Commercial Plate Reader Research Articles

Luminescent Terbium Probe for Time‐Resolved FRET and NSET Binding Assays with Quantum Dots and Gold Nanoparticles

AbstractTime‐gated or time‐resolved FRET (TR‐FRET) assays are important tools in biosensing, bioimaging, drug screening, and molecular diagnostics. Efficient TR‐FRET assays require stable lanthanide complexes with high absorption cross sections, high quantum yields, and long photoluminescence lifetimes. Owing to their challenging synthesis, such complexes are relatively rare and new components are of potential interest when developing TR‐FRET probes. Here, we evaluate the recently developed Tb complex CoraFluor‐1 concerning its analytical performance in terbium‐to‐quantum dot FRET and terbium‐to‐gold nanoparticle NSET assays using the prototypical biological recognition system of streptavidin and biotin. Biological binding was quantifiable at sub‐picomolar concentrations in small sample volumes, with broad applicability demonstrated across three commercial fluorescence plate readers used for time‐resolved, spectrally‐resolved, and clinical bioanalysis. Overall, CoraFluor‐1 provided excellent analytical performance as both FRET and NSET donor, validating its potential for developing new TR‐FRET probes for biosensing and bioimaging.

EP4 Receptor Conformation Sensor Suited for Ligand Screening and Imaging of Extracellular Prostaglandins.

Prostaglandins are important lipid mediators with a wide range of functions in the human body. They act mainly via plasma membrane localized prostaglandin receptors, which belong to the G-protein coupled receptor class. Due to their localized formation and short lifetime, it is important to be able to measure the distribution and abundance of prostaglandins in time and/or space. In this study, we present a Foerster resonance energy transfer (FRET)-based conformation sensor of the human prostaglandin E receptor subtype 4 (EP4 receptor), which was capable of detecting prostaglandin E2 (PGE2)-induced receptor activation in the low nanomolar range with a good signal-to-noise ratio. The sensor retained the typical selectivity for PGE2 among arachidonic acid products. Human embryonic kidney cells stably expressing the sensor did not produce detectable amounts of prostaglandins making them suitable for a coculture approach allowing us, over time, to detect prostaglandin formation in Madin-Darby canine kidney cells and primary mouse macrophages. Furthermore, the EP4 receptor sensor proved to be suited to detect experimentally generated PGE2 gradients by means of FRET-microscopy, indicating the potential to measure gradients of PGE2 within tissues. In addition to FRET-based imaging of prostanoid release, the sensor allowed not only for determination of PGE2 concentrations, but also proved to be capable of measuring ligand binding kinetics. The good signal-to-noise ratio at a commercial plate reader and the ability to directly determine ligand efficacy shows the obvious potential of this sensor interest for screening and characterization of novel ligands of the pharmacologically important human EP4 receptor. SIGNIFICANCE STATEMENT: The authors present a biosensor based on the prostaglandin E receptor subtype 4, which is well suited to measure extracellular prostaglandin E2 (PGE2) concentration with high temporal and spatial resolution. It can be used for the imaging of PGE2 levels and gradients by means of Foerster resonance energy transfer microscopy, and for determining PGE2 release of primary cells as well as for screening purposes in a plate reader setting.

A novel, accurate, and non-invasive liquid biopsy test to measure cellular immune responses as a tool to diagnose early-stage lung cancer: a clinical trials study

IntroductionLung cancer remains the leading cause of death from cancer, worldwide. Developing early detection diagnostic methods, especially non-invasive methods, is a critical component to raising the overall survival rate and prognosis for lung cancer. The purpose of this study is to evaluate two protocols of a novel in vitro cellular immune response test to detect lung cancer. The test specifically quantifies the glycolysis metabolism pathway, which is a biomarker for the activation level of immune cells. It summarizes the results of two clinical trials, where each deploys a different protocol's version of this test for the detection of lung cancer. In the later clinical trial, an improved test protocol is applied.MethodThe test platform is based on changes in the metabolic pathways of the immune cells following their activation by antigenic stimuli associated with Lung cancer. Peripheral Blood Mononuclear Cells are loaded on a multiwell plate together with various lung tumor associated antigens and a fluorescent probe that exhibits a pH-dependent absorption shift. The acidification process in the extracellular fluid is monitored by a commercial fluorescence plate reader device in continuous reading for 3 h at 37 °C to document the fluorescent signal received from each well.ResultsIn the later clinical trial, an improved test protocol was applied and resulted in increased test accuracy. Specificity of the test increased to 94.0% and test sensitivity increased to 97.3% in lung cancer stage I, by using the improved protocol.ConclusionThe improved protocol of the novel cellular immune metabolic response based test detects stage I and stage II of lung cancer with high specificity and sensitivity, with low material costs and fast results.

A smart tablet-phone-based system using dynamic light modulation for highly sensitive colorimetric biosensing

Facile, efficient, and inexpensive biosensing systems are in high demand for biomedical test. In recent years, numerous smartphone-based biosensing systems have been developed to match demand for biomedical test in source-limited environment. However, application of these smartphone-based biosensing systems was limited because of performance gap between the smartphone-based systems and commercial plate readers. In this study, we have developed a smart tablet–phone-based colorimetric plate reader (STPCPR) with intelligent and dynamic light modulation for broad-range colorimetric assays. The STPCPR allows controllable modulation of exciting light in three different color channels that is lack in conventional smartphone-based system. Using optimized exciting modulation, the STPCPR shows higher sensitivities, lower detection limits, and broader detection ranges in test of pigments, proteins, and cells when compared to conventional plate readers and smartphone-based system. Therefore, the developed STPCPR can serve as an ideal next-generation smartphone-based biosensing system for point-of-care colorimetric test in diverse biomedical applications in source-limited environment.

Front Cover: Isothermal Rolling Circle Amplification and Lanthanide‐Based FRET for Femtomolar Quantification of MicroRNA (Anal. Sens. 6/2022)

The front cover picture illustrates rolling circle (RCA) amplification of one microRNA into a long DNA concatemer and its multicolor detection via terbium (Tb) and europium (Eu) Förster resonance energy transfer (FRET). This sensing concept was used to develop wash-free diagnostic assays on two commercial fluorescence plate readers. More information can be found in the Research Article by N. Hildebrandt and co-workers.

Whole-cell bacterial biosensor with the capability to detect red palm weevil, Rhynchophorus ferrugineus, in date palm trees, Phoenix dactylifera: a proof of concept study

The red palm weevil (RPW), Rhynchophorus ferrugineus, is considered a severe pest of palms. Usually, the early stages of infection are without visible signs. An attractive early sensing approach of non-visible infections is based on volatile organic compounds (VOCs). In this study, a whole-cell bacterial biosensor was used for the identification of RPW in date palm (Phoenix dactylifera). The cells are genetically modified to produce light in the presence of general stresses. The bioluminescent bacterial panel is based on three genetically engineered Escherichia coli strains that are sensitive to cytotoxicity (TV1061), genotoxicity (DPD2794), or quorum-sensing (K802NR). The bioluminescent bacterial panel detects the presence of VOCs and a change in the light signal is then generated, reflecting the health status of the date palm tree. The bioreporter bacteria cells are immobilized in calcium alginate tablets and placed in a sealed jar without direct contact with the tested sample, thereby exposing them only to the VOCs in the surrounding air. The immobilized bacteria cells were exposed to the air near infected by RPW or uninfected sugar canes, date palm tree pieces, and on date palm trees. Commercial plate reader was used for signal measurement. The findings show that quorum-sensing was induced by all the tested samples of infected sugar canes, date palm tree pieces, and date palm trees. While, cytotoxicity was induced only by infected date palm tree pieces, and genotoxicity was induced only by infected date palm trees. The bacterial monitoring results enable the identification of specific signatures that will allow a quick and accurate diagnosis.

Cytosolic calcium and membrane potential in articular chondrocytes during parabolic flight

Chondrocytes are the sole resident cells in articular cartilage, which line the articulating bones in joints allowing effortless movements. Chondrocytes are responsible for cartilaginous matrix synthesis, maintenance and degradation. Since these cells are frequently exposed to mechanical loading patterns, it is generally believed that chondrocytes require mechanical stimuli for adequate cartilage homeostasis. In this context, mechanosensitive ion channels are thought to be among the first instances in cellular mechanosensing. Prior studies showed that applied mechanical stimuli increase cytosolic free calcium and hyperpolarization of the cells’ membrane potential. We used an adapted commercial plate reader during a parabolic flight to examine whether cytosolic free calcium and membrane potential are gravity-dependent. Our experiments showed that both cytosolic free calcium and membrane potential remain unchanged in response to short periods of increased or reduced gravity. Careful data analysis also revealed some pitfalls and shortcomings when using a commercial plate reader during a parabolic flight.

Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector.

Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA3) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo.

Anti-inflammatory effect of salt water and chlorhexidine 0.12% mouthrinse after periodontal surgery: a randomized prospective clinical study.

The purpose of this study was to compare the anti-inflammatory efficacy of sodium chloride- and a 0.12% chlorhexidine mouth rinses in patients undergoing minimal invasive periodontal surgery. Forty-seven patients with a diagnosis of periodontitis and indication for access flap procedure were randomly selected. Group A: a sodium chloride (salt)water-based mouth rinse (test group) or group B: a 0.12% chlorhexidine mouth rinse (control group) administered after surgery. Gingival Index (GI) were evaluated in the whole mouth and in the surgical site at baseline (T1), a week later (T2), and 12 weeks (T3) after the treatment. Total MMP activity was measured in GCF using a commercial kit and plate reader. Medians of total MMP activity and GI were compared for time intervals T1 vs. T2, T1 vs. T3, and T2 vs T3 using Friedman tests and Wilcoxon signed rank tests, and were also compared between test and control using Mann-WhitneyU tests at each timepoint. The average GI values showed significant differences between baseline and T2 (p = 0.0005) and baseline and T3 (p = 0.003) in the test group. The sodium chloride-mouth rinse use after periodontal surgery seems to have similar anti-inflammatory properties as CHX mouth rinse and can be used regularly postoperatively after periodontal surgical procedures. The use of salt water mouthwash showed an anti-inflammatory effect similar to CHX 0.12% after minimal invasive periodontal surgery. Salt water mouthwash is accessible to the world population and can contribute on the healing process after periodontal surgery.

Portable and accurate diagnostics for COVID-19: Combined use of the miniPCR thermocycler and a well-plate reader for SARS-CoV-2 virus detection.

The coronavirus disease 2019 (COVID-19) pandemic has crudely demonstrated the need for massive and rapid diagnostics. By the first week of July, more than 10,000,000 positive cases of COVID-19 have been reported worldwide, although this number could be greatly underestimated. In the case of an epidemic emergency, the first line of response should be based on commercially available and validated resources. Here, we demonstrate the use of the miniPCR, a commercial compact and portable PCR device recently available on the market, in combination with a commercial well-plate reader as a diagnostic system for detecting genetic material of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19. We used the miniPCR to detect and amplify SARS-CoV-2 DNA sequences using the sets of initiators recommended by the World Health Organization (WHO) for targeting three different regions that encode for the N protein. Prior to amplification, samples were combined with a DNA intercalating reagent (i.e., EvaGreen Dye). Sample fluorescence after amplification was then read using a commercial 96-well plate reader. This straightforward method allows the detection and amplification of SARS-CoV-2 nucleic acids in the range of ~625 to 2×105 DNA copies. The accuracy and simplicity of this diagnostics strategy may provide a cost-efficient and reliable alternative for COVID-19 pandemic testing, particularly in underdeveloped regions where RT-QPCR instrument availability may be limited. The portability, ease of use, and reproducibility of the miniPCR makes it a reliable alternative for deployment in point-of-care SARS-CoV-2 detection efforts during pandemics.

CCD Based Detector for Detection of Abrin Toxin Activity.

Abrin is a highly potent and naturally occurring toxin produced in the seeds of Abrus precatorius (Rosary Pea) and is of concern as a potential bioterrorism weapon. There are many rapid and specific assay methods to detect this toxic plant protein, but few are based on detection of toxin activity, critical to discern biologically active toxin that disables ribosomes and thereby inhibits protein synthesis, producing cytotoxic effects in multiple organ systems, from degraded or inactivated toxin which is not a threat. A simple and low-cost CCD detector system was evaluated with colorimetric and fluorometric cell-based assays for abrin activity; in the first instance measuring the abrin suppression of mitochondrial dehydrogenase in Vero cells by the MTT-formazan method and in the second instance measuring the abrin suppression of green fluorescent protein (GFP) expression in transduced Vero and HeLa cells. The limit of detection using the colorimetric assay was 10 pg/mL which was comparable to the fluorometric assay using HeLa cells. However, with GFP transduced Vero cells a hundred-fold improvement in sensitivity was achieved. Results were comparable to those using a more expensive commercial plate reader. Thermal inactivation of abrin was studied in PBS and in milk using the GFP-Vero cell assay. Inactivation at 100 °C for 5 min in both media was complete only at the lowest concentration studied (0.1 ng/mL) while treatment at 63 °C for 30 min was effective in PBS but not milk.

An internet of things-based intensity and time-resolved fluorescence reader for point-of-care testing

A miniature internet of things (IoT)-based point-of-care testing (PoCT) fluorescence reader, able to perform both intensity and time-resolved measurements of different fluorescent tags, is presented. This low cost platform has been conceived for performing tests in low-resource and remote settings, displaying versatile performance and yet simple operation. It consists on an external case of 43 × 30 × 42 mm3 (built in a 3D-printer) where all the elements are fixed, including some basic optics (3 lenses and 2 filters), a laser diode and a custom designed Single-Photon Avalanche Diodes (SPADs) camera. Both, the laser and the camera are controlled by a Field Programmable Gate Array (FPGA) with IoT capabilities.The PoCT was validated by detecting Plasmodium antigen in a fluorescent enzyme-linked immunosorbent assay (ELISA) using a fluorescence substrate. The results were compared to those provided in parallel by two commercial fluorescent plate readers. As it will be shown, the PoCT fluorescent readout was more sensitive than its colorimetric counterpart. Furthermore, the PoCT displayed similar signal trends and levels of detection than the bulkier and more expensive commercial fluorescence plate readers. These results demonstrate that the PoCT platform developed could bring the performance of central laboratory assay techniques closer to the end-user level.

Quantitative measurements of inorganic analytes on a digital microfluidics platform

Two methods were studied for selectively measuring the on-chip absorbance of trace sulfate analytes in droplets on a digital microfluidics (DMF) platform. In one method, the direction of measurement was perpendicular to the flat upper and lower surfaces of the DMF platform (vertical), and in the second method, the measurement direction was parallel to the DMF platform surfaces (horizontal). The channel height or the vertical light path length was 0.24 mm, and the droplet diameter was 1 mm. The DMF system employed a silicone oil transport medium whereby a thin, non-uniform oil layer formed between the droplet and the upper/lower plates which was unstable, resulting in randomly formed local oil lenses. The mobile oil lenses caused vertical absorbance measurement errors and uncertainties. The effects of the oil lenses were verified by simulation. Horizontal absorbance measurements were taken with embedded optical fibers (0.2 mm in diameter) aligned over the bottom chip surface in contact with the sides of the droplet, resulting in a horizontal light path length approximately three times that of the vertical light path. Because no oil lenses could form on the droplet’s sides, the stability of repeated horizontal measurements outperformed repeated vertical measurements made on the same droplet and on multiple droplets actuated into the measurement positions. Comparisons were based on measurement standard deviations and limits of detection (LOD). The following LODs and measurement standard deviations were achieved for horizontal measurements of multiple sulfate concentrations in 1.5 µl droplets: 7 ppm for sulfate (0.3–2.7%) and an R2 value of 0.957 from a least square data fit. Measurements on a commercial plate reader gave comparable results (200 µl liquid in each well, LOD equals 11 ppm, CV equals to 0.2–4%), even though the absorbance path was larger (0.7 mm). This LOD value means that the chip could detect 10.5 ng of sulfate. LOD values on vertical measurements were also similar, but large measurement errors from numerous outlier points yielded an R2 value of 0.735 and large average measurement standard deviations (36%).

Quantification of Cellular Deoxyribonucleoside Triphosphates by Rolling Circle Amplification and Förster Resonance Energy Transfer.

The quantification of cellular deoxyribonucleoside triphosphate (dNTP) levels is important for studying pathologies, genome integrity, DNA repair, and the efficacy of pharmacological drug treatments. Current standard methods, such as enzymatic assays or high-performance liquid chromatography, are complicated, costly, and labor-intensive, and alternative techniques that simplify dNTP quantification would present very useful complementary approaches. Here, we present a dNTP assay based on isothermal rolling circle amplification (RCA) and rapid time-gated Förster resonance energy transfer (TG-FRET), which used a commercial clinical plate reader system. Despite the relatively simple assay format, limits of detection down to a few picomoles of and excellent specificity for each dNTP against the other dNTPs, rNTPs, and dUTP evidenced the strong performance of the assay. Direct applicability of RCA-FRET to applied nucleic acid research was demonstrated by quantifying all dNTPs in CEM-SS leukemia cells with and without hydroxyurea or auranofin treatment. Both pharmacological agents could reduce the dNTP production in a time- and dose-dependent manner. RCA-FRET provides simple, rapid, sensitive, and specific quantification of intracellular dNTPs and has the potential to become an advanced tool for both fundamental and applied dNTP research.

1380-P: Multiplex Detection of T1D-Associated Autoantibodies by High-Quantum-Efficiency FRET

We have developed a homogeneous time-resolved Förster Resonance Energy Transfer (TR-FRET) assay for rapid, no-wash multiplexed detection of T1D-associated autoantibodies from small volumes (5-10 μL) of patient plasma or serum. In our assay, separate pools of recombinant antigen are labelled with either a long-lived high-quantum-efficiency terbium cryptate FRET donor or a small-molecule flurophore FRET acceptor, generating potent FRET pairs. With the mean distance between the tips of the two epitope binding regions approximately 12-13 nm and the distance between the edges of each epitope binding arm on the order of 3.5 nm, the pairs are brought into signaling proximity when the twin binding arms of immunoglobulin bind a donor and an acceptor. We used terbium’s tightly banded multi-peak luminescence with large stokes shift and long millisecond-scale lifetime under 350 nm excitation to engineer the assay for detection of four T1D targets at clinically relevant concentrations—GAD 65, IA-2, ZnT8 and insulin—along with a fifth pair as an internal control. We report on the assay’s impressive performance with clinical samples, including the development and implementation of a high-performance crosstalk correction algorithm. The assay described here functions identically in both commercial plate readers and in point-of-care systems, a key feature for highly consistent test results. Disclosure A.A. Metz: Employee; Self; Wave 80 Biosciences, Inc. A.M. Arsham: Consultant; Self; Wave 80 Biosciences. D.J. Laser: Board Member; Self; Wave 80 Biosciences. A.D. Droitcour: Employee; Self; Wave 80 Biosciences. Funding Type 1 Diabetes Foundation

An Open-Source Plate Reader.

Microplate readers are foundational instruments in experimental biology and bioengineering that enable multiplexed spectrophotometric measurements. To enhance their accessibility, we here report the design, construction, validation, and benchmarking of an open-source microplate reader. The system features full-spectrum absorbance and fluorescence emission detection, in situ optogenetic stimulation, and stand-alone touch screen programming of automated assay protocols. The total system costs less than $3500, a fraction of the cost of commercial plate readers, and can detect the fluorescence of common dyes at concentrations as low as ∼10 nM. Functional capabilities were demonstrated in the context of synthetic biology, optogenetics, and photosensory biology: by steady-state measurements of ligand-induced reporter gene expression in a model of bacterial quorum sensing and by flavin photocycling kinetic measurements of a LOV (light-oxygen-voltage) domain photoreceptor used for optogenetic transcriptional activation. Fully detailed guides for assembling the device and automating it using the custom Python-based API (Application Program Interface) are provided. This work contributes a key technology to the growing community-wide infrastructure of open-source biology-focused hardware, whose creation is facilitated by rapid prototyping capabilities and low-cost electronics, optoelectronics, and microcomputers.

Novel non-invasive early detection of lung cancer using liquid immunobiopsy metabolic activity profiles.

Lung cancer is the leading cause of cancer death worldwide. Survival is largely dependent on the stage of diagnosis: the localized disease has a 5-year survival greater than 55%, whereas, for spread tumors, this rate is only 4%. Therefore, the early detection of lung cancer is key for improving prognosis. In this study, we present an innovative, non-invasive, cancer detection approach based on measurements of the metabolic activity profiles of immune system cells. For each Liquid ImmunoBiopsy test, a 384 multi-well plate is loaded with freshly separated PBMCs, and each well contains 1 of the 16 selected stimulants in several increasing concentrations. The extracellular acidity is measured in both air-open and hermetically-sealed states, using a commercial fluorescence plate reader, for approximately 1.5h. Both states enable the measurement of real-time accumulation of 'soluble' versus 'volatile' metabolic products, thereby differentiating between oxidative phosphorylation and aerobic glycolysis. The metabolic activity profiles are analyzed for cancer diagnosis by machine-learning tools. We present a diagnostic accuracy study, using a multivariable prediction model to differentiate between lung cancer and control blood samples. The model was developed and tested using a cohort of 200 subjects (100 lung cancer and 100 controlsubjects), yielding 91% sensitivity and 80% specificity in a 20-fold cross-validation. Our results clearly indicate that the proposed clinical model is suitable for non-invasive early lung cancer diagnosis, and is indifferent to lung cancer stage and histological type.

Low cost bioluminescence imaging as an alternative to in vivo bioassays for quantifying biologically active staphylococcal enterotoxin type E

Staphylococcus aureus is a major causative agent implicated in outbreaks of food poisoning. It acts through the production of a range of toxins including staphylococcal enterotoxin type E which has been associated with foodborne outbreaks in the USA, UK and France. While tests such as ELISA exist to detect the toxin’s molecules, existing methods to distinguish active toxin from inactivated are costly and require the use of live animal testing. In this study we constructed and evaluated a low cost CCD camera device in conjunction with a cell based assay for active SEE employing a genetically engineered T-cell line with a luciferase reporter regulated by nuclear factor of activated T-cells combined with a B-cell line for toxin presentation. The emitted light intensity from the T-cells is proportional to SEE concentration over an 8-log range and can discern the active form of toxin that sickens consumers, from toxin inactivated by heat treatment. The assay was verified in sample food matrices by spiking white grape and peach mango juices as well as apple cider. The low cost imaging device compared favorably against the expensive commercial luminometer plate reader using the same cell based assay. The low cost camera device should improve the availability of active toxin testing, especially in resource poor regions.

GryphSens: A Smartphone-Based Portable Diagnostic Reader for the Rapid Detection of Progesterone in Milk.

Enzyme-linked immunosorbent assay (ELISA) is a popular assay technique for the detection and quantification of various biological substances due its high sensitivity and specificity. More often, it requires large and expensive laboratory instruments, which makes it difficult to conduct when the tests must be performed quickly at the point-of-care (POC). To increase portability and ease of use, we propose a portable diagnostic system based on a Raspberry Pi imaging sensor for the rapid detection of progesterone in milk samples. We designed, assembled, and tested a standalone portable diagnostic reader and validated it for progesterone detection against a standard ELISA assay using a commercial plate reader. The portable POC device yielded consistent results, regardless of differences in the cameras and flashlights between various smartphone devices. An Android application was built to provide front-end access to users, control the diagnostic reader, and display and store the progesterone measurement on the smartphone. The diagnostic reader takes images of the samples, reads the pixel values, processes the results, and presents the results on the handheld device. The proposed POC reader can perform to superior levels of performance as a plate reader, while adding the desirable qualities of portability and ease of use.

Fabrication and optimisation of a fused filament 3D-printed microfluidic platform

A 3D-printed microfluidic device was designed and manufactured using a low cost ($2000) consumer grade fusion deposition modelling (FDM) 3D printer. FDM printers are not typically used, or are capable, of producing the fine detailed structures required for microfluidic fabrication. However, in this work, the optical transparency of the device was improved through manufacture optimisation to such a point that optical colorimetric assays can be performed in a 50 µl device. A colorimetric enzymatic cascade assay was optimised using glucose oxidase and horseradish peroxidase for the oxidative coupling of aminoantipyrine and chromotropic acid to produce a blue quinoneimine dye with a broad absorbance peaking at 590 nm for the quantification of glucose in solution. For comparison the assay was run in standard 96 well plates with a commercial plate reader. The results show the accurate and reproducible quantification of 0–10 mM glucose solution using a 3D-printed microfluidic optical device with performance comparable to that of a plate reader assay.