biomarker PSA, and a sandwich inflammatory cytokine IL-1ß. Serum matrix interference was significantly affected by capillary antibody surface coverage, suggesting for the first time that the main cause of the serum matrix effect is low-affinity serum components (e.g., autoantibodies) competing with high-affinity antigens for the immobilized antibody. Additional experiments carried out with different capillary diameters confirmed the importance of antibody surface coverage in managing matrix interference. Building on these findings, we propose a novel analytical approach where antibody surface coverage and sample incubation times are key for eliminating and/or minimizing serum matrix interference, consisting in bioassay optimization carried out in serum instead of buffer, without compromising the performance of the bioassay or adding extra cost or steps. This will help establishing a new route toward faster development of modern point-of-care tests and effective biosensor development.

Counting bacteria in microfluidic devices: Smartphone compatible 'dip-and-test' viable cell quantitation using resazurin amplified detection in microliter capillary arrays.

Viable bacterial cell counting is fundamental to analytical microbiology and agar plate colony counting remains common yet laborious and slow. Here, we demonstrate two methods for counting bacteria using commercially available microfluidic devices. We show that accurate viable cell counting is possible using simple and easy 'dip and test' arrays of microcapillaries. Colorimetric and fluorescent growth detection both permit viable cell counting in microcapillaries either by limiting dilution into multiple microfluidic compartments using a single endpoint measurement, or alternatively by quantifying growth kinetics. The microcapillary devices are compatible with conventional 96 well plates and multichannel pipettes, expanding each microplate row into 120 individual 1 or 2 µL samples. At limiting dilution, counting the proportion of positive compartments permitted accurate calculation of gram-negative and gram-positive bacteria (E. coli and S. saprophyticus) at concentrations down to as low as 10 CFU/mL with almost 1:1 agreement with agar plate colony counts over four orders of magnitude. A smartphone camera was sufficient to record endpoint images of resazurin growth detection both colorimetrically and fluorescently. Viable cell counting of E. coli and S. saprophyticus was also possible through recording growth kinetics and determining the time taken to detect resazurin conversion. However, only the limiting dilution method remained consistent in the presence of urine matrix, as some interference in growth rate was observed when bacteria were spiked into higher concentrations of normal urine to simulate urinary tract infection patient samples. However, with the limiting dilution counting method endpoint growth was always detected even in the presence of 90% urine matrix, suggesting that this method might permit bacterial pathogen counting directly in clinical samples without agar plating.

Affordable mobile microfluidic diagnostics: minimum requirements for smartphones and digital imaging for colorimetric and fluorometric anti-dengue and anti-SARS-CoV-2 antibody detection.

Background: Miniaturised bioassays permit diagnostic testing near the patient, and the results can be recorded digitally using inexpensive cameras including smartphone and mobile phone cameras. Although digital cameras are now inexpensive and portable, the minimum performance required for microfluidic diagnostic bioassays has not been defined. We present a systematic comparison of a wide range of different digital cameras for capturing and measuring results of microfluidic bioassays and describe a framework to specify performance requirements to quantify immunoassays. Methods: A set of 200 µm diameter microchannels was filled with a range of concentrations of dyes used in colorimetric and fluorometric enzyme immunoassays. These were imaged in parallel using cameras of varying cost and performance ranging from <£30 to >£500. Results: Higher resolution imaging allowed larger numbers of microdevices to be resolved and analysed in a single image. In contrast, low quality cameras were still able to quantify results but for fewer samples. In some cases, an additional macro lens was added to focus closely. If image resolution was sufficient to identify individual microfluidic channels as separate lines, all cameras were able to quantify a similar range of concentrations of both colorimetric and fluorometric dyes. However, the mid-range cameras performed better, with the lowest cost cameras only allowing one or two samples to be quantified per image. Consistent with these findings, we demonstrate that quantitation (to determine endpoint titre) of antibodies against dengue and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses is possible using a wide range of digital imaging devices including the mid-range smartphone iPhone 6S and a budget Android smartphone costing <£50. Conclusions: In conclusion, while more expensive and higher quality cameras allow larger numbers of devices to be simultaneously imaged, even the lowest resolution and cheapest cameras were sufficient to record and quantify immunoassay results.

Exploiting open source 3D printer architecture for laboratory robotics to automate high-throughput time-lapse imaging for analytical microbiology.

Growth in open-source hardware designs combined with the low-cost of high performance optoelectronic and robotics components has supported a resurgence of in-house custom lab equipment development. We describe a low cost (below $700), open-source, fully customizable high-throughput imaging system for analytical microbiology applications. The system comprises a Raspberry Pi camera mounted on an aluminium extrusion frame with 3D-printed joints controlled by an Arduino microcontroller running open-source Repetier Host Firmware. The camera position is controlled by simple G-code scripts supplied from a Raspberry Pi singleboard computer and allow customized time-lapse imaging of microdevices over a large imaging area. Open-source OctoPrint software allows remote access and control. This simple yet effective design allows high-throughput microbiology testing in multiple formats including formats for bacterial motility, colony growth, microtitre plates and microfluidic devices termed 'lab-on-a-comb' to screen the effects of different culture media components and antibiotics on bacterial growth. The open-source robot design allows customization of the size of the imaging area; the current design has an imaging area of ~420 × 300mm, which allows 29 'lab-on-a-comb' devices to be imaged which is equivalent 3480 individual 1µl samples. The system can also be modified for fluorescence detection using LED and emission filters embedded on the PiCam for more sensitive detection of bacterial growth using fluorescent dyes.

Mixing is required for uniform reconstitution of filter-dried protein antigens in a single-injection vaccine formulation.

Ambient temperature filter dried vaccine formulations have been proposed to simultaneously achieve thermostability and offer a ready-to-use immunisation device that combines reconstitution and injection. Vaccine concentration should be uniform at the point of injection, but the uniformity following direct reconstitution of filter-dried vaccines has not been reported. We present here a study of vaccine mixing and release following dissolution of filter-dried model protein and toxoid antigens within a single syringe, filter and needle unit. Release was better for filters made from glass than cellulose. Without additional mixing, uniformity was poor and only 41% of input protein was released from protein filter-dried onto glass fiber. In contrast, adding a simple glass bead and mixing by inversion, 100% release antigen solution was achieved, with uniform concentration at exit from the needle throughout a simulated injection. Adsorption onto alum adjuvant had no detectable effect on vaccine dissolution and mixing. The uniformity and yield of low doses of diphtheria and tetanus toxoid was also improved by mixing, albeit with a lower yield of 60-68%. We conclude that uniformity and mixing should be studied to ensure safety and efficacy of directly reconstituted filter-dried vaccine formulations.

High potency of lipid conjugated TLR7 agonist requires nanoparticulate or liposomal formulation.

Conjugation of small molecule agonists of Toll-like receptor 7 (TLR7) to proteins, lipids, or polymers is known to modulate potency, and the physical form or formulation of these conjugates is likely to have a major effect on their immunostimulatory activity. Here, we studied the effect of formulation on potency of a 1,2­di­(9Z­octadecenoyl)­sn­glycero­3­phosphoethanolamine (DOPE) conjugated TLR7 agonist (DOPE-TLR7a) alongside assessing physical form using Dynamic Light Scattering (DLS), Nanosight Particle Tracking (NTA) analysis and Small Angle X-ray Scattering (SAXS). A very high potency of DOPE-TLR7a conjugate (EC50 around 9 nM) was observed either when prepared by direct dilution from DMSO or when formulated into 400-700 nm large multilamella liposomes containing dimethyldioctadecylammonium bromide salt (DDA) and DOPE. When prepared by dissolution in DMSO followed by dilution in aqueous culture medium, 93 ±â€¯5 nm nanoparticles were formed. Without dilution from solution in DMSO, no nanoparticles were observed and no immunostimulatory activity could be detected without this formulation step. SAXS analysis of the conjugate after DMSO dissolution/water dilution revealed a lamellar order with a layer spacing of 68.7 Å, which correlates with arrangement in groups of 3 bilayers. The addition of another immunostimulatory glycolipid, trehalose­6,6­dibehenate (TDB), to DOPE:DDA liposomes gave no further increase in immunostimulatory activity beyond that provided by incorporating DOPE-TLR7a. Given the importance of nanoparticle or liposomal formulation for activity, we conclude that the major mechanism for increased potency when TLR7 agonists are conjugated to macromolecules is through alteration of physical form.

Covalent immobilisation of antibodies in Teflon-FEP microfluidic devices for the sensitive quantification of clinically relevant protein biomarkers.

This study reports for the first time the sensitive colorimetric and fluorescence detection of clinically relevant protein biomarkers by sandwich immunoassays using the covalent immobilisation of antibodies onto the fluoropolymer surface inside Teflon®-FEP microfluidic devices. Teflon®-FEP has outstanding optical transparency ideal for high-sensitivity colorimetric and fluorescence bioassays, however this thermoplastic is regarded as chemically inert and very hydrophobic. Covalent immobilisation can offer benefits over passive adsorption to plastic surfaces by allowing better control over antibody density, orientation and analyte binding capacity, and so we tested a range of different and novel covalent immobilisation strategies. We first functionalised the inner surface of a 10-bore, 200 µm internal diameter FEP microcapillary film with high-molecular weight polyvinyl alcohol (PVOH) without changing the outstanding optical transparency of the device delivered by the matched refractive index of FEP and water. Glutaraldehyde immobilisation was compared with the use of photoactivated linkers and NHS-ester crosslinkers for covalently immobilising capture antibodies onto PVOH. Three clinically relevant sandwich ELISAs were tested against the cytokine IL-1ß, the myocardial infarct marker cardiac troponin I (cTnI), and the chronic heart failure marker brain natriuretic peptide (BNP). Overall, glutaraldehyde immobilisation was effective for BNP assays, but yielded unacceptable background for IL-1ß and cTnI assays caused by direct binding of the biotinylated detection antibody to the modified PVOH surface. We found NHS-ester groups reacted with APTES-treated PVOH coated fluoropolymers. This facilitated a novel method for capture antibody immobilisation onto fluoropolymer devices using a bifunctional NHS-maleimide crosslinker. The density of covalently immobilised capture antibodies achieved using PVOH/APTES/NHS/maleimide approached levels seen with passive adsorption, and sensitive and quantitative assay performance was achieved using this method. Overall, the PVOH coating provided an excellent surface for controlled covalent antibody immobilisation onto Teflon®-FEP for performing high-sensitivity immunoassays.

Lab on a stick: multi-analyte cellular assays in a microfluidic dipstick.

A new microfluidic concept for multi-analyte testing in a dipstick format is presented, termed "Lab-on-a-Stick", that combines the simplicity of dipstick tests with the high performance of microfluidic devices. Lab-on-a-stick tests are ideally suited to analysis of particulate samples such as mammalian or bacterial cells, and capable of performing multiple different parallel microfluidic assays when dipped into a single sample with results recorded optically. The utility of this new diagnostics format was demonstrated by performing three types of multiplex cellular assays that are challenging to perform in conventional dipsticks: 1) instantaneous ABO blood typing; 2) microbial identification; and 3) antibiotic minimum inhibitory (MIC) concentration measurement. A pressure balance model closely predicted the superficial flow velocities in individual capillaries, that were overestimated by up to one order of magnitude by the Lucas-Washburn equation conventionally used for wicking in cylindrical pores. Lab-on-a-stick provides a cost-effective, simple, portable and flexible multiplex platform for a range of assays, and will deliver a new generation of advanced yet affordable point-of-care tests for global diagnostics.

Evaluating and optimizing oral formulations of live bacterial vaccines using a gastro-small intestine model.

Gastrointestinal (GI) models that mimic physiological conditions in vitro are important tools for developing and optimizing biopharmaceutical formulations. Oral administration of live attenuated bacterial vaccines (LBV) can safely and effectively promote mucosal immunity but new formulations are required that provide controlled release of optimal numbers of viable bacterial cells, which must survive gastrointestinal transit overcoming various antimicrobial barriers. Here, we use a gastro-small intestine gut model of human GI conditions to study the survival and release kinetics of two oral LBV formulations: the licensed typhoid fever vaccine Vivotif comprising enteric coated capsules; and an experimental formulation of the model vaccine Salmonella Typhimurium SL3261 dried directly onto cast enteric polymer films and laminated to form a polymer film laminate (PFL). Neither formulation released significant numbers of viable cells when tested in the complete gastro-small intestine model. The poor performance in delivering viable cells could be attributed to a combination of acid and bile toxicity plus incomplete release of cells for Vivotif capsules, and to bile toxicity alone for PFL. To achieve effective protection from intestinal bile in addition to effective acid resistance, bile adsorbent resins were incorporated into the PFL to produce a new formulation, termed BR-PFL. Efficient and complete release of 4.4×10(7) live cells per dose was achieved from BR-PFL at distal intestinal pH, with release kinetics controlled by the composition of the enteric polymer film, and no loss in viability observed in any stage of the GI model. Use of this in vitro GI model thereby allowed rational design of an oral LBV formulation to maximize viable cell release.

Targeted Activation of Toll-Like Receptors: Conjugation of a Toll-Like Receptor 7 Agonist to a Monoclonal Antibody Maintains Antigen Binding and Specificity.

Therapeutic activation of Toll-like receptors (TLR) has potential for cancer immunotherapy, for augmenting the activity of antitumor monoclonal antibodies (mAbs), and for improved vaccine adjuvants. A previous attempt to specifically target TLR agonists to dendritic cells (DC) using mAbs failed because conjugation led to nonspecific binding and mAbs lost specificity. We demonstrate here for the first time the successful conjugation of a small molecule TLR7 agonist to an antitumor mAb (the anti-hCD20 rituximab) without compromising antigen specificity. The TLR7 agonist UC-1V150 was conjugated to rituximab using two conjugation methods, and yield, molecular substitution ratio, retention of TLR7 activity, and specificity of antigen binding were compared. Both conjugation methods produced rituximab-UC-1V150 conjugates with UC-1V150: rituximab ratio ranging from 1:1 to 3:1 with drug loading quantified by UV spectroscopy and drug substitution ratio verified by MALDI TOF mass spectroscopy. The yield of purified conjugates varied with conjugation method and dropped as low as 31% using a method previously described for conjugating UC-1V150 to proteins, where a bifunctional cross-linker was first reacted with rituximab and second to the TLR7 agonist. We therefore developed a direct conjugation method by producing an amine-reactive UV active version of UC-1V150, termed NHS:UC-1V150. Direct conjugation with NHS:UC-1V150 was quick and simple and gave improved conjugate yields of 65-78%. Rituximab-UC-1V150 conjugates had the expected pro-inflammatory activity in vitro (EC50 28-53 nM) with a significantly increased activity over unconjugated UC-1V150 (EC50 547 nM). Antigen binding and specificity of the rituxuimab-UC-1V150 conjugates was retained, and after incubation with human peripheral blood leukocytes, all conjugates bound strongly only to CD20-expressing B cells while no nonspecific binding to CD20-negative cells was observed. Selective targeting of Toll-like receptor activation directly within tumors or to DC is now feasible.

Enteric coated spheres produced by extrusion/spheronization provide effective gastric protection and efficient release of live therapeutic bacteria.

We present a novel but simple enteric coated sphere formulation containing probiotic bacteria (Lactobacillus casei). Oral delivery of live bacterial cells (LBC) requires live cells to survive firstly manufacturing processes and secondly GI microbicidal defenses including gastric acid. We incorporated live L. casei directly in the granulation liquid, followed by granulation, extrusion, spheronization, drying and spray coating to produce dried live probiotic spheres. A blend of MCC, calcium-crosslinked alginate, and lactose was developed that gave improved live cell survival during manufacturing, and gave excellent protection from gastric acid plus rapid release in intestinal conditions. No significant loss of viability was observed in all steps except drying, which resulted in approximately 1 log loss of viable cells. Eudragit coating was used to protect dried live cells from acid, and microcrystalline cellulose (MCC) was combined with sodium alginate to achieve efficient sphere disintegration leading to rapid and complete bacterial cell release in intestinal conditions. Viability and release of L. casei was evaluated in vitro in simulated GI conditions. Uncoated spheres gave partial acid protection, but enteric coated spheres effectively protected dried probiotic LBC from acid for 2h, and subsequently released all viable cells within 1h of transfer into simulated intestinal fluid.

Multiplexed femtomolar quantitation of human cytokines in a fluoropolymer microcapillary film.

Sensitive quantitation of multiple cytokines can provide important diagnostic information during infection, inflammation and immunopathology. In this study sensitive immunoassay detection of human cytokines IL-1ß, IL-6, IL-12p70 and TNFα is shown for singleplex and multiplex formats using a novel miniaturized ELISA platform. The platform uses a disposable plastic multi-syringe aspirator (MSA) integrating 8 disposable fluoropolymer microfluidic test strips, each containing an array of ten 200 µm mean i.d. microcapillaries coated with a set of monoclonal antibodies. Each MSA device thus performs 10 tests on 8 samples, delivering 80 measurements. Unprecedented levels of sensitivity were obtained with the novel fluoropolymer microfluidic material and simple colorimetric detection in a flatbed scanner. The limit of detection for singleplex detection ranged from 2.0 to 15.0 pg ml(-1), i.e. 35 and 713 femtomolar for singleplex cytokine detection, and the intra- and inter-assay coefficient of variation (CV) remained within 10%. In addition, a triplex immunoassay was developed for measuring IL-1ß, IL-12p70 and TNFα simultaneously from a given sample in the pg ml(-1) range. These assays permit high sensitivity measurement with rapid <15 min assay or detection from undiluted blood serum. The portability, speed and low-cost of this system are highly suited to point-of-care testing and field diagnostics applications.

Portable smartphone quantitation of prostate specific antigen (PSA) in a fluoropolymer microfluidic device.

We present a new, power-free and flexible detection system named MCFphone for portable colorimetric and fluorescence quantitative sandwich immunoassay detection of prostate specific antigen (PSA). The MCFphone is composed by a smartphone integrated with a magnifying lens, a simple light source and a miniaturised immunoassay platform, the Microcapillary Film (MCF). The excellent transparency and flat geometry of fluoropolymer MCF allowed quantitation of PSA in the range 0.9 to 60 ng/ml with<7% precision in 13 min using enzymatic amplification and a chromogenic substrate. The lower limit of detection was further improved from 0.4 to 0.08 ng/ml in whole blood samples with the use of a fluorescence substrate. The MCFphone has shown capable of performing rapid (13 to 22 min total assay time) colorimetric quantitative and highly sensitive fluorescence tests with good %Recovery, which represents a major step in the integration of a new generation of inexpensive and portable microfluidic devices with commercial immunoassay reagents and off-the-shelf smartphone technology.

A lab-in-a-briefcase for rapid prostate specific antigen (PSA) screening from whole blood.

We present a new concept for rapid and fully portable prostate specific antigen (PSA) measurements, termed "lab-in-a-briefcase", which integrates an affordable microfluidic ELISA platform utilising a melt-extruded fluoropolymer microcapillary film (MCF) containing an array of 10 200 µm internal diameter capillaries, a disposable multi-syringe aspirator (MSA), a sample tray pre-loaded with all of the required immunoassay reagents, and a portable film scanner for colorimetric signal digital quantification. Each MSA can perform 10 replicate microfluidic immunoassays on 8 samples, allowing 80 measurements to be made in less than 15 minutes based on semi-automated operation, without the need of additional fluid handling equipment. The assay was optimised for the measurement of a clinically relevant range of PSA of 0.9 to 60.0 ng ml(-1) in 15 minutes with CVs on the order of 5% based on intra-assay variability when read using a consumer flatbed film scanner. The PSA assay performance in the MSA remained robust in undiluted or 1 : 2 diluted human serum or whole blood, and the matrix effect could simply be overcome by extending sample incubation times. The PSA "lab-in-a-briefcase" is particularly suited to a low-resource health setting, where diagnostic labs and automated immunoassay systems are not accessible, by allowing PSA measurement outside the laboratory using affordable equipment.