Microfluidic Separation Research Articles

Toward a mechanistic understanding of microfluidic droplet-based extraction and separation of lanthanides

Droplet-based microfluidic extraction is a promising way for effective lanthanides extraction due to its outstanding mass transfer performance. The separation process can be greatly enhanced with the droplet-based microfluidic extraction technique. However, the interactions between mass transfer, microfluidic dynamics and extraction kinetics are still unclear, which has hindered further manipulation on microfluidic extraction to boost extraction performance. In this study, the mechanisms of microfluidic droplet-based extraction and separation intensification of lanthanides are for the first time unveiled by using a numerical simulation model. The limiting factors for the performance of droplet-based microfluidic extraction are identified through a model-based parametric analysis. The numerical analyses provide a comprehensive understanding of droplet-based microfluidic extraction systems and offer operation and optimization guidelines for future research in this area.

Deterministic displacement of particles and oil droplets in a cross-flow microsieve module

Our investigation aims to apply Deterministic Lateral Displacement (DLD) to separate (deformable) particles or droplets from dispersions on industrial scale. DLD is a promising technique because it can separate particles smaller than the pores. Previous work shows how to manipulate the critical particle diameter in a sieve-based lateral displacement system by modifying the hydrodynamics. In this study, we apply this fundamental understanding of the DLD separation principle to deterministically displace particles in a cross-flow microsieve module. First, two-dimensional simulations of the fluid dynamics in this cross-flow module were performed to investigate the hydrodynamic conditions required for particle displacement. Next, these simulations were compared with the flow fields visualized in the experimental setup. In addition, high speed recordings confirmed deterministic displacement of particles and oil droplets over the microsieve surface. Last, the systems performance was evaluated by measuring the transmission of rigid PMMA particles and deformable hexadecane droplets and the particle size distribution for different operation conditions. These results clearly demonstrate that the DLD principle can be effectively applied in a cross-flow microsieve module. With this, the application of this microfluidic separation principle to separate particles or droplets (1–20 µm) from dispersions on industrial scale has become realistic.

Abstract A21: High-throughput isolation of circulating tumor cells (CTCs) from non-small cell lung cancer (NSCLC) patients for personalized treatments

Abstract It has been recognized that circulating tumor cells (CTCs) shed by a primary tumor can be a valuable surrogate to tissue biopsies. Our project goal is to develop a CTC-based liquid biopsy platform as a noninvasive method for identifying primary and secondary mutations in non-small lung cancer to direct personalized therapies. The high-throughput, label-free and size-based inertial microfluidic separation device “Labyrinth” developed in the Nagrath lab has been used for simultaneous isolation of epithelial (EpCs) and mesenchymal (EMTCs) CTCs from lung cancer patients. The total channel length of Labyrinth is 637 mm, with 500 mm in width and 100 mm in height. The device consists of complex of small and high ratio of curvature structures (11 long loops and sharp right-angle 56 corners) to enhance differential focusing of both CTCs and white blood cells into separate fluid streams in the outlet area. The high recoveries and advantage of size-based biomarker independent separation are crucial for the downstream applications including molecular characterization of CTCs (mutation and mRNA profiling) and expansion of CTCs in vitro. Labyrinth was optimized and tested for inertial separation of cancer cells using the human lung cancer cell line PC-9. The percentage of recovery and purity was >93% with 2400 µL/min flow rate. We have enrolled 15 metastatic patients for this study and isolated CTCs from baseline (n=15) and post-therapy follow-up visits (range 1-4). To be able to detect different subpopulations of CTCs including EpCs and EMTCs from patients, we have employed immunofluorescence to label EpCAM (for EpCs) and Vim (for EMTCs), in addition to PanCK (tumor marker) and CD45 (leukocyte marker). CTCs were defined as EpCAM+/-/ Vim+/-/ PanCK+/ CD45-. All 15 patients, with different mutations/fusion, including EGFR, RET, ROS1, and ALK, had detectable CTCs at all visits, including 156±175 (at baseline, n=15), 141±133 (at visit 1, n=14), 255±218 (at visit 2, n=8), 84±81 (at visit 3, n=4), and 157±177 per mL (at visit 4, n=2). Patients with EGFR mutants had an average of 112±97 total CTCs per mL at their baseline and 66±57 after they were treated with either erlotinib or osimertanib. Patients with ROS1 fusion had slightly higher total CTCs per mL at their baseline (185±250) and during treatment with crizotinib (120±113) compared to EGFR mutant patients. To expand patient-derived CTCs for ex vivo drug testing, three different CTC culture conditions were tested: a plain, a “Fibronectin coated” (2D), and a “Matrigel-collagen-irradiated mice fibroblast coated” (3D) 48-well plate. After 6 weeks in culture, the growth rate was significantly higher in the 3D model. For CTC expansion, the enrichment methods require further optimization prior to adopting the 3D culture model for future samples. The cell surface marker independent microfluidic device, Labyrinth, isolated heterogeneous CTC subpopulations that were characterized and expanded in vitro. This could allow ex vivo drug testing to direct patient-specific therapies. Citation Format: Mina Zeinali, Arthi Nadhan, Anvya Mathur, Maggie Lee, Wei Huang, Mathias Hafner, Nithya Ramnath, Sunitha Nagrath. High-throughput isolation of circulating tumor cells (CTCs) from non-small cell lung cancer (NSCLC) patients for personalized treatments [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A21.

Advection Flows-Enhanced Magnetic Separation for High-Throughput Bacteria Separation from Undiluted Whole Blood.

A major challenge to scale up a microfluidic magnetic separator for extracorporeal blood cleansing applications is to overcome low magnetic drag velocity caused by viscous blood components interfering with magnetophoresis. Therefore, there is an unmet need to develop an effective method to position magnetic particles to the area of augmented magnetic flux density gradients while retaining clinically applicable throughput. Here, a magnetophoretic cell separation device, integrated with slanted ridge-arrays in a microfluidic channel, is reported. The slanted ridges patterned in the microfluidic channels generate spiral flows along the microfluidic channel. The cells bound with magnetic particles follow trajectories of the spiral streamlines and are repeatedly transferred in a transverse direction toward the area adjacent to a ferromagnetic nickel structure, where they are exposed to a highly augmented magnetic force of 7.68 µN that is much greater than the force (0.35 pN) at the side of the channel furthest from the nickel structure. With this approach, 91.68% ± 2.18% of Escherichia coli (E. coli) bound with magnetic nanoparticles are successfully separated from undiluted whole blood at a flow rate of 0.6 mL h-1 in a single microfluidic channel, whereas only 23.98% ± 6.59% of E. coli are depleted in the conventional microfluidic device.

Sheathless Focusing and Separation of Microparticles Using Tilted-Angle Traveling Surface Acoustic Waves.

Sheathless focusing and separation of microparticles is an important preprocessing step in various biochemical assays in which enriched sample isolation is critical. Most of the previous microfluidic particle separation techniques have used sheath flows to achieve efficient sample focusing. The sheath flow dilutes the analyte and requires additional microchannels and accurate flow control. We demonstrated a tilted-angle traveling surface acoustic wave (taTSAW)-based sheathless focusing and separation of particles in a continuous flow. The proposed device consists of a piezoelectric substrate with a pair of interdigitated transducers (IDTs) deposited at two different angles relative to the flow direction. A Y-shaped polydimethylsiloxane (PDMS) microchannel having one inlet and two outlet ports was positioned on top of the IDTs such that the acoustic energy coupling into the fluid was maximized and wave attenuation by the PDMS walls was minimized. The two IDTs independently produced high-frequency taTSAWs, which propagated at ±30° with respect to the flow direction and imparted a direct acoustic radiation force onto the target particles. A sample mixture of 4.8 and 3.2 μm particles was focused and then separated by the actuation of the IDTs at 194 and 136 MHz frequencies, respectively, without using an additional sheath flow. The proposed taTSAW-based particle separation device offered a high purity >99% at the both outlets over a wide range of flow speeds (up to 83.3 mm/s).

Magnetic activated cell sorting (MACS) pipette tip for immunomagnetic bacteria separation

Magnetic activated cell sorting is a well-established technology to sort target cells from heterogeneous cell populations based on their specific surface receptors. However, conventional bulk separators rely on irregular and dense ferromagnetic matrices that can cause variances in separation purity and recovery. Recent microfluidic separators typically use narrow and two-dimensional laminar flows that can lead to low separation throughput. Here, we present a magnetizable micropipette tip incorporating a ferromagnetic matrix of nickel meshes that have regular and rectangular micropores. The functional pipette tip can be transiently magnetized by permanent magnets, thereby enabling the efficient capture and release of target cells by simple pipetting. We performed parametric studies to investigate optimal separation protocols such as the number of mesh layers and pipetting repetitions. We then applied the functional pipette tip for separation of bacterial cells from whole blood, confirming that the separation quality is sufficient for downstream cell culture and analysis. The magnetizable pipette tip provides high-throughput separation, processing 1 mL of blood within 7 min, and high-recovery separation, recovering ≈90.5% of the bacterial cells spiked into blood.

Motorized smart pipette for handheld operation of a microfluidic blood plasma separator

Simple and institutionalized testing of circulating biomarkers is essential for rapid cancer diagnosis and prognosis. However, blood sample preparation typically requires time-consuming separation steps and bulky, costly equipment. Although microfluidic separators have been developed for rapid and simple blood separation, their fluidic accessories such as syringe pumps still need to be miniaturized at a lower price before translating the separators into clinical practice. Here, we present a motorized smart pipette as a portable, cost-effective, and controllable pumping tool that can generate stable and high-pressure conditions suitable for high-throughput microfluidic blood separators. The pumping technology is based on a miniature air pump and its feedback control using an open-source microcontroller board. We characterized the pumping performance (controllability and stability) of the proposed technology using a pressure divider and a bang-bang feedback control method in a range of flow rate (166.5–529.0 μL/min). Additionally, we demonstrated the clinical utility of the pumping technology by rapidly separating blood plasma from whole blood and successfully recovering cancer cell-derived exosomes spiked in blood. We achieved high-throughput (≈422.3 μL/min) and high-purity (≈99.8%) plasma separation without hemolysis, recovering ≈90.5% of cancer cell-derived exosomes spiked in blood. With the advantages of high usability and versatility, this method can potentially accelerate the widespread adoption and clinical translation of microfluidics-based liquid biopsy technologies.

Abrasive jet turning of glass and PMMA rods and the micro-machining of helical channels

This paper investigates the use of air-driven abrasive jets as a lathe and as a means of machining helical patterns into rotating rods. The first part of the paper presents a model for the prediction of material removal during the machining of rotating and translating ductile (PMMA) and brittle (glass) rods using abrasive jet micromachining (AJM). The model predictions were in good agreement with the experimental measurements, showing that, for a given particle speed, the volumetric removal was only a function of the abrasive dose received by the surface, and was unaffected by the rod diameter, its rotational speed, and the axial feed speed. The second part of the paper presents a new experimental procedure for machining helical channels in glass and PMMA rods using a cylindrical steel spring as a mask. Such helical channels may have applications in microfluidic devices where they can be used to aid liquid mixing and the separation of particles from a flow. The use of spring masks at various pitches and the control of the exposure time of the jet to the workpiece made it possible to fabricate helical channels with a wide range of aspect ratios, from 0.09 to 1.2. High curvature, which is favorable in the microfluidic separation of particles, was achieved by machining rods of small diameter. In addition to microfluidic applications, the models and techniques can potentially be used to machine threads in glass and other difficult to machine materials.

Negative Selection by Spiral Inertial Microfluidics Improves Viral Recovery and Sequencing from Blood.

In blood samples from patients with viral infection, it is often important to separate viral particles from human cells, for example, to minimize background in performing viral whole genome sequencing. Here, we present a microfluidic device that uses spiral inertial microfluidics with continuous circulation to separate host cells from viral particles and free nucleic acid. We demonstrate that this device effectively reduces white blood cells, red blood cells, and platelets from both whole blood and plasma samples with excellent recovery of viral nucleic acid. Furthermore, microfluidic separation leads to greater viral genome coverage and depth, highlighting an important application of this device in processing clinical samples for viral genome sequencing.

Label-free extraction of extracellular vesicles using centrifugal microfluidics.

Extracellular vesicles (EVs) play an important role as active messengers in intercellular communication and distant microenvironment modeling. Increasingly, these EVs are recognized as important biomarkers for clinical diagnostics. However, current isolation methods of EVs are time-consuming and ineffective due to the high diffusive characteristics of nanoparticles coupled with fluid flow instability. Here, we develop a microfluidic CEntrifugal Nanoparticles Separation and Extraction (µCENSE) platform for the rapid and label-free isolation of microvesicles. By utilizing centrifugal microhydrodynamics, we subject the nanosuspensions between 100 nm and 1000 nm to a unique fluid flow resulting in a zonal separation into different outlets for easy post-processing. Our centrifugal platform utilizes a gentle and efficient size-based separation without the requirements of syringe pump and other accessories. Based on our results, we report a high separation efficiency of 90% and an extraction purity of 85% within a single platform. Importantly, we demonstrate high EV extraction using a table top centrifuge within a short duration of eight minutes. The simple processes and the small volume requirement further enhance the utility of the platform. With this platform, it serves as a potential for liquid biopsy extraction and point-of-care diagnostics.

A fast massively parallel two-phase flow solver for microfluidic chip simulation

This work presents a parallel finite element solver of incompressible two-phase flow targeting large-scale simulations of three-dimensional dynamics in high-throughput microfluidic separation devic...

Microfluidic electrophoretic non-enzymatic kanamycin assay making use of a stirring bar functionalized with gold-labeled aptamer, of a fluorescent DNA probe, and of signal amplification via hybridization chain reaction.

The authors describe an enzyme-free aptamer-based assay for the determination of the model antibiotic kanamycin (Kana). The method is making use of (a) microfluidic chip electrophoresis; (b) a stirring bar carrying a gold-labeled aptamer probe, and (c) the hybridization chain reaction (HCR) for signal amplification. Firstly, a stirring bar (length: 1cm; diameter: 0.2mm) was modified with a large amount of duplex DNA and then hybridized with aptamer and its partially complementary chains (cDNA). In the presence of Kana, the binding between the Kana and aptamer unwinds the duplex structures and releases a corresponding amount of cDNA into the supernatant. The released cDNA triggers the HCR in the presence of H1 and H2 DNA hairpin to produce a large amount of duplex DNA chains with different lengths. At the same time, the amounts of H1 and H2 are reduced. The decreased signal of H1/H2 after several HCR cycles can be used to quantify kana in the 1pg·mL-1 to 10ng·mL-1, with a detection limit of 0.29pg·mL-1. The signal is generated by reading the fluorescence, best at excitation/emission maxima of 470/525nm. The whole detection process takes 3min only. The assay was employed to the detection of Kana in spiked milk and fish samples. Results are consistent with those of an enzyme linked immunosorbent assay. The assay has high throughput, high selectivity, and high amplification capability. Graphical abstract Schematic of a stirring bar functionalized with gold-labeled aptamer acting as the capture probe. It can capture the target and release primer simultaneously. The primer triggers the hybridization chain reaction inducing the consumption of H1 and H2. After a certain reaction time, the mixture is injected into the MCE platform for microfluidic electrophoretic separation and fluorometric detection.

Ion-neutral Clustering of Bile Acids in Electrospray Ionization Across UPLC Flow Regimes.

Bile acid authentic standards were used as model compounds to quantitatively evaluate complex in-source phenomenon on a UPLC-ESI-TOF-MS operated in the negative mode. Three different diameter columns and a ceramic-based microfluidic separation device were utilized, allowing for detailed descriptions of bile acid behavior across a wide range of flow regimes and instantaneous concentrations. A custom processing algorithm based on correlation analysis was developed to group together all ion signals arising from a single compound; these grouped signals produce verified compound spectra for each bile acid at each on-column mass loading. Significant adduction was observed for all bile acids investigated under all flow regimes and across a wide range of bile acid concentrations. The distribution of bile acid containing clusters was found to depend on the specific bile acid species, solvent flow rate, and bile acid concentration. Relative abundancies of each cluster changed non-linearly with concentration. It was found that summing all MS level (low collisional energy) ions and ion-neutral adducts arising from a single compound improves linearity across the concentration range (0.125-5 ng on column) and increases the sensitivity of MS level quantification. The behavior of each cluster roughly follows simple equilibrium processes consistent with our understanding of electrospray ionization mechanisms and ion transport processes occurring in atmospheric pressure interfaces. Graphical Abstract ᅟ.

Detection of sepsis in patient blood samples using CD64 expression in a microfluidic cell separation device.

A microfluidic affinity separation device was developed for the detection of sepsis in critical care patients. An affinity capture method was developed to capture cells based on changes in CD64 expression in a single, simple microfluidic chip for sepsis detection. Both sepsis patient samples and a laboratory CD64+ expression model were used to validate the microfluidic assay. Flow cytometry analysis showed that the chip cell capture had a linear relationship with CD64 expression in laboratory models. The Sepsis Chip detected an increase in upregulated neutrophil-like cells when the upregulated cell population is as low as 10% of total cells spiked into commercially available aseptic blood samples. In a proof of concept study, blood samples obtained from sepsis patients within 24 hours of diagnosis were tested on the chip to further validate its performance. On-chip CD64+ cell capture from 10 patient samples (619 ± 340 cells per chip) was significantly different from control samples (32 ± 11 cells per chip) and healthy volunteer samples (228 ± 95 cells per chip). In addition, the on-chip cell capture has a linear relationship with CD64 expression indicating our approach can be used to measure CD64 expression based on total cell capture on Sepsis Chip. Our method has proven to be sensitive, accurate, rapid, and cost-effective. Therefore, this device is a promising detection platform for neutrophil activation and sepsis diagnosis.

Continuous removal of small nonviable suspended mammalian cells and debris from bioreactors using inertial microfluidics.

Removing nonviable cells from a cell suspension is crucial in biotechnology and biomanufacturing. Label-free microfluidic cell separation devices based on dielectrophoresis, acoustophoresis, and deterministic lateral displacement are used to remove nonviable cells. However, their volumetric throughputs and test cell concentrations are generally too low to be useful in typical bioreactors in biomanufacturing. In this study, we demonstrate the efficient removal of small (<10 μm) nonviable cells from bioreactors while maintaining viable cells using inertial microfluidic cell sorting devices and characterize their performance. Despite the size overlap between viable and nonviable cell populations, the devices demonstrated 3.5-28.0% dead cell removal efficiency with 88.3-83.6% removal purity as well as 97.8-99.8% live cell retention efficiency at 4 million cells per mL with 80% viability. Cascaded and parallel configurations increased the cell concentration capacity (10 million cells per mL) and volumetric throughput (6-8 mL min-1). The system can be used for the removal of small nonviable cells from a cell suspension during continuous perfusion cell culture operations.

Microfluidic affinity separation chip for selective capture and release of label-free ovarian cancer exosomes.

Exosomes are nanoscale vesicles found in many bodily fluids which play a significant role in cell-to-cell signaling and contain biomolecules indicative of their cells of origin. Recently, microfluidic devices have provided the ability to efficiently capture exosomes based on specific membrane biomarkers, but releasing the captured exosomes intact and label-free for downstream characterization and experimentation remains a challenge. We present a herringbone-grooved microfluidic device which is covalently functionalized with antibodies against general and cancer exosome membrane biomarkers (CD9 and EpCAM) to isolate exosomes from small volumes of high-grade serous ovarian cancer (HGSOC) serum. Following capture, intact exosomes are released label-free using a low pH buffer and immediately neutralized downstream to ensure their stability. Characterization of captured and released exosomes was performed using fluorescence microscopy, nanoparticle tracking analysis, flow-cytometry, and SEM. Our results demonstrate the successful isolation of intact and label-free exosomes, indicate that the amount of both total and EpCAM+ exosomes increases with HGSOC disease progression, and demonstrate the downstream internalization of isolated exosomes by OVCAR8 cells. This device and approach can be utilized for a nearly limitless range of downstream exosome analytical and experimental techniques, both on and off-chip.

High purity and viability cell separation of a bacterivorous jakobid flagellate based on a steep velocity gradient induced soft inertial force.

Cell separation is one of the key limiting factors for precise analysis of non-axenic microbial lab cultures or environmental samples, and it remains a challenge to isolate target cells with high purity and viability via high-throughput cell sorting. During the past decade, hydrodynamic microfluidic platforms have attracted great attention in cell preparation for their high efficiency, robust performance and low cost. Here, we employ the use of a low-velocity sheath flow with high viscosity near the wall and a high-velocity sheath flow with low viscosity on the other side of the sample flow in a soft inertial separation chip. This not only prevents hard interactions between cells and chip walls but, in comparison to previous inertial separation methods, generates a significant increase in deflection of large cells while keeping the small ones in the original flow. We first conducted experiments on a mixture of small and large fluorescent particles (1.0 and 9.9 μm, respectively) and removed over 99% of the small particles. The separation efficiency was then tested on a culture of a bacterivorous jakobid flagellate, Seculamonas ecuadoriensis fed on the live bacterium, Klebsiella sp. Using our microfluidic chip, over 94% of live bacteria were removed while maintaining high jakobid cell viability. For comparison, we also conducted size-based cell sorting of the same culture using flow cytometry, which is widely used as a rapid and automated separation tool. Compared with the latter, our chip showed more than 40% higher separation efficiency. Thus, our device provides high purity and viability for cell separation of a sensitive cell sample (jakobid cells). Potentially, the method can be further used for applications in diagnostics, biological analyses and environmental assessment of mixed microbial samples.

Rapid and effective enrichment of mononuclear cells from blood using acoustophoresis

Effective separation methods for fractionating blood components are needed for numerous diagnostic and research applications. This paper presents the use of acoustophoresis, an ultrasound based microfluidic separation technology, for label-free, gentle and continuous separation of mononuclear cells (MNCs) from diluted whole blood. Red blood cells (RBCs) and MNCs behave similar in an acoustic standing wave field, compromising acoustic separation of MNC from RBC in standard buffer systems. However, by optimizing the buffer conditions and thereby changing the acoustophoretic mobility of the cells, we were able to enrich MNCs relative to RBCs by a factor of 2,800 with MNC recoveries up to 88%. The acoustophoretic microchip can perform cell separation at a processing rate of more than 1 × 105 cells/s, corresponding to 5 µl/min undiluted whole blood equivalent. Thus, acoustophoresis can be easily integrated with further down-stream applications such as flow cytometry, making it a superior alternative to existing MNC isolation techniques.

545P - Next-generation sequencing of circulating tumor cells isolated from peripheral blood of patients with head and neck, and gastrointestinal cancer

Background: Recently, precision medicine has gained attention for the prediction of therapeutic strategies in individual patients. Because the biological behavior of tumor cells has changed over time according to the pressures from treatment, real-time monitoring of tumor biology is needed to provide suitable information for choosing the most effective therapy for each patient. Circulating tumor cells (CTCs) can reflect current tumor status from primary sites and metastases from the bloodstream without invasive testing. In the current CTCs capture platforms of flow cytometry, gradient centrifugation, filtration, and droplets have been employed. A novel label-free inertial microfluidics approach (MFA) based on biomechanical properties has been developed at National Singapore University (Hou et al, Sci Rep 2013), and is able to capture CTCs independent of EpCAM expression. To identify genomic alterations in CTCs as basic data for precision medicine, we investigated the genomic profiling of CTCs using next-generation sequencing (NGS). Methods: Participants in this prospective study comprised 31 patients with advanced head and neck cancer (HN), esophageal cancer (EC), gastric cancer (GC), or colorectal cancer (CRC). CTCs were isolated using an MFA technique from 5 ml of whole blood. NGS was performed after whole-genome amplification (WGA) of DNA extracted from CTCs. Results: The underlying pathology was HN in 11 patients, EC in 8 patients, GC in 1 patient, and CRC in 11 patients. CTCs were detected from the peripheral blood of all patients (median number of CTCs, 14.5/ml; range, 3-133/ml). The most frequently detected mutations in CTCs were to APC, EGFR, RB1 and SMAD4 (10.5%) in HN and EC, and TP53 (27.2%), MET, RB1, and SMAD4 (18.2%) in CRC. NGS was successfully performed using WGA-treated DNA from CTCs and related material. Conclusions: We were able to effectively capture CTCs from patients with HN, EC, GC and CRC, and successfully performed NGS of CTCs using a microfluidic separation system without antibodies. Another trial is now ongoing to assess correlations between emergence of gene mutations in CTCs and changes in therapeutic effect during molecular-targeted therapy. Legal entity responsible for the study: Division of Early Detection for Cancer, National Cancer Center Research Institute Funding: None Disclosure: All authors have declared no conflicts of interest.

Microfluidic Separation Coupled to Mass Spectrometry for Quantification of Peanut Allergens in a Complex Food Matrix.

Peanut is an important food allergen, but it cannot currently be reliably detected and quantified in processed foods at low levels. A level of 3 mg protein/kg is increasingly being used as a reference dose above which precautionary allergen labeling is applied to food products. Two exemplar matrices (chocolate dessert and chocolate bar) were prepared and incurred with 0, 3, 10, or 50 mg/kg peanut protein using a commercially available lightly roasted peanut flour ingredient. After simple buffer extraction employing an acid-labile detergent, multiple reaction monitoring (MRM) experiments were used to assess matrix effects on the detection of a set of seven peptide targets derived from peanut allergens using either conventional or microfluidic chromatographic separation prior to mass spectrometry. Microfluidic separation provided greater sensitivity and increased ionization efficiency at low levels. Individual monitored transitions were detected in consistent ratios across the dilution series, independent of matrix. The peanut protein content of each sample was then determined using ELISA and the optimized MRM method. Although other peptide targets were detected with three transitions at the 50 mg/kg peanut protein level in both matrices, only Arah2(Q6PSU2)147-155 could be quantified reliably and only in the chocolate dessert at 10 mg/kg peanut protein. Recoveries were consistent with ELISA analysis returning around 30-50% of the incurred dose. MS coupled with microfluidic separation shows great promise as a complementary analytical tool for allergen detection and quantification in complex foods using a simple extraction methodology.