Centrifugal microfluidic system for colorimetric sample-to-answer detection of viral pathogens.

We describe a microfluidic system for conducting thermal lysis, polymerase chain reaction (PCR) amplification, hybridization, and colorimetric detection of foodborne viral organisms in a sample-to-answer format. The on-chip protocol entails 24 steps which are conducted by a centrifugal platform that allows for actuating liquids pneumatically during rotation and so facilitates automation of the workflow. The microfluidic cartridge is fabricated from transparent thermoplastic polymers and accommodates assay components along with an embedded micropillar array for detection and read-out. A panel of oligonucleotide primers and probes has been developed to perform PCR and hybridization assays that allows for identification of five different viruses, including pathogens such as norovirus and hepatitis A virus (HAV) in a multiplexed format using digoxigenin-labelled amplicons and immunoenzymatic conversion of a chromogenic substrate. Using endpoint detection, we demonstrate that the system can accurately and repetitively (n = 3) discriminate positive and negative signals for HAV at 350 genome copies per µL. As part of the characterization and optimization process, we show that the implementation of multiple (e.g., seven) micropillar arrays in a narrow fluidic pathway can lead to variation (up to 50% or more) in the distribution of colorimetric signal deriving from the assay. Numerical modeling of flow behaviour was used to substantiate these findings. The technology-by virtue of automation-can provide a pathway toward rapid detection of viral pathogens, shortening response time in food safety surveillance, compliance, and enforcement as well as outbreak investigations.

Automated sample-to-answer centrifugal microfluidic system for rapid molecular diagnostics of SARS-CoV-2.

Testing for SARS-CoV-2 is one of the most important assets in COVID-19 management and mitigation. At the onset of the pandemic, SARS-CoV-2 testing was uniquely performed in central laboratories using RT-qPCR. RT-qPCR relies on trained personnel operating complex instrumentation, while time-to-result can be lengthy (e.g., 24 to 72 h). Now, two years into the pandemic, with the surge in cases driven by the highly transmissible Omicron variant, COVID-19 testing capabilities have been stretched to their limit worldwide. Rapid antigen tests are playing an increasingly important role in quelling outbreaks by expanding testing capacity outside the realm of clinical laboratories. These tests can be deployed in settings where repeat and rapid testing is essential, but they often come at the expense of limited accuracy and sensitivity. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) provides a number of advantages to SARS-CoV-2 testing in standard laboratories and at the point-of-need. In contrast to RT-qPCR, RT-LAMP is performed at a constant temperature, which circumvents the need for thermal cycling and translates into a shorter analysis time (e.g., <1 h). In addition, RT-LAMP is compatible with colorimetric detection, facilitating visualization and read-out. However, even with these benefits, RT-LAMP is not yet clinically deployed at its full capacity. Lack of automation and integration of sample preparation, such as RNA extraction, limits the sensitivity and specificity of the method. Furthermore, the need for cold storage of reagents complicates its use at the point of need. The developments presented in this work address these limitations: We describe a fully automated SARS-CoV-2 detection method using RT-LAMP, which also includes up-front lysis and extraction of viral RNA, performed on a centrifugal platform with active pneumatic pumping, a disposable, all-polymer-based microfluidic cartridge and lyophilized reagents. We demonstrate that the limit of detection of the RT-LAMP assay itself is 0.2 copies per µL using N and E genes as target sequences. When combined with integrated RNA extraction, the assay sensitivity is 0.5 copies per µL, which is highly competitive to RT-qPCR. We tested the automated assay using 12 clinical swab specimens from patients and were able to distinguish positive and negative samples for SARS-CoV-2 within 60 min, thereby obtaining 100% agreement with RT-qPCR results.

Use of Polymer Micropillar Arrays as Templates for Solid-Phase Immunoassays.

We investigate the use of periodic micropillar arrays produced by high-fidelity microfabrication with cyclic olefin polymers for solid-phase immunoassays. These three-dimensional (3D) templates offer higher surface-to-volume ratios than two-dimensional substrates, making it possible to attach more antibodies and so increase the signal obtained by the assay. Micropillar arrays also provide the capacity to induce wicking, which is used to distribute and confine antibodies on the surface with spatial control. Micropillar array substrates are modified by using oxygen plasma treatment, followed by grafting of (3-aminopropyl)triethoxysilane for binding proteins covalently using glutaraldehyde as a cross-linker. The relationship between microstructure and fluorescence signal was investigated through variation of pitch (10-50 µm), pillar diameter (5-40 µm), and pillar height (5-57 µm). Our findings suggest that signal intensity scales proportionally with the 3D surface area available for performing solid-phase immunoassays. A linear relationship between fluorescence intensity and microscale structure can be maintained even when the aspect ratio and pillar density both become very high, opening the possibility of tuning assay response by design such that desired signal intensity is obtained over a wide dynamic range compatible with different assays, analyte concentrations, and readout instruments. We demonstrate the versatility of the approach by performing the most common immunoassay formats-direct, indirect, and sandwich-in a qualitative fashion by using colorimetric and fluorescence-based detection for a number of clinically relevant protein markers, such as tumor necrosis factor alpha, interferon gamma (IFN-γ), and spike protein of severe acute respiratory syndrome coronavirus 2. We also show quantitative detection of IFN-γ in serum using a fluorescence-based sandwich immunoassay and calibrated samples with spike-in concentrations ranging from 50 pg/mL to 5 µg/mL, yielding an estimated limit of detection of ∼1 pg/mL for arrays with high micropillar density (11561 per mm2) and aspect ratio (1:11.35).

On-the-Fly Phase Transition and Density Changes of Aqueous Two-Phase Systems on a Centrifugal Microfluidic Platform.

This paper describes on-the-fly physical property changes of aqueous two-phase systems (ATPS) in microfluidic devices. The properties and phases of the ATPS are modulated on-demand by using a centrifugal microfluidic device filled with poly(ethylene glycol) (PEG) and dextran (DEX) solutions. By use of the centrifugal force and active pneumatic controls provided by a centrifugal microfluidic platform (CMP), PEG-DEX mixtures are manipulated and processed inside simple thermoplastic microfluidic devices. First, we experimentally demonstrate an on-chip ATPS transition from two phases to a single phase and vice versa by dynamically changing the concentration of the solution to bring ATPS across the binodal curve. We also demonstrate a density modulation scheme by introducing an ATPS solution mixed with sodium diatrizoate hydrate, which allows to increase the liquid density. By adding precisely metered volumes of water, we spontaneously change the density of the solution on the CMP and show that density marker microbeads fall into the solution according to their corresponding densities. The measured densities of ATPS show a good agreement with densities of microbeads and analytical plots. The results presented in this paper highlight the tremendous potential of CMPs for performing complex on-chip processing of ATPS. We anticipate that this method will be useful in applications such as microparticle-based plasma protein analysis and blood cell fractionation.

Multifunctional magnetic nanoparticle cloud assemblies for in situ capture of bacteria and isolation of microbial DNA.

We investigate the formation of suspended magnetic nanoparticle (MNP) assemblies (M-clouds) and their use for in situ bacterial capture and DNA extraction. M-clouds are obtained as a result of magnetic field density variations when magnetizing an array of micropillars coated with a soft ferromagnetic NiP layer. Numerical simulations suggest that the gradient in the magnetic field created by the pillars is four orders of magnitude higher than the gradient generated by the external magnets. The pillars therefore serve as the sole magnetic capture sites for MNPs which accumulate on opposite sides of each pillar facing the magnets. Composed of loosely aggregated MNPs, the M-cloud can serve as a porous capture matrix for target analyte flowing through the array. The concept is demonstrated by using a multifunctional M-cloud comprising immunomagnetic NPs (iMNPs) for capture of Escherichia coli O157:H7 from river water along with silica-coated NPs for subsequent isolation and purification of microbial DNA released upon bacterial lysis. Confocal microscopy imaging of fluorescently labeled iMNPs and E. coli O157:H7 reveals that bacteria are trapped in the M-cloud region between micropillars. Quantitative assessment of in situ bacterial capture, lysis and DNA isolation using real-time polymerase chain reaction shows linear correlation between DNA output and input bacteria concentration, making it possible to confirm E. coli 0157:H7 at 103 cells per mL. The M-cloud method further provides one order of magnitude higher DNA output concentrations than incubation of the sample with iMNPs in a tube for an equivalent period of time (e.g., 10 min). Results from assays performed in the presence of Listeria monocytogenes (at 106 cells per mL each) suggest that non-target organisms do not affect on-chip E. coli capture, DNA extraction efficiency and quality of the eluted sample.

Real-time monitoring of bead-based DNA hybridization in a microfluidic system: study of amplicon hybridization behavior on solid supports.

DNA hybridization phenomena occurring on solid supports are not understood as clearly as aqueous phase hybridizations and mathematical models cannot predict some empirically obtained results. Ongoing research has identified important parameters but remains incomplete to accurately account for all interactions. It has previously been shown that the length of the overhanging (dangling) end of the target DNA strand following hybridization to the capture probe is correlated to interactions with the complementary strand in solution which can result in unbinding of the target and its release from the surface. We have developed an instrument for real-time monitoring of DNA hybridization on spherical particles functionalized with oligonucleotide capture probes and arranged in the form of a tightly packed monolayer bead bed inside a microfluidic cartridge. The instrument is equipped with a pneumatic module to mediate displacement of fluid on the cartridge. We compared this system to both conventional (passive) and centrifugally-driven (active) microfluidic microarray hybridization on glass slides to establish performance levels for the detection of single nucleotide polymorphisms. The system was also used to study the effect of the dangling end's length in real-time when the immobilized target DNA is exposed to the complementary strand in solution. Our findings indicate that increasing the length of the dangling end leads to desorption of target amplicons from bead-bound capture probes at a rate approaching that of the initial hybridization process. Finally, bead bed hybridization was performed with Streptococcus agalactiae cfb gene amplicons obtained from randomized clinical samples, which allowed for identification of group B streptococci within 5-15 min. The methodology presented here is useful for investigating competitive hybridization mechanisms on solid supports and to rapidly validate the suitability of microarray capture probes.

Evaporation-Driven Water-in-Water Droplet Formation.

We present new observations of aqueous two-phase system (ATPS) thermodynamic and interfacial phenomena that occur inside sessile droplets due to water evaporation. Sessile droplets that contain polymeric solutions, which are initially in equilibrium in a single phase, are observed at their three-phase liquid-solid-air contact line. As evaporation of a sessile droplet proceeds, we find that submicron secondary water-in-water (W/W) droplets emerge spontaneously at the edges of the mother sessile droplet due to the resulting phase separation from water evaporation. To understand this phenomenon, we first study the secondary W/W droplet formation process on different substrate materials, namely, glass, polycarbonate (PC), thermoplastic elastomer (TPE), poly(dimethylsiloxane)-coated glass slide (PDMS substrate), and Teflon-coated glass slide (Teflon substrate), and show that secondary W/W droplet formation arises only in lower-contact-angle substrates near the three-phase contact line. Next, we characterize the size of the emergent secondary W/W droplets as a function of time. We observe that W/W drops are formed, coalesced, aligned, and trapped along the contact line of the mother droplet. We demonstrate that this W/W multiple emulsion system can be used to encapsulate magnetic particles and blood cells, and achieve size-based separation. Finally, we show the applicability of this system for protein sensing. This is the first experimental observation of evaporation-induced secondary W/W droplet generation in a sessile droplet. We anticipate that the phenomena described here may be applicable to some biological assay applications, for example, biomarker detection, protein sensing, and point-of-care diagnostic testing.

Centrifugal microfluidic lab-on-a-chip system with automated sample lysis, DNA amplification and microarray hybridization for identification of enterohemorrhagic Escherichia coli culture isolates.

The development of technology for the rapid, automated identification of bacterial culture isolates can help regulatory agencies to shorten response times in food safety surveillance, compliance, and enforcement as well as outbreak investigations. While molecular methods such as polymerase chain reaction (PCR) enable the identification of microbial organisms with high sensitivity and specificity, they generally rely on sophisticated instrumentation and elaborate workflows for sample preparation with an undesirably high level of hands-on engagement. Herein, we describe the design, operation and performance of a lab-on-a-chip system integrating thermal lysis, PCR amplification and microarray hybridization on the same cartridge. The assay is performed on a centrifugal microfluidic platform that allows for pneumatic actuation of liquids during rotation, making it possible to perform all fluidic operations in a fully-automated fashion without the need for integrating active control elements on the microfluidic cartridge. The cartridge, which is fabricated from hard and soft thermoplastic polymers, is compatible with high-volume manufacturing (e.g., injection molding). Chip design and thermal interface were both optimized to ensure efficient heat transfer and allow for fast thermal cycling during the PCR process. The integrated workflow comprises 14 steps and takes less than 2 h to complete. The only manual steps are related to loading of the sample and reagents on the cartridge as well as fluorescence imaging of the microarray. On-chip lysis and PCR amplification both provided results comparable to those obtained by bench-top instrumentation. The microarray, incorporating a panel of oligonucleotide probes for multiplexed detection of seven enterohemorrhagic E. coli priority serotypes, was implemented on a cyclic olefin copolymer substrate using a novel activation scheme that involves the conversion of hydroxyl groups (derived from oxygen plasma treatment) into reactive cyanate ester using cyanogen bromide. On-chip hybridization was demonstrated in a non-quantitative fashion using fluorescently-labelled gene markers for E. coli O157:H7 (rfbO157, eae, vt1, and vt2) obtained through a multiplexed PCR amplification step.

Methylation Specific Multiplex Droplet PCR using Polymer Droplet Generator Device for Hematological Diagnostics.

A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, along with a thermoplastic elastomer (TPE) microfluidic droplet generation device. Epigenetic markers are used for WBC subtyping which is of important prognostic value in different diseases. This is achieved through the quantification of DNA methylation patterns of specific CG-rich regions in the genome (CpG loci). In this paper, bisulfite-treated DNA from peripheral blood mononuclear cells (PBMCs) is encapsulated in droplets with mdPCR reagents including primers and hydrolysis fluorescent probes specific for CpG loci that correlate with WBC sub-populations. The multiplex approach allows for the interrogation of many CpG loci without the need for separate mdPCR reactions, enabling more accurate parametric determination of WBC sub-populations using epigenetic analysis of methylation sites. This precise quantification can be extended to different applications and highlights the benefits for clinical diagnosis and subsequent prognosis.

Buoyancy-driven step emulsification on pneumatic centrifugal microfluidic platforms.

We present here a new method for controlling the droplet size in step emulsification processes on a centrifugal microfluidic platform, which, in addition to the centrifugal force, uses pneumatic actuation for fluid displacement. We highlight the importance of the interplay between buoyancy effects and the flow rate at the step junction, and provide a simple analytical model relating these two quantities to the size of the droplets. Numerical models as well as experiments with water-in-oil emulsions are performed in support of the proposed model.

Polymer Micropillar Arrays for Colorimetric DNA Detection.

We describe the use of periodic micropillar arrays, produced from cyclic olefin copolymer using high-fidelity microfabrication, as templates for colorimetric DNA detection. The assay involves PCR-amplified gene markers for E. coli O157:H7 (rfbO157, eae, vt1, and vt2) incorporating a detectable digoxigenin label, which is revealed through an immunoenzymatic process following hybridization with target-specific oligonucleotide capture probes. The capacity of micropillar arrays to induce wicking is used to distribute and confine capture probes with spatial control, making it possible to achieve a uniform signal while allowing multiple, independent probes to be arranged in close proximity on the same substrate. The kinetic profile of color pigment formation on the surface was followed using absorbance measurements, showing maximum signal increase between 20 and 60 min of reaction time. The relationship between microstructure and colorimetric signal was investigated through variation of geometric parameters, such as pitch (10-50 µm), pillar diameter (5-40 µm), and height (16-48 µm). Our findings suggest that signal intensity is largely influenced by the edges of the pillars and less by their height such that it deviates from a linear relationship when both aspect ratio and pillar density become very high. A theoretical model used to simulate the changes in surface composition at the molecular level suggests that differences in the temporal and spatial accumulation of assay components account for this observation.

Epigenetic subtyping of white blood cells using a thermoplastic elastomer-based microfluidic emulsification device for multiplexed, methylation-specific digital droplet PCR.

Epigenetic markers attract increasing attention for the study of phenotypic variations, which has led to the investigation of cell-lineage DNA methylation patterns that correlate with human leukocyte populations for obtaining counts of white blood cell (WBC) subsets. Current methods of DNA methylation analysis involve genome sequencing or loci-specific quantitative PCR (qPCR). Herein, a multiplexed digital droplet PCR (ddPCR) workflow for determining epigenetic-based WBC differential count is described for the first time. A microfluidic emulsification device fabricated from a commercially available thermoplastic elastomer (e.g., Mediprene) promotes customizability and cost-effectiveness of the methodology, which are prerequisites for translation into clinical and point-of-care diagnostics. Bisulfite-treated DNA from peripheral blood mononuclear cells and whole blood is encapsulated in droplets with ddPCR reagents containing primers and fluorescent hydrolysis probes specific for CpG loci correlated with WBC sub-population types. The method enables multiplexed detection of various methylation sites within a single droplet. Both qPCR and immunofluorescence staining (IF) were conducted to validate the capacity of the ddPCR methodology to accurately determine WBC sub-populations using epigenetic analysis of methylation sites. ddPCR results correlated closely to cell proportions obtained using IF, whereas qPCR significantly underestimated these values for both high and low copy number gene targets.

From cellular lysis to microarray detection, an integrated thermoplastic elastomer (TPE) point of care Lab on a Disc.

We present an all-thermoplastic integrated sample-to-answer centrifugal microfluidic Lab-on-Disc system (LoD) for nucleic acid analysis. The proposed CD system and engineered platform were employed for analysis of Bacillus atrophaeus subsp. globigii spores. The complete assay comprised cellular lysis, polymerase chain reaction (PCR) amplification, amplicon digestion, and microarray hybridization on a plastic support. The fluidic robustness and operating efficiency of the assay were ensured through analytical optimization of microfluidic tools enabling beneficial implementation of capillary valves and accurate control of all flow timing procedures. The assay reliability was further improved through the development of two novel microfluidic strategies for reagents mixing and flow delay on the CD platform. In order to bridge the gap between the proof-of-concept LoD and production prototype demonstration, low-cost thermoplastic elastomer (TPE) was selected as the material for CD fabrication and assembly, allowing the use of both, high quality hot-embossing and injection molding processes. Additionally, the low-temperature and pressure-free assembly and bonding properties of TPE material offer a pertinent solution for simple and efficient loading and storage of reagents and other on-board components. This feature was demonstrated through integration and conditioning of microbeads, magnetic discs, dried DNA buffer reagents and spotted DNA array inserts. Furthermore, all microfluidic functions and plastic parts were designed according to the current injection mold-making knowledge for industrialization purposes. Therefore, the current work highlights a seamless strategy that promotes a feasible path for the transfer from prototype toward realistic industrialization. This work aims to establish the full potential for TPE-based centrifugal system as a mainstream microfluidic diagnostic platform for clinical diagnosis, water and food safety, and other molecular diagnostic applications.

All-thermoplastic nanoplasmonic microfluidic device for transmission SPR biosensing.

Early and accurate disease diagnosis still remains a major challenge in clinical settings. Biomarkers could potentially provide useful tools for the detection and monitoring of disease progression, treatment safety and efficacy. Recent years have witnessed prodigious advancement in biosensor development with research directed towards rapid, real-time, label-free and sensitive biomarker detection. Among emerging techniques, nanoplasmonic biosensors pose tremendous potential to accelerate clinical diagnosis with real-time multiplexed analysis, rapid and miniaturized assays, low sample consumption and high sensitivity. In order to translate these technologies from the proof-of-principle concept level to point of care clinical diagnosis, integrated, portable devices having small footprint cartridges that house low-cost disposable consumables are sought. Towards this goal, we developed an all-polymeric nanoplasmonic microfluidic (NMF) transmission surface plasmon resonance (SPR) biosensor. The device was fabricated in thermoplastics using a simple, single step and cost-effective hot embossing technique amenable to mass production. The novel 3D hierarchical mold fabrication process enabled monolithic integration of blazed nanogratings within the detection chambers of a multichannel microfluidic system. Consequently, a single hard thermoplastic bottom substrate comprising plasmonic and fluidic features allowed integration of active fluidic elements, such as pneumatic valves, in the top soft thermoplastic cover, increasing device functionality. A simple and compact transmission-based optical setup was employed with multiplexed end-point or dual-channel kinetic detection capability which did not require stringent angular accuracy. The sensitivity, specificity and reproducibility of the transmission SPR biosensor was demonstrated through label-free immunodetection of soluble cell-surface glycoprotein sCD44 at clinically relevant picomolar to nanomolar concentrations.

Designed biointerface using near-infrared quantum dots for ultrasensitive surface plasmon resonance imaging biosensors.

The surface plasmon resonance imaging chip biointerface is fully designed using near-infrared (NIR) quantum dots (QDs) for the enhancement of surface plasmon resonance imaging (SPRi) signals in order to extend their application for medical diagnostics. The measured SPRi detection signal following the QD binding to the surface was amplified 25-fold for a 1 nM concentration of single-stranded DNA (ssDNA) and 50-fold for a 1 µg/mL concentration of prostate-specific antigen (PSA), a cancer biomarker, thus substantiating their wide potential to study interactions of a diverse set of small biomolecules. This significant enhancement is attributed to the QD's mass-loading effect and spontaneous emission coupling with propagating surface plasmons, which allowed the SPRi limit of detection to be reduced to 100 fM and 100 pg/mL for ssDNA and PSA, respectively. Furthermore, this study illustrates the potential of SPRi to be easily integrated with fluorescent imaging for advanced correlative surface-interaction analysis.

Nanostructured digital microfluidics for enhanced surface plasmon resonance imaging.

The advances in genomics and proteomics have unveiled an exhaustive catalogue of biomarkers that can potentially be used as diagnostic and prognostic indicators of genetic and infectious diseases. Current thrust in biosensor development is towards rapid, real-time, label-free and highly sensitive detection of the indicative biomarkers. While surface plasmon resonance imaging (SPRi) biosensors could potentially be the best suited candidate for biomarker-based diagnosis, important milestones need to be reached. Commercially available SPRi instrumentation is currently limited by the flow-cell technology to serial-sample processing and has limited sensitivity for the detection of markers present at low concentration. In this paper, we have implemented an approach to enhance sample handling and increase the sensitivity of the SPRi detection technique. We have developed a digital microfluidic platform with an integrated nanostructured biosensor interface that allows for rapid, ultra-low volume, sensitive, and automated on-chip SPRi detection of DNA hybridization reactions. Through the exploitation of electromagnetic properties of nanofabricated periodic gold nanoposts, SPRi signal was increased by 200% with the estimated limit of detection of 500 pM (90 attomoles). Using the versatile fluidic manipulation provided by the digital microfluidics, rapid and parallel target identification was achieved on multiple array elements within 1 min using 180 nL sample volume. By delivering multiple target analytes in individually addressable low volume droplets, without external pumps and fluidic interconnects, the overall assay time, cost and complexity was reduced. The proposed platform allows extreme versatility in the manipulation of precious low volume samples which makes this technology very suitable for diagnostic applications.

Integration and detection of biochemical assays in digital microfluidic LOC devices.

The ambition of lab-on-a-chip (LOC) systems to achieve chip-level integration of a complete analytical process capable of performing a complex set of biomedical protocols is hindered by the absence of standard fluidic components able to be assembled. As a result, most microfluidic platforms built to date are highly specialized and designed to fulfill the requirements of a single particular application within a limited set of operations. Electrowetting-on-dielectric (EWOD) digital microfluidic technology has been recently introduced as a new methodology in the quest for LOC systems. Herein, unit volume droplets are manipulated along electrode arrays, allowing a microfluidic function to be reduced to a set of basic operations. The highly reprogrammable architecture of these systems can satisfy the needs of a diverse set of biochemical assays and ensure reconfigurability, flexibility and portability between different categories of applications and requirements. While important progress was made over past years in the fabrication, miniaturization and function programming of the basic EWOD fluidic operations, the success of this technology will in great part depend on the ability of researchers to couple or integrate digital microfluidics to detection approaches that can make the system competitive for LOC applications. The detection techniques should be able to circumvent the limitations of hydrophobic surfaces and exploit the advantages of the array format, high droplet transport speeds and rapid mixing schemes. This review provides an in-depth look at recent developments for the coupling and integration of detection techniques with digital microfluidic platforms for bio-chemical applications.

Nanoimprinted plastic substrates for enhanced surface plasmon resonance imaging detection.

Periodic nanostructures fabricated by Nanoimprint Litography (NIL) in low-cost plastic substrates and coated with thin gold film were explored for enhanced surface plasmon resonance imaging (SPRi) detection. Rigorous coupled-wave analysis was used to model the SPRi response of these nanostructured surfaces. Two-dimensional nanogratings and nanogrooves were fabricated on Zeonor 1060R(TM) by NIL and followed by metal deposition. The detection of refractive index changes in the dielectric layer due to bulk medium change, DNA immobilization and DNA hybridization events were monitored using SPRi to assess the corresponding signal amplification. The results indicate target-dependent sensitivity enhancement which is maximized for the detection of biomolecular binding events. The 500 nm period nanogrooves provided a 4 times SPR signal amplification compared to the conventional uniform gold film on SF-11 glass for DNA hybridization detection. Our work demonstrates that the use of nanoimprinted plastic substrates provides a low-cost solution for the SPR-based detection with sensitivity that meets the requirements in practical diagnostic applications.

Biochip functionalization using electrowetting-on-dielectric digital microfluidics for surface plasmon resonance imaging detection of DNA hybridization.

This work reports on a dynamically configurable micro-array surface plasmon resonance biochip platform. The platform comprises a digital electrowetting-on-dielectric (EWOD) microfluidic device tailored to surface plasmon resonance imaging (SPRi). We demonstrate its application for simultaneous immobilization of different DNA probes at the designated detection sites on-chip from sub-microL volume solutions in combination with multichannel label-free real-time detection of subsequent hybridization reactions. Successful on-chip DNA probe dilution and immobilization is also demonstrated using SPRi hybridization detection. Furthermore, active control of the immobilized probe density and orientation is achieved under an applied potential using the electric interface of the EWOD device. For low probe densities, under negative applied potential, the DNA hybridization efficiency is enhanced compared to passive probe immobilization, yielding a two-fold SPR signal increase within only 8min of hybridization. EWOD microfluidic platform coupled with SPRi promises to dramatically increase the speed of detection and quantification of biomolecular interactions while reducing reagent consumption. The proposed system would enable the development of high-throughput, rapid and ultrasensitive detection of biomolecules beyond DNA microarray applications.

Two-dimensional droplet-based surface plasmon resonance imaging using electrowetting-on-dielectric microfluidics.

This article presents a multichannel droplet-based surface plasmon resonance platform. The platform comprises a digital electrowetting-on-dielectric (EWOD) microfluidic device coupled to surface plasmon resonance imaging (SPRi). SPRi is now a well-established detection technique that enables in-situ monitoring of multiple reactions occurring at the surface of the chip without the use of labels. Currently, the limiting factor in the application of SPRi for high-throughput applications is the flow-cell technology which relies on sequential sample processing within the continuous fluid flow. An original solution compared to the continuous flow-cell technology is proposed to increase the capability of existing SPRi technology. A parallel SPRi detection of different samples on the surface is achieved using the array-based digital microfluidic device.