Intracellular Amyloid-ß Detection from Human Brain Sections Using a Microfluidic Immunoassay in Tandem with MALDI-MS.

Alzheimer's disease (AD) currently affects more than 30 million people worldwide. The lack of understanding of AD's physiopathology limits the development of therapeutic and diagnostic tools. Soluble amyloid-ß peptide (Aß) oligomers that appear as intermediates along the Aß aggregation into plaques are considered among the main AD neurotoxic species. Although a wealth of data are available about Aß from in vitro and animal models, there is little known about intracellular Aß in human brain cells, mainly due to the lack of technology to assess the intracellular protein content. The elucidation of the Aß species in specific brain cell subpopulations can provide insight into the role of Aß in AD and the neurotoxic mechanism involved. Here, we report a microfluidic immunoassay for in situ mass spectrometry analysis of intracellular Aß species from archived human brain tissue. This approach comprises the selective laser dissection of individual pyramidal cell bodies from tissues, their transfer to the microfluidic platform for sample processing on-chip, and mass spectrometric characterization. As a proof-of-principle, we demonstrate the detection of intracellular Aß species from as few as 20 human brain cells.

MIMAS: microfluidic platform in tandem with MALDI mass spectrometry for protein quantification from small cell ensembles.

Understanding cell-to-cell variation at the molecular level provides relevant information about biological phenomena and is critical for clinical and biological research. Proteins carry important information not available from single-cell genomics and transcriptomics studies; however, due to the minute amount of proteins in single cells and the complexity of the proteome, quantitative protein analysis at the single-cell level remains challenging. Here, we report an integrated microfluidic platform in tandem with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the detection and quantification of targeted proteins from small cell ensembles (> 10 cells). All necessary steps for the assay are integrated on-chip including cell lysis, protein immunocapture, tryptic digestion, and co-crystallization with the matrix solution for MALDI-MS analysis. We demonstrate that our approach is suitable for protein quantification by assessing the apoptotic protein Bcl-2 released from MCF-7 breast cancer cells, ranging from 26 to 223 cells lysed on-chip (8.75 nL wells). A limit of detection (LOD) of 11.22 nM was determined, equivalent to 5.91 × 107 protein molecules per well. Additionally, the microfluidic platform design was further improved, establishing the successful quantification of Bcl-2 protein from MCF-7 cell ensembles ranging from 8 to 19 cells in 4 nL wells. The LOD in the smaller well designs for Bcl-2 resulted in 14.85 nM, equivalent to 3.57 × 107 protein molecules per well. This work shows the capability of our approach to quantitatively assess proteins from cell lysate on the MIMAS platform for the first time. These results demonstrate our approach constitutes a promising tool for quantitative targeted protein analysis from small cell ensembles down to single cells, with the capability for multiplexing through parallelization and automation.

Electric Triggering for Enhanced Control of Droplet Generation.

Serial femtosecond crystallography (SFX) is a powerful technique that uses X-ray free-electron lasers (XFEL) to determine structures of biomolecular complexes. Specifically, it benefits the study of atomic resolution structures of large membrane protein complexes and time-resolved reactions with crystallography. One major drawback of SFX studies with XFELs is the consumption of large amounts of a protein crystal sample to collect a complete X-ray diffraction data set for high-resolution crystal structures. This increases the time and resources required for sample preparation and experimentation. The intrinsic pulsed nature of all current X-ray sources is a major reason why such large amounts of sample are required. Any crystal sample that is delivered in the path of the X-ray beam during its "off-time" is wasted. To address this large sample consumption issue, we developed a 3D printed microfluidic system with integrated metal electrodes for water-in-oil droplet generation to dynamically create and manipulate aqueous droplets. We demonstrate on-demand droplet generation using DC potentials and the ability to tune the frequency of droplet generation through the application of AC potentials. More importantly, to assist with the synchronization of droplets and XFEL pulses, we show that the device can induce a phase shift in the base droplet generation frequency. This novel approach to droplet generation has the potential to reduce sample waste by more than 95% for SFX experiments with XFELs performed with liquid jets and can operate under low- and high-pressure liquid injection systems.

Thermally induced all-optical inverter and dynamic hysteresis loops in graphene oxide dispersions.

We experimentally study the temporal dynamics of amplitude-modulated laser beams propagating through a water dispersion of graphene oxide sheets in a fiber-to-fiber U-bench. Nonlinear refraction induced in the sample by thermal effects leads to both phase reversing of the transmitted signals and dynamic hysteresis in the input-output power curves. A theoretical model including beam propagation and thermal lensing dynamics reproduces the experimental findings.

Modular droplet injector for sample conservation providing new structural insight for the conformational heterogeneity in the disease-associated NQO1 enzyme.

Droplet injection strategies are a promising tool to reduce the large amount of sample consumed in serial femtosecond crystallography (SFX) measurements at X-ray free electron lasers (XFELs) with continuous injection approaches. Here, we demonstrate a new modular microfluidic droplet injector (MDI) design that was successfully applied to deliver microcrystals of the human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin. We investigated droplet generation conditions through electrical stimulation for both protein samples and implemented hardware and software components for optimized crystal injection at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Stanford Linac Coherent Light Source (LCLS). Under optimized droplet injection conditions, we demonstrate that up to 4-fold sample consumption savings can be achieved with the droplet injector. In addition, we collected a full data set with droplet injection for NQO1 protein crystals with a resolution up to 2.7 Å, leading to the first room-temperature structure of NQO1 at an XFEL. NQO1 is a flavoenzyme associated with cancer, Alzheimer's and Parkinson's disease, making it an attractive target for drug discovery. Our results reveal for the first time that residues Tyr128 and Phe232, which play key roles in the function of the protein, show an unexpected conformational heterogeneity at room temperature within the crystals. These results suggest that different substates exist in the conformational ensemble of NQO1 with functional and mechanistic implications for the enzyme's negative cooperativity through a conformational selection mechanism. Our study thus demonstrates that microfluidic droplet injection constitutes a robust sample-conserving injection method for SFX studies on protein crystals that are difficult to obtain in amounts necessary for continuous injection, including the large sample quantities required for time-resolved mix-and-inject studies.

Microwave synthesis of upconverting nanoparticles with bis(2-ethylhexyl) adipate.

Despite efforts to develop effective upconverting nanoparticle (UCNP) synthesis methods, there is still a need for approaches that are accessible and up-scalable while reproducibly providing fine control of UCNP size, crystallinity, and luminescence. This work presents a one-pot microwave-assisted strategy for synthesizing NaYF4:Yb3+/Er3 UCNPs. A premixed rare earth (RE) solution in oleic acid (OA) was used to enhance repeatability while testing various synthesis conditions. The stock solution aliquots were mixed with OA and bis(2-ethylhexyl) adipate (BEHA), a polycarboxylic ester with a high boiling point, high thermal stability, and moderately polar character that facilitated rapid microwave heating at an average rate (room temperature to 300 °C) up to 60 °C min-1. Combinations of BEHA concentration and high-temperature reaction time were identified for consistently producing cubic and hexagonal UCNPs with narrow size distributions in the tens and hundreds of nanometers. After washing, the resulting UCNPs were dispersible in aqueous media without further processing. This straightforward, accessible, and repeatable microwave-assisted synthesis method holds potential for scaling up the production of UCNPs with well-defined size and crystallinity.

Electrically stimulated droplet injector for reduced sample consumption in serial crystallography.

With advances in X-ray free-electron lasers (XFELs), serial femtosecond crystallography (SFX) has enabled the static and dynamic structure determination for challenging proteins such as membrane protein complexes. In SFX with XFELs, the crystals are typically destroyed after interacting with a single XFEL pulse. Therefore, thousands of new crystals must be sequentially introduced into the X-ray beam to collect full data sets. Because of the serial nature of any SFX experiment, up to 99% of the sample delivered to the X-ray beam during its "off-time" between X-ray pulses is wasted due to the intrinsic pulsed nature of all current XFELs. To solve this major problem of large and often limiting sample consumption, we report on improvements of a revolutionary sample-saving method that is compatible with all current XFELs. We previously reported 3D-printed injection devices coupled with gas dynamic virtual nozzles (GDVNs) capable of generating samples containing droplets segmented by an immiscible oil phase for jetting crystal-laden droplets into the path of an XFEL. Here, we have further improved the device design by including metal electrodes inducing electrowetting effects for improved control over droplet generation frequency to stimulate the droplet release to matching the XFEL repetition rate by employing an electrical feedback mechanism. We report the improvements in this electrically triggered segmented flow approach for sample conservation in comparison with a continuous GDVN injection using the microcrystals of lysozyme and 3-deoxy-D-manno-octulosonate 8-phosphate synthase and report the segmented flow approach for sample injection applied at the Macromolecular Femtosecond Crystallography instrument at the Linear Coherent Light Source for the first time.

Co-flow injection for serial crystallography at X-ray free-electron lasers.

Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macro-molecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX.

Micropreparative isoelectric focusing protein separation in a suspended drop.

IEF protein binary separations were performed in a 12-µL drop suspended between two palladium electrodes, using pH gradients created by electrolysis of simple buffers at low voltages (1.5-5 V). The dynamics of pH gradient formation and protein separation were investigated by computer simulation and experimentally via digital video microscope imaging in the presence and absence of pH indicator solution. Albumin, ferritin, myoglobin, and cytochrome c were used as model proteins. A drop containing 2.4 µg of each protein was applied, electrophoresed, and allowed to evaporate until it splits to produce two fractions that were recovered by rinsing the electrodes with a few microliters of buffer. Analysis by gel electrophoresis revealed that anode and cathode fractions were depleted from high pI and low pI proteins, respectively, whereas proteins with intermediate pI values were recovered in both fractions. Comparable data were obtained with diluted bovine serum that was fortified with myoglobin and cytochrome c.

Oligonucleotide sensor based on magnetic capture and photoligation of upconverting nanoparticles in solid surfaces.

In this work, we present a luminescence platform that can be used as point of care system for determining the presence and concentration of specific oligonucleotide sequences. This sensor exhibited a limit of detection as low as 50 fM by means of: (i) the use of single-stranded DNA (ssDNA) functionalized magnetic microparticles that captured and concentrated ssDNA-upconverting nanoparticles (ssDNA-UCNPs) on a solid support, when the target sequence (miR-21-5p DNA-analogue) was in the sample, and (ii) a photoligation reaction that covalently linked the ssDNA-UCNPs and the ssDNA magnetic microparticles, allowing stringent washes. The presented sensor showed a similar limit of detection when the assays were conducted in samples containing total miRNA extracted from human serum, demonstrating its suitability for detecting small specific oligonucleotide sequences under real-like conditions. The strategy of combining UCNPs, magnetic microparticles, and a photoligation reaction provides new insight into low-cost, rapid, and ultra-sensitive detection of oligonucleotide sequences.

Segmented flow generator for serial crystallography at the European X-ray free electron laser.

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported.

Separation Phenomena in Tailored Micro- and Nanofluidic Environments.

Separations of bioanalytes require robust, effective, and selective migration phenomena. However, due to the complexity of biological matrices such as body fluids or tissue, these requirements are difficult to achieve. The separations field is thus constantly evolving to develop suitable methods to separate biomarkers and fractionate biospecimens for further interrogation of biomolecular content. Advances in the field of microfabrication allow the tailored generation of micro- and nanofluidic environments. These can be exploited to induce interactions and dynamics of biological species with the corresponding geometrical features, which in turn can be capitalized for novel separation approaches. This review provides an overview of several unique separation applications demonstrated in recent years in tailored micro- and nanofluidic environments. These include electrokinetic methods such as dielectrophoresis and electrophoresis, but also rather nonintuitive ratchet separation mechanisms, continuous flow separations, and fractionations such as deterministic lateral displacement, as well as methods employing entropic forces for separation.

Control of upconversion luminescence by gold nanoparticle size: from quenching to enhancement.

Metallic nanostructures have the potential to modify the anti-Stokes emission of upconverting nanoparticles (UCNPs) by coupling their plasmon resonance with either the excitation or the emission wavelength of the UCNPs. In this regard gold nanoparticles (AuNPs) have often been used in sensors for UCNP luminescence quenching or enhancement, although systematic studies are still needed in order to design optimal UCNP-AuNP based biosensors. Amidst mixed experimental evidence of quenching or enhancement, two key factors arise: the nanoparticle distance and nanoparticle size. In this work, we synthesize AuNPs of different sizes to assess their influence on the luminescence of UCNPs. We find that strong luminescence quenching due to resonance energy transfer is preferentially achieved for small AuNPs, peaking at an optimal size. A further increase in the AuNP size is accompanied by a reduction of luminescence quenching due to an incipient plasmonic enhancement effect. This enhancement counterbalances the luminescence quenching effect at the biggest tested AuNP size. The experimental findings are theoretically validated by studying the decay rate of the UCNP emitters near a gold nanoparticle using both a classical phenomenological model and the finite-difference time-domain method. Results from this study establish general guidelines to consider when designing sensors based on UCNPs-AuNPs as donor-quencher pairs, and suggest the potential of plasmon-induced luminescence enhancement as a sensing strategy.

3D printed droplet generation devices for serial femtosecond crystallography enabled by surface coating.

The role of surface wetting properties and their impact on the performance of 3D printed microfluidic droplet generation devices for serial femtosecond crystallography (SFX) are reported. SFX is a novel crystallography method enabling structure determination of proteins at room temperature with atomic resolution using X-ray free-electron lasers (XFELs). In SFX, protein crystals in their mother liquor are delivered and intersected with a pulsed X-ray beam using a liquid jet injector. Owing to the pulsed nature of the X-ray beam, liquid jets tend to waste the vast majority of injected crystals, which this work aims to overcome with the delivery of aqueous protein crystal suspension droplets segmented by an oil phase. For this purpose, 3D printed droplet generators that can be easily customized for a variety of XFEL measurements have been developed. The surface properties, in particular the wetting properties of the resist materials compatible with the employed two-photon printing technology, have so far not been characterized extensively, but are crucial for stable droplet generation. This work investigates experimentally the effectiveness and the long-term stability of three different surface treatments on photoresist films and glass as models for our 3D printed droplet generator and the fused silica capillaries employed in the other fluidic components of an SFX experiment. Finally, the droplet generation performance of an assembly consisting of the 3D printed device and fused silica capillaries is examined. Stable and reproducible droplet generation was achieved with a fluorinated surface coating which also allowed for robust downstream droplet delivery. Experimental XFEL diffraction data of crystals formed from the large membrane protein complex photosystem I demonstrate the full compatibility of the new injection method with very fragile membrane protein crystals and show that successful droplet generation of crystal-laden aqueous droplets intersected by an oil phase correlates with increased crystal hit rates.

Future microfluidic and nanofluidic modular platforms for nucleic acid liquid biopsy in precision medicine.

Nucleic acid biomarkers have enormous potential in non-invasive diagnostics and disease management. In medical research and in the near future in the clinics, there is a great demand for accurate miRNA, mRNA, and ctDNA identification and profiling. They may lead to screening of early stage cancer that is not detectable by tissue biopsy or imaging. Moreover, because their cost is low and they are non-invasive, they can become a regular screening test during annual checkups or allow a dynamic treatment program that adjusts its drug and dosage frequently. We briefly review a few existing viral and endogenous RNA assays that have been approved by the Federal Drug Administration. These tests are based on the main nucleic acid detection technologies, namely, quantitative reverse transcription polymerase chain reaction (PCR), microarrays, and next-generation sequencing. Several of the challenges that these three technologies still face regarding the quantitative measurement of a panel of nucleic acids are outlined. Finally, we review a cluster of microfluidic technologies from our group with potential for point-of-care nucleic acid quantification without nucleic acid amplification, designed to overcome specific limitations of current technologies. We suggest that integration of these technologies in a modular design can offer a low-cost, robust, and yet sensitive/selective platform for a variety of precision medicine applications.

Discrete microfluidics with electrochemical detection.

A droplet-based electrochemical digital magnetofluidics system has been developed. The system relies on the magnetic movement, in air, of different aqueous microdroplets containing magnetic microparticles--serving as the 'sample', 'blank', 'wash' and 'reagent' solutions--into and out of a three-electrode assembly. The movement of all droplets was controlled using the magnetic fields generated by three separate external magnets positioned below the superhydrophobic surface. Square-wave voltammetry was used for rapid measurements of dopamine in multiple successive microdrops with minimal cross talk. The ability of the droplet-based electrochemical microfluidic system to manipulate microliter solutions was also illustrated in bioassays of glucose, involving the merging of enzyme (GOx) and substrate droplets, followed by chronoamperometric measurements of the hydrogen peroxide product in the merged droplet. Variables of the new electrochemical digital magnetomicrofluidic technique were examined and optimized. The new droplet-based electrochemical microfluidic system offers a promising platform for automated clinical diagnostics and drug discovery.