Reagent Reservoirs Research Articles

Electrochemical Diffusion Study in Poly(Ethylene Glycol) Dimethacrylate-Based Hydrogels.

Hydrogels are of great importance for functionalizing sensors and microfluidics, and poly(ethylene glycol) dimethacrylate (PEG-DMA) is often used as a viscosifier for printable hydrogel precursor inks. In this study, 1-10 kDa PEG-DMA based hydrogels were characterized by gravimetric and electrochemical methods to investigate the diffusivity of small molecules and proteins. Swelling ratios (SRs) of 14.43-9.24, as well as mesh sizes ξ of 3.58-6.91 nm were calculated, and it was found that the SR correlates with the molar concentration of PEG-DMA in the ink (MCI) (SR = 0.1127 × MCI + 8.3256, R2 = 0.9692) and ξ correlates with the molecular weight (Mw) (ξ = 0.3382 × Mw + 3.638, R2 = 0.9451). To investigate the sensing properties, methylene blue (MB) and MB-conjugated proteins were measured on electrochemical sensors with and without hydrogel coating. It was found that on sensors with 10 kDa PEG-DMA hydrogel modification, the DPV peak currents were reduced to 92 % for MB, 73 % for MB-BSA, and 23 % for MB-IgG. To investigate the diffusion properties of MB(-conjugates) in hydrogels with 1-10 kDa PEG-DMA, diffusivity was calculated from the current equation. It was found that diffusivity increases with increasing ξ. Finally, the release of MB-BSA was detected after drying the MB-BSA-containing hydrogel, which is a promising result for the development of hydrogel-based reagent reservoirs for biosensing.

Programmable droplet microfluidics for complex multistep bioassays

AbstractProgrammable droplet microfluidics (PDM) refers to a microfluidic device that integrates the functionality of a series of droplets with distinct spatial locations in a designated temporal order. PDM streamlines the intricate workflow of complex bioassays by enabling programmable and macroscopic droplet displacements, in which the droplets serve as reservoirs for reagents, microvalves for liquid insulation, and in some cases micropumps for mass transportation. As these droplets are intangible structures, the need for expensive microfabrication procedures is eliminated. Furthermore, the parallelization of the droplet series provides flexibility in controlling the throughput of the microfluidic analysis system. This paper provides a comprehensive review of PDMs enabled by various microfluidic mechanisms, including magnetism actuation, relative liquid displacement, capillary suction and sequential microdisplacement. Additionally, important applications of PDM systems for nucleic acid detection, immunoassay, drug testing, and sample recovery are also introduced. In conclusion, PDM demonstrates its potential as a highly advantageous tool for executing intricate multistep bioassays on a microfluidic platform. These technologies have exhibited superiority over their traditional counterparts in terms of size reduction, automation, and low sample/reagent consumption.

An automated parallel multi-channel chromatographic system for isotopic analysis - Demonstration considering Sr.

A fully automated, closed-column chromatographic system with parallel multi-channel has been developed. This system is established with seven reagent reservoirs, one multi-channel syringe pump, eight 10-port valves, forty sample tubes, 40 columns, and a fraction collection tray. Four samples can be purified simultaneously at a time, and 40 samples can be purified in one batch. Each sample can be purified by an independent channel, avoiding cross-contamination. The sample tubes can be flipped upside down for automatic cleaning, which eliminates the residue of samples. Moreover, the fraction collection tray can collect up to 104 different target components. The key performance of the system has been investigated. The results show that the sample tubes are well-cleaned, the bubble does not affect the chemical behavior of columns, the consistency of the parallel channels is excellent and the blank of the system is negligible. The system was demonstrated by the purification of Sr from reference materials (BCR-2, JB-2, JB-3, and NIST SRM 987). The recoveries of Sr are better than 89.4% and the blank of the whole procedure is less than 200 pg. The Sr isotope values agree well with the reference values.

Modular automated microfluidic cell culture platform reduces glycolytic stress in cerebral cortex organoids

Organ-on-a-chip systems combine microfluidics, cell biology, and tissue engineering to culture 3D organ-specific in vitro models that recapitulate the biology and physiology of their in vivo counterparts. Here, we have developed a multiplex platform that automates the culture of individual organoids in isolated microenvironments at user-defined media flow rates. Programmable workflows allow the use of multiple reagent reservoirs that may be applied to direct differentiation, study temporal variables, and grow cultures long term. Novel techniques in polydimethylsiloxane (PDMS) chip fabrication are described here that enable features on the upper and lower planes of a single PDMS substrate. RNA sequencing (RNA-seq) analysis of automated cerebral cortex organoid cultures shows benefits in reducing glycolytic and endoplasmic reticulum stress compared to conventional in vitro cell cultures.

Salivary uric acid sensor using the fordable “Finger‐Powered” microfluidic device

AbstractHere, we report a novel biosensing system using a finger‐powered microfluidic device, which is suitable for daily self‐testing of uric acid in saliva. To realize the rapid determination of salivary uric acid, the biosensing system measures directly from the paper‐based saliva sampling device. The microfluidic device has the diaphragm pump, the reagent reservoir, and the reaction cell in which the electrochemical uric acid biosensor is embedded. Once the microfluidic chip has been folded, the finger‐powered microfluidic device prepares the reagent containing the saliva, which is ready to measure. A wireless potentiostat for electrochemical measurement of uric acid with the biosensor was also developed. Combining these elements together, the whole process from saliva sampling to determination was completed in less than a minute (Sensitivity: 0.03 nA/μM). The measurement results were also consistent with that measured using a commercially available uric acid determination kit. Hence, our method is expected to have the potential to enhance the use of biochemical information in the field of healthcare IoT.

Salivary Uric Acid Sensor using The Fordable ‘Finger-Powered' Microfluidic Device

Here, we report a novel biosensing system using a finger-powered microfluidic device, which is suitable for daily self-testing of uric acid in saliva. To realize the rapid determination of salivary uric acid, the biosensing system measures directly from the paper-based saliva sampling device. The microfluidic device has the diaphragm pump, the reagent reservoir, and the reaction cell in which the electrochemical uric acid biosensor is embedded. Once the microfluidic chip has been folded, the finger-powered microfluidic device prepares the reagent containing the saliva, which is ready to measure. A wireless potentiostat for electrochemical measurement of uric acid with the biosensor was also developed. Combining these elements together, the whole process from saliva sampling to determination was completed in less than a minute (Sensitivity: 0.03 nA/µM). The measurement results were also consistent with that measured using a commercially available uric acid determination kit. Hence, our method is expected to have the potential to enhance the use of biochemical information in the field of healthcare IoT.

Interconnectable solid-liquid protein extraction unit and chip-based dilution for multiplexed consumer immunodiagnostics

While consumer-focused food analysis is upcoming, the need for multiple sample preparation and handling steps is limiting. On-site and consumer-friendly analysis paradoxically still requires laboratory-based and skill-intensive sample preparation methods. Here, we present a compact, inexpensive, and novel prototype immunosensor combining sample preparation and on-chip reagent storage for multiplex allergen lateral flow immunosensing. Our comprehensive approach paves the way for personalized consumer diagnostics. The prototype allows for handheld solid-liquid extraction, pipette-free on-chip dilution, and adjustment of sample concentrations into the appropriate assay dynamic working range. The disposable and interconnectable homogenizer unit allows for the extraction and 3D-sieve based filtration of allergenic proteins from solid bakery products in 1 min. The homogenizer interconnects with a 3D-printed unibody lab-on-a-chip (ULOC) microdevice, which is used to deliver precise volumes of sample extract to a reagent reservoir. The reagent reservoir is implemented for on-chip storage of carbon nanoparticle labeled antibodies and running buffer for dilution. The handheld prototype allows for total homogenization of solid samples, solid-liquid protein extraction, 3D-printed sieve based filtration, ULOC-enabled dilution, mixing, transport, and smartphone-based detection of hazelnut and peanut allergens in solid bakery products with limited operational complexity. The multiplex lateral flow immunoassay (LFIA) detects allergens as low as 0.1 ppm in real bakery products, and the system is already consumer-operable, demonstrating its potential for future citizen science approaches. The designed system is suitable for a wide range of analytical applications outside of food safety, provided an LFIA is available.

Smartphone-Based Microfluidic Colorimetric Sensor for Gaseous Formaldehyde Determination with High Sensitivity and Selectivity.

Formaldehyde is one of the most dangerous air pollutants, which can cause sick building syndrome. Thus, it is very crucial to precisely determine formaldehyde with a low cost and simple operation. In this paper, a smartphone-based microfluidic colorimetric sensor is devised for gaseous formaldehyde determination with high sensitivity and selectivity. Specifically, a novel microfluidic chip is proposed based on the 4-aminohydrazine-5-mercapto-1,2,4-triazole (AHMT) method to determine formaldehyde; the chip consists of two reagent reservoirs, one reaction reservoir and a mixing column. In this design to prevent the fluid from flowing out while letting the gas molecule in, a hydrophobic porous poly tetra fluoroethylene (PTFE) membrane is put on the top of the reaction reservoir. Using the microfluidic chip sensor, a smartphone-based formaldehyde determination system is developed, which makes the measuring process automated and simple. As per the experiment results, the limit-of-detection (LOD) of the system is as low as 0.01 ppm, which is much lower than the maximum exposure concentration (0.08 ppm) recommended by the World Health Organization (WHO). Moreover, the sensor is hardly affected by acetaldehyde, volatile organic compounds (VOCs) or acidic-alkaline, which shows great selectivity. Finally, the performance of the proposed sensor is verified by using it for the determination of formaldehyde in a newly decorated house.

P092 Validation of the automated NGSGO library preparation workflow for HLA typing on the BIOMEK 4000

Aim The GenDx NGSgo workflow for HLA typing using Next-generation sequencing (NGS) consists of four practical steps: amplification, library preparation, sequencing and data analysis. Out of these four steps the library preparation is the most labor intensive and most interspersed by incubations. To decrease hands-on time and reduce the risk of human error, NGSgo library preparation can be automated from start to finish using a liquid handler workstation. Here we describe the results of the automation of the NGSgo library preparation for Illumina on a Beckman Coulter Biomek 4000 workstation. The workflow was fully automated from amplicon pool to finalized library, making it completely hands-off to accommodate overnight runs and allow the operator to step away from the machine. Methods The Biomek setup included a thermal cycler, 96-well plate magnet, two peltiers devices for cooling 96-well plates and 1.5 ml tubes, a gripper tool, reagent reservoirs and liquid waste disposal. Pipetting was performed by two pipet tools. This setup allowed for flexible processing of 4 to 24 samples per run, with any number of loci. Per run only three boxes of pipetting tips were used for 24 samples. Panels with variable numbers of samples and replicate panels were processed with the Biomek. Subsequently, the pooled libraries were sequenced on a MiSeq system and the resulting data was analyzed with NGSengine software (GenDx). Results MiSeq run parameters were indicative of a high-quality sequencing runs. Read depth, mappability and noise levels of the sequence data presented here showed that the automated workflow resulted in high quality reads and accurate typings. Results from the replicate panel showed a 100% typing match. Conclusions – NGSgo library preparation for 4 to 24 samples can be performed on the Biomek 4000 platform, without manual intervention. – The automated workflow results in high-quality reproducible libraries. – Performing automated NGSgo library preparation as described here reduces hands-on time from 1.5 h to 10 min as compared to manual execution. B. Luiken: 5. Employee; Company/Organization; GenDx. E. Tavkin: 5. Employee; Company/Organization; Beckman Coulter. S. Parakh: 5. Employee; Company/Organization; Beckman Coulter. J. Lu: 5. Employee; Company/Organization; Beckman Coulter. A. Jackson: 5. Employee; Company/Organization; Beckman Coulter. S. van Wageningen: 5. Employee; Company/Organization; GenDx. M. Penning: 5. Employee; Company/Organization; GenDx.

Fabrication of a Lab-on-Chip Device Using Material Extrusion (3D Printing) and Demonstration via Malaria-Ab ELISA.

Additive manufacturing, such as fused deposition modeling (FDM), has been increasingly employed to produce microfluidic platforms due to ease of use, wide distribution of affordable 3D printers and relatively inexpensive materials for printing. In this work, we discuss fabrication and testing of an FDM-printed fully automated colorimetric enzyme-linked immunosorbent assay (ELISA) designed to detect malaria. The detection platform consists of a disposable 3D-printed fluidic cartridge (with elastomeric silicone domes on top of reagent-storage reservoirs) and a nondisposable frame with servomotors and electronic controls such as an Arduino board and a rechargeable battery. The system is controlled by a novel interface where a music file (so-called “song”) is sent to the Arduino board, where the onboard program converts the set of frequencies into action of individual servomotors to rotate their arms a certain amount, thus depressing specific elastomeric domes atop reagent reservoirs and displacing the specific reagents into the detection wells, where bioassay steps are executed. Another of the distinguished characteristics of the demonstrated system is its ability to aspirate the fluid from the detection wells into the waste reservoir. Therefore, the demonstrated automated platform has the ability to execute even the most complex multi-step assays where dilution and multiple washes are required. Optimization of 3D-printer settings and ways to control leakages typical of FDM-printed fluidic systems are also discussed.

Abstract 2244: Development of an automated device for size-based enrichment and isolation of circulating tumor cells in lung cancer patients

Abstract Background and Purpose: Circulating tumor cells (CTCs) are relatively rare cells defined as tumor cells circulating in the peripheral blood of patients with solid tumors. Diagnosis utilizing CTCs is expected to help guide decision-making for precision cancer medicine. We developed an automated microcavity array (MCA) system to detect CTCs based on the differences in size and deformability between tumor cells and normal blood cells. Here we evaluated its performance using preclinical spike-in model and blood samples from non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) patients. Material and method: The automated MCA system consists of components such as chambered cartridge containing micro metal filter, reagent and waste reservoirs, and peristaltic pump. To evaluate the recovery of CTCs, preclinical experiments using NSCLC cells, NCI-H820, A549, NCI-H441 and NCI-H23 spiked into peripheral whole blood from healthy volunteers were performed. For clinical evaluation, 6 mL of peripheral whole blood was collected from 50 advanced lung cancer patients prior to the initiation of chemotherapy and from 10 healthy donors. Samples were collected in an EDTA-containing tube and were processed within 3 hours of blood draw. Recovered cells on the filter were then fixed, permeabilized, and stained automatically and high-resolution fluorescent images were obtained using fluorescence microscope. We defined CTC as DAPI-positive, cytokeratin-positive and CD45-negative cell. Results: Results of the preclinical study showed that up to 90% of spiked-in tumor cells were recovered, confirming that the detection sensitivity by this automated device is on par with that by previous manual detection procedure. Demographics of 50 lung cancer patients enrolled in clinical study were as follows: median age 72 (range, 48 to 85); male 66%; stage III/IV 12/88%; NSCLC/SCLC 78/22%. Cells defined as CTC were detected in 2 cases out of 10 healthy volunteers, of which CTC count was 1 and 2 / 6 mL, respectively. Three or more CTCs were detected in 71% of patients with advanced lung cancer (39 out of 50) and five or more CTCs were detected in 52% of patients (26 out of 50) (median CTC count 13.5). Among stage IV NSCLC patients, patients with extrathoracic metastasis tend to have more CTCs than in those with intrathoracic metasitasis (median CTC count, 8 versus 4, p = 0.058). A head-to-head comparison between CellSearch system and our system was conducted in NSCLC patients, showing the superiority of our system (median CTC count, 0 versus 11.25, p = 0.0001, n = 17). Conclusions Our results suggest that the automated MCA device has a clinical potential for CTCs diagnosis towards precision medicine in lung cancer. This device also enables higher throughput owing to its automated procedure. Further clinical evaluation including the detection of PD-L1 expression will be performed in an expansion cohort. Citation Format: Satomi Yagi, Yasuhiro Koh, Hiroaki Akamatsu, Woong Kim, Ayaka Tanaka, Kuninobu Kanai, Atsushi Hayata, Ryota Shibaki, Masayuki Higuchi, Hisashige Kanbara, Takashi Kikuchi, Keiichiro Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Development of an automated device for size-based enrichment and isolation of circulating tumor cells in lung cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2244.

Low Cost 3D Printed Biosensor Arrays for Biomedical Diagnostics

Development and fabrication of bioanalytical devices by 3D printing offers revolutionary new routes to low cost clinical diagnostic devices for molecular measurements. Relevant to future protein-based cancer diagnostics, we describe here our development of prototype protein immunoarray devices using desktop Fused Deposition Modeling (FDM) and stereolithographic 3D printers. All these system feature sensitive electro-optical detection by a method called electrochemiluminescence (ECL). Our first 3D-printed immunoarray features screen-printed sensors in which manual manipulations enable gravity flow reagent delivery for measurement of 3 proteins at detection limits of 0.3 to 0.5 pg/mL. ECL detection is achieved in an open channel on integrated disposable screen-printed sensor elements. We then address the issue of printing and processing optically clear plastic using a stereolithographic printer to build a closed ECL detection chamber. Finally, we describe a prototype 3D-printed microprocessor-controlled enclosed microfluidic ECL immunoarray featuring reagent reservoirs, micropumps and clear plastic detection chamber with printed nanowells for ECL emission.

The fertile physics of chemical gardens

Many a child has enjoyed watching the gardens grow; many a physicist has puzzled over the transformation of self-organized, nonequilibrium patterns into permanent structures.

Labware, Lab Supplies, and Microplates

Labware, Lab Supplies, and Microplates

Enrichment of tumor-initiating breast cancer cells within a mammosphere-culture microdevice

We report for the first time a microdevice that enables the selective enrichment, culture, and identification of tumor-initiating cells on native polydimethylsiloxane (PDMS). For nearly a decade, researchers have identified tumor-initiating breast cancer cells within heterogeneous populations of breast cancer cells by utilizing low-attachment serum-free culture conditions, which lead to the formation of spheroidal colonies (mammospheres) that are enriched for tumor-initiating cells. However, the utility of this assay has been limited by difficulties in combining this culture-plate-based technique with other cellular and molecular analyses. Integrating the mammosphere technique into a microsystem can enable it to be combined directly with a number of functions, such as cell sorting, drug screens, and molecular assays. In this work, we demonstrate mammosphere culture within a PDMS microdevice. We first prove that a native hydrophobic PDMS surface is as effective as commercial low-attachment plates at selectively promoting the formation of mammospheres. We then experimentally assess the PDMS microdevice. Time-lapse images of mammosphere formation within the microdevice show that mammospheres form from single cells or small clusters of cells. Following formation of the mammospheres, it is desirable to evaluate the cells within the spheroids for enrichment of tumor initiating cells. To perform assays such as this (which require the loading and rinsing of reagents) without flushing the cells (which are in suspension) from the device, the culture chamber is separated from a reagent reservoir by a commercially available microporous membrane, and thus reagents are exchanged between the reservoir and the culture chamber by diffusion only. Using this capability, we verify that the mammospheres are enriched for tumor initiating cells by staining aldehyde dehydrogenase activity, a cancer stem cell marker. To the best of our knowledge, this is the first assay that enables the direct observation of tumor-initiating cells within a suspended mammosphere.

Surface immobilized hydrogels as versatile reagent reservoirs for microarrays

The accuracy of antibody-based microarrays depends on eliminating or at least minimizing the effect of cross-reactive components. In this work, a method to create a versatile, compartmentalized storage system for antibodies and soluble microarray reagents is described. These containers are made of hydrogel plugs immobilized on a slide facing the corresponding sub-array on a printed surface.The hydrogels were polymerized using a 4% w/v N,N-dimethylacrylamide and 3% w/v bisacrylamide solution, thus generating large pore sizes to allow the facile transfer of intact, functional biomolecules. The hydrogel microenvironments can be desiccated and rehydrated with a desired solution, and they can store reagents in a dry form.These hydrogels were shown to provide limits of detection similar to those obtained by conventional incubation conditions in an assay for HIV p24 antigen. Moreover, they prevent cross-reactivity issues in the detection of Staphylococcus aureus enterotoxins A and B and promote the accurate quantification of interleukin 10 by a microarray-integrated calibration curve.

An innovative sample-to-answer polymer lab-on-a-chip with on-chip reservoirs for the POCT of thyroid stimulating hormone (TSH)

A new sample-to-answer polymer lab-on-a-chip, which can perform immunoassay with minimum user intervention through on-chip reservoirs for reagents and single-channel assay system, has been designed, developed and successfully characterized as a point-of-care testing (POCT) cartridge for the detection of thyroid stimulating hormone (TSH). Test results were obtained within 30 minutes after a sample was dropped into the POCT cartridge. The analyzed results of TSH showed a linear range of up to 55 μIU mL(-1) with the limit of detection (LOD) of 1.9 μIU mL(-1) at the signal-to-noise ratio (SNR) of 3. The reagents stored in the on-chip reservoirs maintained more than 97% of their initial volume for 120 days of storage time while the detection antibody retained its activity above 98% for 120 days. The sample-to-answer polymer lab-on-a-chip developed in this work using the mass-producible and low-cost polymer is well suited for the point-of-care testing of rapid in vitro diagnostics (IVD) of TSH.

The ALGADEC device: A semi-automated rRNA biosensor for the detection of toxic algae

In order to facilitate the monitoring of toxic algae, a multiprobe chip and a semi-automated rRNA biosensor for the detection of toxic algae in the field were developed. The biosensor consisted of a multiprobe chip with an array of 16 gold electrodes for the detection of upto 14 target species. The multiprobe chip was placed inside an automated hybridization chamber, which was in turn placed inside a portable waterproof case with reservoirs for different reagents. A peristaltic pump transferred the reagents across the flow cell (hybridization chamber) containing the multiprobe chip. For use of the device by laypersons, a lysis protocol was successfully developed so that manual rRNA isolation was no longer required. Only water sample filtration has to be done manually. The stand-alone system was evaluated using total rRNA isolated from Alexandrium minutum. The device processed automatically the main steps of the analysis and completed the electrochemical detection of toxic algae in less than 2h in comparison to other routine monitoring methods that need at least up to one day for analysis.

A surface topography assisted droplet manipulation platform for biomarker detection and pathogen identification

This paper reports a droplet microfluidic, sample-to-answer platform for the detection of disease biomarkers and infectious pathogens using crude biosamples. The platform exploited the dual functionality of silica superparamagnetic particles (SSP) for solid phase extraction of DNA and magnetic actuation. This enabled the integration of sample preparation and genetic analysis within discrete droplets, including the steps of cell lysis, DNA binding, washing, elution, amplification and detection. The microfluidic device was self contained, with all reagents stored in droplets, thereby eliminating the need for fluidic coupling to external reagent reservoirs. The device incorporated unique surface topographic features to assist droplet manipulation. Pairs of micro-elevations were created to form slits that facilitated efficient splitting of SSP from droplets. In addition, a compact sample handling stage, which integrated the magnet manipulator, the droplet microfluidic device and a Peltier thermal cycler, was built for convenient droplet manipulation and real-time detection. The feasibility of the platform was demonstrated by analysing ovarian cancer biomarker Rsf-1 and detecting Escherichia coli with real time polymerase chain reaction and real time helicase dependent amplification.

3D Rapid Prototyping Technology (RPT) as a powerful tool in microfluidic development

A novel method, a 3D printing technique, in particular, acrylic photopolymer material-based Rapid Prototyping (RPT) have been used to 1) fabricate molds for PDMS (PolyDiMethylSiloxane) casting; 2) fabricate RPT-based microfluidics and 3) fabricate RPT-based research instrument platforms. 3D RPT-based molds have been fabricated in order to cast a PDMS flow cell for a Surface Plasmon Resonance (SPR) instrument, and to cast a PDMS chamber for cell lysis in a nanobiological sensor. 3D printing has been utilized to create and test several acrylic photopolymer resin-based prototypes for different microfluidic structures (chaotic mixers, reagent and buffer reservoirs, fluid homogenizers) to be deployed for gynecological cervical sample preparation. Besides, RPT technique has been used also to fabricate platform elements for various microfluidic applications.