Droplet Platform Research Articles

Facile syringe filter-enabled bacteria separation, enrichment, and buffer exchange for clinical isolation-free digital detection and characterization of bacterial pathogens in urine.

The development of accelerated methods for pathogen identification (ID) and antimicrobial susceptibility testing (AST) for infectious diseases is necessary to facilitate evidence-based antibiotic therapy and reduce clinical overreliance on broad-spectrum antibiotics. Towards this end, droplet-based microfluidics has unlocked remarkably rapid diagnostic assays with single-cell and single-molecule resolution. Yet, droplet platforms invariably rely on testing purified bacterial samples that have been clinically isolated after lengthy (>16 h) plating. While plating-based clinical isolation is important for enriching and separating out bacteria from background in clinical samples and also facilitating buffer exchange, it creates a diagnostic bottleneck that ultimately precludes droplet-based methods from achieving significantly accelerated times-to-result. To alleviate this bottleneck, we have developed facile syringe filter-enabled strategies for bacterial separation, enrichment, and buffer exchange from urine samples. By selecting appropriately sized filter membranes, we separated bacterial cells from background particulates in urine samples and achieved up to 91% bacterial recovery after such 1-step filtration. When interfaced with droplet-based detection of bacterial cells, 1-step filtration improved the limit of detection for bacterial ID and quantification by over an order of magnitude. We also developed a facile buffer exchange strategy to prepare bacteria in urine samples for droplet-based AST that achieved up to 10-fold bacterial enrichment during buffer exchange. Our filtration strategies, can be easily integrated into droplet workflows, enable clinical isolation-free sample-to-answer ID and AST, and significantly accelerate the turnaround of standard infectious disease diagnostic workflows.

A droplet platform capable of handling dissimilar liquids and its application for separation of bacteria from blood.

For passive droplet generation, multiple parameters such as the fluid viscosities and flow rates of the continuous and discrete phases correlate to each other, raising relevant control difficulties. In the current study, a droplet platform that is capable of handling dissimilar liquids is proposed. Through combining oscillatory flow and electric charge, synchronized generation and forced coalescence of different droplets can be achieved. Its application for the separation of E. coli from blood is tested, which leads to a high capture efficiency with less sample and within a shorter time than usual.

An ultra high-efficiency droplet microfluidics platform using automatically synchronized droplet pairing and merging.

Droplet microfluidics systems hold great promise in their ability to conduct high-throughput assays for a broad range of life science applications. Despite their promise in the field and capability to conduct complex liquid handling steps, currently, most droplet microfluidic systems used for real assays utilize only a few droplet manipulation steps connected in series, and are often not integrated together on a single chip or platform. This is due to the fact that linking multiple sequential droplet functions within a single chip to operate at high efficiency over long periods of time remains technically challenging. Considering sequential manipulation is often required to conduct high-throughput screening assays on large cellular and molecular libraries, advancements in sequential operation and integration are required to advance the field. This current limitation greatly reduces the type of assays that can be realized in a high-throughput droplet format and becomes more prevalent in large library screening applications. Here we present an integrated multi-layer droplet microfluidic platform that can handle large numbers of droplets with high efficiency and minimum error. The platform combines two-photon photolithography-fabricated curved microstructures that allow high-efficiency (99.9%) re-flow of droplets and a unique droplet cleaving that automatically synchronizes paired droplets enabling high-efficiency (99.9%) downstream merging. We demonstrate that this method is applicable to a broad range of droplet sizes, including relatively large droplet sizes (hundreds of micrometers in diameter) that are typically more difficult to manipulate with high efficiency, yet are required in many cell assay applications requiring large organisms or multiple incubation steps. The utility of this highly efficient integrated droplet microfluidic platform was demonstrated by conducting a mock antibiotic screening assay against a bacterial pathogen. The approach and system presented here provides new avenues for the realization of ultra-high-efficiency multi-step droplet microfluidic systems with minimal error.

A high-throughput system combining microfluidic hydrogel droplets with deep learning for screening the antisolvent-crystallization conditions of active pharmaceutical ingredients.

Crystallization of active pharmaceutical ingredients (APIs) is a crucial process in the pharmaceutical industry due to its great impact in drug efficacy. However, conventional approaches for screening the optimal crystallization conditions of APIs are usually time-consuming, labor-intensive and expensive. Recently, droplet microfluidic technology has offered an alternative strategy for high-throughput screening of crystallization conditions. Despite its many advantages such as low sample consumption, reduced operation time, increased throughput, etc., some challenges remain to be solved, such as instability of droplets in the long-term and tedious efforts required for extracting useful information from massive data. To solve these problems, a high-throughput system that combined microfluidic hydrogel droplets with deep learning was proposed for the first time to screen the antisolvent-crystallization conditions of APIs. In this system, stable hydrogel droplets containing different concentrations of indomethacin, its solvent and antisolvent were generated on a chip. Crystals of indomethacin with different morphologies were formed in hydrogel droplets, and their optical images were captured by a camera. Then, deep learning was applied to identify the hundreds of indomethacin crystal images and successfully classify the crystal morphologies in a short time; a ternary phase diagram was drawn by combining the experimental results with the recognition results of crystal morphologies, and was used to guide the scale-up preparations of indomethacin crystals as desired. This system, which integrated high throughput preparation, characterization and data analysis, is also useful for screening the crystallization conditions and processes of semiconductors, catalysts, agrochemicals, proteins and other specialty chemicals.

Label-Free Detection of Multiplexed Metabolites at Single-Cell Level via a SERS-Microfluidic Droplet Platform

Single-cell metabolomics could be used to discover the chemical strategies of cells for coping with chemical or environmental stress because metabolomics provides a more immediate and dynamic picture of cell functionality. However, these small-molecule metabolites are the most difficult to measure because they characterize rapid metabolic dynamics, structural diversity, and incapacity to achieve signal amplification or labeling. In order to solve above problems, we presented a surface-enhanced Raman scattering (SERS)-microfluidic droplet platform to realize the label-free simultaneous analysis of multiplexed metabolites at the single-cell level via a versatile magnetic SERS substrate composed by silver nanoparticles (AgNPs, 30 nm)-decorated 400 nm Fe3O4 magnetic microspheres. This metal-magnetic composite substrate is beneficial to efficient adsorption of single cell metabolites, fast separation from complex matrixes, and high SERS sensitivity. Also, the endocytosis effect can be fully prohibited due to its relatively large size. This work achieves label-free, nondestructive, simultaneous determination of three single-cell metabolites, pyruvate, adenosine triphosphate (ATP), and lactate, owing to their intrinsic SERS fingerprints. The "hotspots" effect induced by the magnetic aggregation of Fe3O4@AgNPsallows highly sensitive SERS detection. Encapsulating metabolites in such a limited and isolated droplet accelerates the process of diffusion and adsorption equilibriums. Activation with saponin for metabolites was assessed on different cell lines. The SERS-microdroplet platform is a powerful tool for exploring single-cell heterogeneity at the metabolic level.

Use of a Droplet Platform To Optimize Pd-Catalyzed C–N Coupling Reactions Promoted by Organic Bases

Recent advances in Pd-catalyzed carbon–nitrogen cross-coupling have enabled the use of soluble organic bases instead of insoluble or strong inorganic bases that are traditionally employed. The sing...

Automated Femtoliter Droplet-Based Determination of Oil–Water Partition Coefficient

The oil-water partition coefficient of organic compounds is an essential parameter for the determination of their behaviors in environments, food, drug delivery, and biomedical systems, just to name a few. In this work, we establish a highly efficient approach to quantify the partition/distribution coefficient using surface femtoliter droplets. In our approach, droplets of 1-octanol were produced on the surface of a solid substrate in contact with the flow of an aqueous solution of the analyte. The analyte was rapidly enriched in the droplets from the flow and reached the partition equilibrium in a few seconds. The entire procedure was automated by continuous solvent exchange, and the analyte partition in the droplets was quantified from the in situ UV-vis spectrum collected by a microspectrophotometer. Our approach was validated for several substances with the octanol-water partition/distribution coefficient ranging from -1.5 to 4, where our results were in good agreement with the values reported in the literature. This method took ∼3 min to detect one analyte with the volume of the organic solvent at ∼50 μL. Thus, our surface droplet platform can greatly minimize the consumption of both solvent and analytes and can shorten the time for the determination of the partition of new compounds, which overcomes the drawbacks of the traditional shake-flask method and presents excellent reproducibility, high accuracy, cost-effectiveness, and labor-saving operation. The highly efficient micro/nanoextraction, partition, and real-time detection enabled by the surface droplets has the potential for many other high-throughput applications.

Customizing droplet contents and dynamic ranges via integrated programmable picodroplet assembler

Droplet microfluidic technology is becoming increasingly useful for high-throughput and high-sensitivity detection of biological and biochemical reactions. Most current droplet devices function by passively discretizing a single sample subject to a homogeneous or random reagent/reaction condition into tens of thousands of picoliter-volume droplets for analysis. Despite their apparent advantages in speed and throughput, these droplet devices inherently lack the capability to customize the contents of droplets in order to test a single sample against multiple reagent conditions or multiple samples against multiple reagents. In order to incorporate such combinatorial capability into droplet platforms, we have developed the fully Integrated Programmable Picodroplet Assembler. Our platform is capable of generating customized picoliter-volume droplet groups from nanoliter-volume plugs which are assembled in situ on demand. By employing a combination of microvalves and flow-focusing-based discretization, our platform can be used to precisely control the content and volume of generated nanoliter-volume plugs, and thereafter the content and the effective dynamic range of picoliter-volume droplets. Furthermore, we can use a single integrated device for continuously generating, incubating, and detecting multiple distinct droplet groups. The device successfully marries the precise control and on-demand capability of microvalve-based platforms with the sensitivity and throughput of picoliter droplet platforms in a fully automated monolithic device. The device ultimately will find important applications in single-cell and single-molecule analyses.

Abstract 3540: A complete solution for high throughput single cell targeted multiomic DNA and RNA sequencing for cancer research

Abstract Recent advancements in single cell analysis technologies are now able to provide insights into genomic DNA content, RNA expression and protein surface markers. In bulk assays, the effect of genetic variation on gene expression would be masked by the heterogeneity inherent in tumor cells. Additionally, for cancer immunotherapy studies that rely on gene editing, single cell resolution is necessary to minimize possible off target effects. However, the ability to simultaneously interrogate multiple intracellular analytes, such as genomic DNA and RNA, have proved difficult to implement in a high throughput single cell workflow. We report the development of a complete solution that enables both targeted genomic DNA and RNA sequencing from individual cells. This workflow relies on the Tapestri microfluidic droplet platform, where up to 20,000 cells can be sequenced in each run. Leveraging proprietary cell barcoding, novel primer design strategies and enzymatic manipulation of cellular contents, DNA and RNA multiplex targeted sequencing panels provide for independent barcoded sequence information from both overlapping mRNA and corresponding genomic DNA regions. In addition, amplification primers can be designed to target separate gDNA and non-overlapping RNA transcript. Sequencing is followed by an integrated analysis solution that assigns the reads from both the gDNA and RNA to each cell. Feasibility of the targeted nucleic acid workflow has been shown with inputs from mixed cancer cell lines. A targeted sequencing panel with overlapping mRNA and gDNA regions was designed covering oncogenes, tumor suppressor genes, and known fusions. Expected SNVs and indels were detected and gene expression measured for thousands of cells per run with high cell recovery. This complete solution for single cell multiomics on the Tapestri platform has the power to quantitatively and unambiguously link genotypic and phenotypic data, giving insight into cancer progression. Citation Format: Dalia Dhingra, Kaustubh Gokhale, Nianzhen Li, Pedro Mendez, Shu Wang, Manimozhi Manivannan, Adam Sciambi, Keith Jones, Charlie Silver, Dennis Eastburn, David Ruff. A complete solution for high throughput single cell targeted multiomic DNA and RNA sequencing for cancer research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3540.

PS975 HIGH‐RESOLUTION CLONAL ANALYSIS OF TUMORS WITH SIMULTANEOUS SINGLE‐CELL DNA SEQUENCING AND CELL SURFACE PHENOTYPING

Background:Intra‐tumor heterogeneity is a common feature of many blood cancers and is correlated with poor clinical outcomes. Single‐cell DNA sequencing can unambiguously resolve the genetic heterogeneity of tumors missed with traditional bulk next generation sequencing (NGS). Despite the advantages of single‐cell DNA sequencing, previous studies have relied upon laborious, expensive and low‐throughput approaches that are not scalable for routine analysis of tumors. Additionally, to provide maximum insight into disease progression, clonality of mutations within a tumor would also be correlated with cell lineage.Aims:We sought to demonstrate how single‐cell analysis of both cell surface markers as well as variant calling across hundreds of disease relevant loci can provide unprecedented insight into tumor architecture.Methods:To enable high‐resolution profiling of intra‐tumor heterogeneity, we previously developed a unique microfluidic droplet platform that can accurately identify genome variants at hundreds of disease relevant loci across 10,000 individual cells per sample run. This platform has a rapid, robust and easy to use workflow that, for the first time, enables routine analysis of genetic heterogeneity for hematologic malignancies.Results:Here, we report our continued work developing and applying targeted single‐cell sequencing panels for AML and other hematologic malignancies such as CLL. The high‐throughput single‐cell approach we developed has consistently revealed rare tumor cells at diagnosis and remission (MRD) that are critically important with respect to therapy response and disease progression. Additionally, for the first time, we also demonstrate integrated multi‐omic capability on our platform by enabling single‐cell measurement of cell surface protein expression levels together with genomic variant identification in a single NGS readout. In our modified workflow, cell lines, PBMCs or bone marrow aspirates are stained with a pool of monoclonal antibodies (e.g., CD34, CD33, CD19, CD45, CD30 and CD3) that are conjugated with oligonucleotides containing unique protein identifying tags. The sequencing data is analyzed with a bioinformatic pipeline that quantifies antibody levels associated genotyping data from a myeloid specific targeted sequencing panel on a cell‐by‐cell basis. With this approach the need for upstream flow sorting of cells can often be eliminated and the well‐characterized catalog of hematology cell surface markers employed to more directly correlate phenotype and cell‐state information of single cells to mutational burden.Summary/Conclusion:The multi‐omic capability we present, together with other improvements to the platform, make our technology ideally suited to fully resolve clonal architecture of hematologic malignancies. Correlation of mutation status with phenotype using high‐throughput single‐cell multi‐omics is a powerful approach with the potential to better predict disease progression and inform therapy selection.

Distinguishing cancer cell lines at asingle living cell level via detection of sialic acid by dual-channel plasmonic imaging and by using a SERS-microfluidic droplet platform.

A high-throughput, dual-channel single cell analytical method is described for the detection of sialic acid (SA) on single cell based on the use of microfluidic droplets integrated with plasmonic imaging and surface-enhanced Raman spectroscopy (SERS) with theassistance of a multifunctional metal nanoparticle-based probe. The multifunctional plasmonic nanoprobe was prepared by modifying silver nanoparticles (AgNPs) with 4-mercaptophenylboronic acid (MPBA) that both warrants SA recognition and acts as a Raman reporter. This nanoprobe is a high-contrast indicator under bright field imaging due to the strong energy loss feature of AgNPs, and also owns possesses astrong SERS enhancement capability toward MPBA. Cells incubated with the plasmonic nanoprobes were isolated in water-in-oil droplets and then were re-dispersed in a chamber array chip. High-precision profiles of SA on asingle cell in one droplet were obtained by the bright field imaging and imageprocessing. The SA expression levels on different cell lines (MCF-7, HepG2, SGC and BNL.CL2) traced by SERS spectroscopy were compared. The statistical data among different cell lines confirm that the SA expression levels on cancer cells are much higher than that on normal cells. Single cell analysis further revealed that the cell-to-cell variations are more obvious in cancer cell lines. This study provides a valuable tool for understanding glycan-related biochemical processes. Graphical abstract A high-throughput, dual-channel microfluidic droplet platform succeeded in distinguishing different cancer cell lines at single living cell level integrated with plasmonic imaging and surface-enhanced Raman spectroscopy with assistance of a multifunctional metal nanoparticle-based probe.

High efficiency single-cell capture based on microfluidics for single cell analysis

A microfluidic droplet platform based on flow resistance has been designed to generate, capture and analyze a single cell. In this device, a single cell was encapsulated in micro-droplet form and positioned precisely in arrays of sloped channels on a polydimethylsiloxane–glass chip. The droplets were formed at the ‘T-junction’ and trapped by the ‘main–bypass’ construction. The single cell capture efficiency was up to 60%. Besides, the device caused no harm to living cells as the microdroplet formed here provided a relatively stable micro-environment. The device had the advantages of reagent economy, passive driving, and minimal cell damage, making it a powerful tool for single-cell analysis.

Ultrasensitive and Simultaneous Detection of Two Cytokines Secreted by Single Cell in Microfluidic Droplets via Magnetic-Field Amplified SERS.

A surface-enhanced Raman scattering (SERS)-microfluidic droplet platform for the rapid, ultrasensitive and simultaneous detection of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) secreted by a single cell is presented. The high-throughput water-in-oil droplets containing an individual cell along with four kinds of immune-particles (antibody-conjugated silver nanoparticles or magnetic beads, AgNPs@Ab1 and MNs@Ab2) in each were achieved by a cross-typed microfluidic chip, and then they were captured by a collection channel array for SERS measurements. In the appearance of cytokines secreted by one cell, AgNPs@Ab1 can be linked onto the surface of MNs@Ab2 through the immune-recognition to form an immune-sandwich, which makes the "turn on" SERS signal of the Raman reporters previously laid on the surface of MNs due to the adjacent AgNPs. Furthermore, the second SERS signal amplification is from the magneticfield-induced spontaneous collection effect, which brings 75 times enhancement for SERS signal. Additionally, the encapsulation of cytokines in an isolated droplet permits an accumulation effect of targets with time. Owing to the dual signal enhancement and the accumulation effect, such few cytokines secreted by a single cell become detectable and a limit of detection is achieved as 1.0 fg/mL in one droplet. By using this ultrasensitive SERS-microdroplet method, the VEGF and IL-8 secretions from several cells in one droplet were explored and the data show that the cell-cell interaction may promote angiogenesis of cancer cells through the up-regulation of VEGF and IL-8.

Functional Femtoliter Droplets for Ultrafast Nanoextraction and Supersensitive Online Microanalysis.

A universal femtoliter surface droplet-based platform for direct quantification of trace of hydrophobic compounds in aqueous solutions is presented. Formation and functionalization of femtoliter droplets, concentrating the analyte in the solution, are integrated into a simple fluidic chamber, taking advantage of the long-term stability, large surface-to-volume ratio, and tunable chemical composition of these droplets. In situ quantification of the extracted analytes is achieved by surface-enhanced Raman scattering (SERS) spectroscopy by nanoparticles on the functionalized droplets. Optimized extraction efficiency and SERS enhancement by tuning droplet composition enable quantitative determination of hydrophobic model compounds of rhodamine 6G, methylene blue, and malachite green with the detection limit of 10-9 to 10-11 m and a large linear range of SERS signal from 10-9 to 10-6 m of the analytes. The approach addresses the current challenges of reproducibility and the lifetime of the substrate in SERS measurements. This novel surface droplet platform combines liquid-liquid extraction and highly sensitive and reproducible SERS detection, providing a promising technique in current chemical analysis related to environment monitoring, biomedical diagnosis, and national security monitoring.

Ultrahigh-throughput functional profiling of microbiota communities

Microbiome spectra serve as critical clues to elucidate the evolutionary biology pathways, potential pathologies, and even behavioral patterns of the host organisms. Furthermore, exotic sources of microbiota represent an unexplored niche to discover microbial secondary metabolites. However, establishing the bacterial functionality is complicated by an intricate web of interactions inside the microbiome. Here we apply an ultrahigh-throughput (uHT) microfluidic droplet platform for activity profiling of the entire oral microbial community of the Siberian bear to isolate Bacillus strains demonstrating antimicrobial activity against Staphylococcus aureus Genome mining allowed us to identify antibiotic amicoumacin A (Ami) as responsible for inhibiting the growth of S. aureus Proteomics and metabolomics revealed a unique mechanism of Bacillus self-resistance to Ami, based on a subtle equilibrium of its deactivation and activation by kinase AmiN and phosphatase AmiO, respectively. We developed uHT quantitative single-cell analysis to estimate antibiotic efficacy toward different microbiomes and used it to determine the activity spectra of Ami toward human and Siberian bear microbiota. Thus, uHT microfluidic droplet platform activity profiling is a powerful tool for discovering antibiotics and quantifying external influences on a microbiome.

Label-Free Sensing in Microdroplet-Based Microfluidic Systems

Droplet microfluidic systems have evolved as fluidic platforms that use much less sample volume and provide high throughput for biochemical analysis compared to conventional microfluidic devices. The variety of droplet fluidic applications triggered several detection techniques to be applied for analysis of droplets. In this review, we focus on label-free droplet detection techniques that were adapted to various droplet microfluidic platforms. We provide a classification of most commonly used droplet platform technologies. Then we discuss the examples of various label-free droplet detection schemes implemented for these platforms. While providing the research landscape for label-free droplet detection methods, we aim to highlight the strengths and shortcomings of each droplet platform so that a more targeted approach can be taken by researchers when selecting a droplet platform and a detection scheme for any given application.

Droplet microfluidics for high-sensitivity and high-throughput detection and screening of disease biomarkers.

Biomarkers are nucleic acids, proteins, single cells, or small molecules in human tissues or biological fluids whose reliable detection can be used to confirm or predict disease and disease states. Sensitive detection of biomarkers is therefore critical in a variety of applications including disease diagnostics, therapeutics, and drug screening. Unfortunately for many diseases, low abundance of biomarkers in human samples and low sample volumes render standard benchtop platforms like 96-well plates ineffective for reliable detection and screening. Discretization of bulk samples into a large number of small volumes (fL-nL) via droplet microfluidic technology offers a promising solution for high-sensitivity and high-throughput detection and screening of biomarkers. Several microfluidic strategies exist for high-throughput biomarker digitization into droplets, and these strategies have been utilized by numerous droplet platforms for nucleic acid, protein, and single-cell detection and screening. While the potential of droplet-based platforms has led to burgeoning interest in droplets, seamless integration of sample preparation technologies and automation of platforms from biological sample to answer remain critical components that can render these platforms useful in the clinical setting in the near future. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > Diagnostic Nanodevices Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease.

Micro-droplet arrays for micro-compartmentalization using an air/water interface.

Here we present a water-in-air droplet platform for micro-compartmentalization for single molecule guided synthesis and analysis consisting of a flow-system hosting dense arrays of aqueous microdroplets on a glass surface surrounded by air. The droplets are formed in a few seconds by passing a waterfront over the array of hydrophilic spots surrounded by a hydrophobic coating, thus forming a micro-droplet array (MDA). The droplet volumes are tunable from approximately 50 femtoliter to 20 picoliter by adjusting the size of the hydrophilic spots. MDAs consisting of femtoliter volume droplets were stable for more than 24 hours in air at 37 °C in a reversibly sealed flow-system, thus allowing us to perform assays that require long incubations in the droplets. Using differently fluorescing liquids, it was further shown that droplets can be reformed on the same MDA several times by passing a new liquid plug over the surface, and that fluorescence from one reaction can be washed away with little to no carry-over, hence allowing for multistep reactions to be carried out on the system. The MDA created by an air/water interface supported digital immunoassays as was demonstrated by measuring the Aβ42 peptide in cerebrospinal fluid of Alzheimers patients and control patients. To demonstrate a two step droplet assay, first, histidine tagged peptides were expressed in the droplets and bound to the droplet-enclosed surface. Subsequently, the his-tagged peptides were detected using enzyme-conjugated antibodies in a second droplet generation step. As such, the chip demonstrates features necessary for library preparations for high throughput screening applications.

Automatic magnetic manipulation of droplets on an open surface using a superhydrophobic electromagnet needle

In this paper, we present a novel magnetic droplet manipulation platform that exploits a superhydrophobic electromagnet needle (EMN) to perform complex liquid manipulations on an open surface. By switching or adjusting the EMN power supply, the platform can simply and flexibly perform a full range of droplet manipulations, including droplet transport, fusion, mixing, magnetic beads (MBs) extraction, and droplet dispensing. Owing to its superhydrophobic coating, the EMN can directly contact a magnetic droplet and then exert a strong magnetic force on the paramagnetic beads suspended in it for effective droplet manipulation without the wetting or contamination problems. This offers particular advantages to multi-step biochemical assays. We also investigated the effects of the MBs concentrations, droplet volume, hydrophilic pattern size and current amplitude applied to the EMN on the droplet kinematics. The EMN-based droplet manipulation is facile, cost-effective, and programmable, and will significantly enhance the functionality of magnetic droplet platforms, and thus broaden their applicability to numerous fields of life science.

Single-step immunoassays and microfluidic droplet operation: Towards a versatile approach for detection of amyloid-β peptide-based biomarkers of Alzheimer’s disease

In this study, single-step magnetic-beads based immunoassays was developed and for the first time adapted to microfluidic droplet operation for sequential determination of well-established amyloid-β peptide biomarkers (i.e. monomeric Aβ 1–42 and Aβ 1–40) for molecular diagnosis of Alzheimer’s disease (AD). With the developed sandwich assay protocol, the capture antibodies (i.e. monoclonal anti-Aβ antibodies that are specific for N-terminus of Aβ 1–42 and Aβ 1–40) grafted onto magnetic beads and the detection antibodies (i.e. beta amyloid 1–16 monoclonal antibody labeled with horseradish peroxidase) can simultaneously bind to monomeric β peptides in a single step. Aβ 1–42 and Aβ 1–40 in cerebrospinal fluid (CSF) samples were successfully detected using the developed batchwise immunoassay approach. With the aim at expanding the spectrum of traced Aβ peptides beyond Aβ 1–42 and Aβ 1–40 for more precise diagnostics of AD, the developed sandwich assay was for the first time coupled as a downstream module to peptide fractionation and collection using capillary isoelectric focusing. Demonstration of this methodological combination was carried out with Aβ 1–40, Aβ 1–40 and Aβ 5–40. The immunoassay in batchwise format was downscaled into a purpose-made microfluidic droplet platform allowing significant sample volume reduction and higher throughput. Using a series of 4 programmable magnetic tweezers to manipulate a train of nano-scale confined droplets containing magnetic beads, sample, washing and detection solutions, a sequence of 8 analyses could be realized within 45min. This droplet based immunoassay was realized in a dedicated platform integrating an in-house-made light emitting diode (LED)-based fluorescent detector, replacing conventional microscopic setup so as to significantly reduce the construction cost and simplify the detection protocol. Using this microfluidic configuration coupled with LED-based detection, information on both Aβ 1–42 and Aβ 1–40 concentrations can be collected in a single sequence with less than 1μL of sample.