Reagent Chambers Research Articles

A novel nucleic acid extraction system integrating a switching valve-assisted microfluidic cartridge and a constant pressure pump

BackgroundNucleic acid extraction (NAE) is essential in molecular diagnostics, genetic engineering, and DNA sequencing. While advances in switching valve-assisted, self-contained microfluidic (S2M) cartridges have improved the NAE processes, challenges persist in streamlining workflows, reducing processing times, and enhancing multi-target detection. Achieving these objectives requires precise control of on-chip fluid dynamics and efficient transfer of nucleic acid solutions (NAS) to detection areas. However, research on novel S2M cartridge designs and compatible fluid control systems is still limited. ResultThis study presented an innovative NAE system that integrated an S2M cartridge, a constant pressure (CP) pump, a metal heating bath, a rotating lever, and a magnet. The S2M cartridge contained the reagents for both NAE and detection, while the CP pump provided stable, precise, and rapid fluid delivery, outperforming traditional peristaltic and injection pumps, especially for handling small volumes and reducing pulsation. A custom rotating lever connects reagent chambers, facilitating mixing, cleaning, elution, and NAS transfer. This system showed advantages over manual methods, achieving all processes in 20 min. The CP pump-assisted S2M cartridge enabled rapid MBs re-suspension with a recovery rate of 80 %. The system's sensitivity, repeatability, and ease of use were also confirmed. The NAE system can extract Escherichia coli DNA (50 CFU mL^-1) from food and serum samples and viral DNA (HBV, HCV, HIV) at 200 copies mL^-1 from serum samples. SignificanceThis study highlighted the need for an efficient fluid system in the S2M cartridge for stable NAE. By streamlining NAE and facilitating on-chip reagent preparation, the system enhanced point-of-care diagnostics. Although manual steps remain, the CP pump effectively managed fluid processes, paving the way for automated pathogen detection. Moreover, when combined with visual detection reagents, the NAE system showed promise as a direct molecular diagnostic tool.

Design and Fabrication of a 3D-Printed Microfluidic Immunoarray for Ultrasensitive Multiplexed Protein Detection.

Microfluidic technology has revolutionized device fabrication by merging principles of fluid dynamics with technologies from chemistry, physics, biology, material science, and microelectronics. Microfluidic systems manipulate small volumes of fluids to perform automated tasks with applications ranging from chemical syntheses to biomedical diagnostics. The advent of low-cost 3D printers has revolutionized the development of microfluidic systems. For measuring molecules, 3D printing offers cost-effective, time, and ease-of-designing benefits. In this paper, we present a comprehensive tutorial for design, optimization, and validation for creating a 3D-printed microfluidic immunoarray for ultrasensitive detection of multiple protein biomarkers. The target is the development of a point of care array to determine five protein biomarkers for aggressive cancers. The design phase involves defining dimensions of microchannels, reagent chambers, detection wells, and optimizing parameters and detection methods. In this study, the physical design of the array underwent multiple iterations to optimize key features, such as developing open detection wells for uniform signal distribution and a flap for covering wells during the assay. Then, full signal optimization for sensitivity and limit of detection (LOD) was performed, and calibration plots were generated to assess linear dynamic ranges and LODs. Varying characteristics among biomarkers highlighted the need for tailored assay conditions. Spike-recovery studies confirmed the assay's accuracy. Overall, this paper showcases the methodology, rigor, and innovation involved in designing a 3D-printed microfluidic immunoarray. Optimized parameters, calibration equations, and sensitivity and accuracy data contribute valuable metrics for future applications in biomarker analyses.

Digitizing protocols into single reactors for the one-pot synthesis of nanomaterials

Digitizing protocols into single reactors for the one-pot synthesis of nanomaterials

A simple 3D printed microfluidic device for point-of-care analysis of urinary uric acid

Point-of-care testing (POCT) technology allows scientists to monitor and diagnose diseases at the patient site, much faster than classical lab-based methods. Herein, a rapid, simple, and sensitive 3D printed microfluidic device integrated with smartphone-based on-chip detection is described for POCT quantification of urinary uric acid. The device includes two circular inputs each connected to a microliter-scale chamber, separated by an integrated porous membrane, located between the sample and reagent chambers. The microfluidic device was fabricated from a transparent photopolymer using a 3D printer, in a single run. The concentration of uric acid was determined based on a chromogenic reaction in which ferrous ion, produced via the reduction of ferric ion by the analyte, complexed with 1,10-phenanthroline, and the color was recorded by a smartphone. Response surface methodology including a central composed design was utilized to evaluate the experimental parameters and subsequent introduction of a multivariate model to describe the experimental conditions. Under the optimum conditions, the calibration curve was linear over the concentration range of 30–600 mg L−1. The limit of detection was determined to be 10.5 mg L−1. The microfluidic device was successfully utilized for the recovery and quantification of uric acid in the urine, with recoveries ranging from 91.7 to 99.7%.

Mechanical Timer-Actuated Fluidic Dispensing System: Applications to an Automated Multistep Lateral Flow Immunoassay with High Sensitivity.

In this study, we present a fluidic dispensing system that can automate the sequential fluidic delivery of multiple reagents for lateral flow assays. Highly sensitive assays typically require multiple solution-based sequences, including washing steps and signal amplification. However, implementation of these types of sequences on an automated and highly sensitive point-of-care testing (POCT) platform remains challenging. Our platform consists of two disposable cartridges with reagent chambers and a test strip and an instrument that has a mechanical timer to actuate the cam-follower-gear components. The timer rotation sequentially shifts the position of the chambers and loads the reagents to the test paper strip. The dispensing intervals are controlled at a variation of <1% within a total actuation time of 60 min. Unlike other POCT devices, the timing of fluid delivery in our timer-actuated platform is not dependent on the selection of substrates and reagents, and the unique approach to fluidic delivery results in no reagent overlap or carryover, minimal reagent loss, and highly accurate fluidic timing control for highly sensitive solution-based assays. As a model application, the proposed platform applies a gold enhancement solution to amplify the detection signal and detect prostate-specific antigen with a limit of detection of 86 pg/mL within 27 min. This platform provides an opportunity for solution-based POCT applications with high sensitivity, thereby satisfying the requirement for user-friendly operations in resource-limited settings.

Ultraviolet-induced in situ gold nanoparticles for point-of-care testing of infectious diseases in loop-mediated isothermal amplification.

The present study investigated ultraviolet-induced in situ gold nanoparticles (AuNPs) coupled with loop-mediated isothermal amplification (LAMP) for the point-of-care testing (POCT) of two major infectious pathogens, namely, Coronavirus (COVID-19) and Enterococcus faecium (E. faecium spp.). In the process, gold ions in a gold chloride (HAuCl4) solution were reduced using trisodium citrate (Na3Ct), a reducing agent, and upon UV illumination, red-colored AuNPs were produced in the presence of LAMP amplicons. The nitrogenous bases of the target deoxyribonucleic acid (DNA) acted as a physical support for capturing gold ions dissolved in the sample. The high affinity of gold with the nitrogenous bases enabled facile detection within 10 min, and the detection limit of COVID-19 plasmid DNA was as low as 42 fg μL-1. To ensure POCT, we designed a portable device that contained arrays of reagent chambers and detection chambers. In the portable device, colorimetric reagents such as HAuCl4 and Na3Ct were contained in the reagent chambers; these reagents were subsequently transferred to the detection chambers where LAMP amplicons were present and thus allowed convenient sample delivery and multiplex detection. Owing to the high sensitivity of the in situ AuNPs, simplicity of portable device fabrication, and rapid colorimetric detection, we strongly believe that the fabricated portable device could serve as a kit for rapid POCT for instantaneous detection of infectious diseases, and could be readily usable at the bedside.

Scalable 3D printing method for the manufacture of single-material fluidic devices with integrated filter for point of collection colourimetric analysis

Assembly and bonding are major obstacles in manufacturing of functionally integrated fluidic devices. Here we demonstrate a single-material 3D printed device with an integrated porous structure capable of filtering particulate matter for the colourimetric detection of iron from soil and natural waters. Selecting a PolyJet 3D printer for its throughput, integrated filters were created exploiting a phenomenon occurring at the interface between the commercially available build material (Veroclear-RGD810) and water-soluble support material (SUP707). The porous properties were tuneable by varying the orientation of the print head relative to the channel and by varying the width of the build material. Porous structures ranging from 100 to 200 μm in thickness separated the sample and reagent chambers, filtering particles larger than 15 μm in diameter. Maintaining the manufacturing throughput of the Polyjet printer, 221 devices could be printed in 1.5 h (∼25 s per device). Including the 12 h post-processing soak in sodium hydroxide to remove the solid support material, the total time to print and process 221 devices was 13.5 h (3.6 min per device), with a material cost of $2.50 each. The applicability of the fluidic device for point of collection analysis was evaluated using colourimetric determination of iron from soil slurry and environmental samples. Following the reduction of Fe3+ to Fe2+ using hydroxylammonium chloride, samples were introduced to the fluidic device where particulate matter was retained by the filter, allowing for particulate-free imaging of the red complex formed with 1,10-phenanthroline using a smartphone camera. The calibration curve ranged from of 1–100 mg L−1 Fe2+ and good agreement (95%) was obtained between the point of collection device and Sector Field ICP-MS.

Exploiting phase change materials in tunable passive heating system for low-resource point-of-care diagnostics

We present a passive heating system that allows controlled and localized on-chip heating, showing a huge potential for point-of-care (POC) diagnostic applications in low-resource settings. By coupling the thermal regulation properties of organic phase change materials (PCMs) to the exothermic crystallization reaction of supercooled sodium acetate trihydrate (SAT), on-chip temperature control is achieved without requiring external power supply. This enables the performance of sensitive and specific diagnostic tests in the field, which are currently restricted to laboratory environments. The presented system consists of 3 stacked microfluidic chambers (SAT, PCM, reagent), fabricated by means of a simple layer-by-layer manufacturing method using low-cost plastics. We showed that the SAT heat source was tunable between 35 and 55 °C by varying the salt fraction in the solution. A PCM was integrated between the SAT and reagent chamber to maintain temperature constant over time. Additionally, the introduction of this thermal buffer layer, increased the system its robustness to fluctuating ambient temperatures. Moreover, to facilitate the PCM and SAT chamber design optimization, a finite element model (FEM) was built to simulate the heat generation and transfer through the chip. With the optimized PCM-SAT system, a constant temperature between 37 and 42 °C was maintained for at least 15 min at different ambient temperatures (25–35 °C), typical conditions for a recombinase polymerase amplification assay. The ability to have simple control over both the heat release from the heat source and the regulation temperature of the PCM combined with the developed FEM, makes this system flexible to a variety of isothermal assay requirements. Moreover, the low-cost and simple fabrication method makes the system compatible with most passive microfluidic technologies (i.e., paper-based and capillary microfluidics), opening up many opportunities for integrated POC platforms.

Rapid and Automated Detection of Six Contaminants in Milk Using a Centrifugal Microfluidic Platform with Two Rotation Axes.

Antibiotic residues and illegal additives are among the most common contaminants in milk and other dairy products, and they have become essential public health concerns. To ensure the safety of milk, rapid and convenient screening methods are highly desired. Here, we integrated microarray technology into a microfluidic device to achieve rapid, sensitive, and fully automated detection of chloramphenicol, tetracyclines, enrofloxacin, cephalexin, sulfonamides, and melamine in milk on a centrifugal microfluidic platform with two rotation axes. All the liquid reagent for the immunoassay was prestored in the reagent chambers of the microdevice and can be released on demand. The whole detection can be automatically accomplished within 17 min, and the limits of detection were defined as 0.92, 1.01, 1.83, 1.14, 1.96, and 7.80 μg/kg for chloramphenicol, tetracycline (a typical drug of tetracyclines), enrofloxacin, cephalexin, sulfadiazine (a typical drug of sulfonamides), and melamine, respectively, satisfying the national standards for maximum residue limits in China. Raw milk samples were used to test the performance of the current immunoassay system, and the recovery rates in the repeatability tests ranged from 80 to 111%, showing a good performance. In summary, the immunoassay system established in this study can simultaneously detect six contaminants of four samples in a fully automated, cost-effective, and easy-to-use manner and thus has great promise as a screening tool for food safety testing.

An integrated microfluidic loop-mediated isothermal amplification platform for koi herpesvirus detection

An integrated microfluidic loop-mediated isothermal amplification (LAMP) platform consisting of a microfluidic chip and a portable operating system is proposed for koi herpesvirus (KHV) detection applications. The microfluidic chip is fabricated using a CO2 laser system and comprises a sample chamber, two reagent chambers, a mixing chamber, a serpentine channel, a reaction chamber, and three flow rectifiers based on PDMS membranes. The main components of the operating system include a power source, a time relay, a microfluidic chip holder, four gas pressure valves, a microheater, and a temperature controller. In the proposed detection method, the KHV sample and LAMP reagent are loaded into the sample and reagent chambers, respectively, and are mixed under the effects of an external gas pressure driving force. The mixed solution is then heated at a temperature of approximately 63 °C for 60 min in order to replicate the KHV sample. Finally, the replicated sample is mixed with SYBR Green I dye in order to facilitate fluorescence-based KHV detection. The feasibility of the proposed microfluidic platform is confirmed by comparing the detection results obtained for a sample containing 10 ng/μL KHV with those obtained using a gel electrophoresis PCR-based method. The experimental results shows that the proposed LAMP platform has a KHV detection limit of around 3 × 10−2 ng/μL. Overall, the results presented in this study show that the proposed microfluidic platform provides a compact, reliable and portable tool for KHV detection purposes.

One-Step Fabrication of a Microfluidic Device with an Integrated Membrane and Embedded Reagents by Multimaterial 3D Printing.

One of the largest impediments in the development of microfluidic-based smart sensing systems is the manufacturability of integrated, complex devices. Here we propose multimaterial 3D printing for the fabrication of such devices in a single step. A microfluidic device containing an integrated porous membrane and embedded liquid reagents was made by 3D printing and applied for the analysis of nitrate in soil. The manufacture of the integrated, sealed device was realized as a single print within 30 min. The body of the device was printed in transparent acrylonitrile butadiene styrene (ABS) and contained a 400 μm wide structure printed from a commercially available composite filament. The composite filament can be turned into a porous material through dissolution of a water-soluble material. Liquid reagents were integrated by briefly pausing the printing before resuming for sealing the device. The devices were evaluated by the determination of nitrate in a soil slurry containing zinc particles for the reduction of nitrate to nitrite using the Griess reagent. Using a consumer digital camera, the linear range of the detector response ranged from 0 to 60 ppm, covering the normal range of nitrate in soil. To ensure that the sealing of the reagent chamber is maintained, aqueous reagents should be avoided. When using the nonaqueous reagent, the multimaterial device containing the Griess reagent could be stored for over 4 days but increased the detection range to 100-500 ppm. Multimaterial 3D printing is a potentially new approach for the manufacture of microfluidic devices with multiple integrated functional components.

A paper-based microfluidic Dot-ELISA system with smartphone for the detection of influenza A

An automated, portable, and integrated paper-based microfluidic system has been developed for influenza A detection with smartphone at point-of-care (POC) settings. The low-cost paper-based microfluidic chip consists of a reagent storage and reaction modules. The storage module, which consists of a couple of reagent chambers with dispensation channels, is responsible for reagent storage and release. The reaction module consists of an absorbent pad and a nitrocellulose (NC) membrane which is functionalized with specific monoclonal antibodies on a test and control spots for immunoassay detection. Microfluidic Dot-ELISA is performed when the dispensed reagent flows through the NC membrane at a controllable speed and reaches the absorbent pad because of the gravity and capillary force without active pumping. A smartphone is used to capture image from the NC membrane with its own camera and process the image with an intelligent algorithm of custom application software which is developed with Java. With a smartphone, the detection result can be displayed and transmitted to other medical agencies if necessary. Experimental results show that, compared with the traditional methods, more convenient and efficient influenza A detection can be achieved with the developed paper-based POC microfluidic chip with the assistance of smartphone.

A microfluidic device for antimicrobial susceptibility testing based on a broth dilution method

Bacterial resistance to antimicrobial compounds is increasing at a faster rate than the development of new antibiotics; this represents a critical challenge for clinicians worldwide. Normally, the minimum inhibitory concentration of an antibiotic, the dosage at which bacterial growth is thwarted, provides an effective quantitative measure for antimicrobial susceptibility testing, and determination of minimum inhibitory concentration is conventionally performed by either a serial broth dilution method or with the commercially available Etest® (Biomerieux, France) kit. However, these techniques are relatively labor-intensive and require a significant amount of training. In order to reduce human error and increase operation simplicity, a simple microfluidic device that can perform antimicrobial susceptibility testing automatically via a broth dilution method to accurately determine the minimum inhibitory concentration was developed herein. As a proof of concept, wild-type (ATCC 29212) and vancomycin-resistant Enterococcus cells were incubated at five different vancomycin concentrations on-chip, and the sample injection, transport, and mixing processes occurred within five reaction chambers and three reagent chambers via the chip's automatic dispensation and dilution functions within nine minutes. The minimum inhibitory concentration values measured after 24h of antibiotic incubation were similar to those calculated using Etest®. With its high flexibility, reliability, and portability, the developed microfluidic device provides a simple method for antimicrobial susceptibility testing in an automated format that could be implemented for clinical and point-of-care applications.

Fully automated molecular diagnosis by a novel cartridge-based platform

A novel cartridge-based platform was developed to perform fully automated sample preparation, amplification, and analysis of nucleic acids. The cartridge consists of three layers: multiple reagent chambers, a mixing reservoir, and elutes and waste chambers. The fluidic manipulation and solution mixing inside the cartridge were controlled by motor systems. After loading a sample specimen, each reagent solution in the chambers of the cartridge was sequentially injected into the mixing reservoir. Extracted nucleic acids were captured by magnetic beads and transferred into a small eluates chamber. Automated sample preparation and isothermal amplification of nucleic acids for pathogenic bacteria were demonstrated in this all-in one cartridge platform. This cartridge system provides rapid and reproducible sample preparation in comparison with manual methods. In addition, real-time detection of isothermal amplification using the implemented photometric system was compared with that of using a commercial polymerase chain reaction (PCR) device. This system has the potential to become a more reliable diagnostic tool for fast detection of contagious, foodborne, and emergency point-of-care (POC) diseases.

Abstract 367: Microfluidic devices for the interrogation of single circulating tumor cells

Abstract Introduction: Genetic and phenotypic characterization of Circulating Tumor Cells (CTC) offer the opportunity for a “real time liquid biopsy”. However, heterogeneity and rarity of CTC command the need for individual cell characterization. Following an enrichment procedure of CTC from blood, the identification, isolation and manipulation of single CTC for further analysis without cell loss remains challenging. Here, we present microfluidic devices for parallel single cell whole genome amplification (psc WGA) and parallel probing of drug response of single cancer cells (psc probing). Method: Microfluidic devices were designed using AUTOCAD software, and fabricated using PDMS multilayer soft-lithography. Cells from the SKBR-3 and MCF-7 breast cancer cell lines were used in the devices and identified using fluorescence microscopy after immunofluorescence staining. For pscWGA, the GE Single Cell GenomiPhi DNA Amplification kit was used under isothermal conditions. Results: In the 1st scWGA device, single cancer cells were addressed in 16 individual reaction chambers, subsequently lyzed, and their DNA amplified on a chip. We successfully amplified DNA of single cancer cells in a ca. 23 nanoliter reaction volume. 1,000-fold amplified DNAs were validated using qPCR targeting a set of genes on different chromosomes. For WGA of CTC present in a large number of other cells, we developed a 2nd scWGA platform by combining a self-sorting microwell cell sorter and a microfluidic device. After filtration of a cell suspension using a microwell plate, cancer cells were identified using fluorescence microscopy at the bottom of the plate. Cells of interest were subsequently punched into the open-well structures of the microfluidic device for further analysis. The lysis and WGA reaction buffers were loaded using peristaltic pumping of integrated micro-valves. After cell lysis, DNA was amplified in the open-well reaction chamber. For validation ∼ 100 ng of DNA was pipetted out of the well. We also developed microfluidic devices to study drug-dose response of single cancer cells. This device is capable of capturing single cells, dosing various concentrations of drugs and exposing the cells to the drugs. We optimized the capturing efficiency using different sizes of beads (3 μm, 6 μm, and 15 μm) as well as MCF-7 cells. We demonstrated that single cancer cells could be exposed to different drug candidates in the reagent chambers and their response measured. Conclusion: We successfully developed various microfluidic devices for individual cell characterization to be applied for CTC analysis. For genetic make-up, whole genome amplification of single cells either in suspension or in a self-sorting microwell plate was demonstrated. On-chip cell lysis and DNA amplification were performed and validated by qPCR targeting specific genes. In addition microfluidic devices were designed and tested to investigate single cell response to cancer drugs. Citation Format: Yoonsun Yang, Hoon Suk Rho, Joost F. Swennenhuis, Michiel Stevens, Arjan GJ Tibbe, Séverine Le Gac, Han Gardeniers, Leon WMM Terstappen. Microfluidic devices for the interrogation of single circulating tumor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 367. doi:10.1158/1538-7445.AM2015-367

Micro packaged MEMS pressure sensor for intracranial pressure measurement**Project supported by the National Natural Science Foundation of China (Nos. 61025021, 61434001), and the ‘Thousands Talents’ Program for Pioneer Researchers and Its Innovation Team, China.

This paper presents a micro packaged MEMS pressure sensor for intracranial pressure measurement which belongs to BioMEMS. It can be used in lumbar puncture surgery to measure intracranial pressure. Miniaturization is key for lumbar puncture surgery because the sensor must be small enough to allow it be placed in the reagent chamber of the lumbar puncture needle. The size of the sensor is decided by the size of the sensor chip and package. Our sensor chip is based on silicon piezoresistive effect and the size is 400 × 400 μm2. It is much smaller than the reported polymer intracranial pressure sensors such as liquid crystal polymer sensors. In terms of package, the traditional dual in-line package obviously could not match the size need, the minimal size of recently reported MEMS-based intracranial pressure sensors after packaging is 10 × 10 mm2. In this work, we are the first to introduce a quad flat no-lead package as the package form of piezoresistive intracranial pressure sensors, the whole size of the sensor is minimized to only 3 × 3 mm2. Considering the liquid measurement environment, the sensor is gummed and waterproof performance is tested; the sensitivity of the sensor is 0.9 × 10−2 mV/kPa.

Single-use thermoplastic microfluidic burst valves enabling on-chip reagent storage.

A simple and reliable method for fabricating single-use normally closed burst valves in thermoplastic microfluidic devices is presented, using a process flow that is readily integrated into established workflows for the fabrication of thermoplastic microfluidics. An experimental study of valve performance reveals the relationships between valve geometry and burst pressure. The technology is demonstrated in a device employing multiple valves engineered to actuate at different inlet pressures that can be generated using integrated screw pumps. On-chip storage and reconstitution of fluorescein salt sealed within defined reagent chambers are demonstrated. By taking advantage of the low gas and water permeability of cyclic olefin copolymer, the robust burst valves allow on-chip hermetic storage of reagents, making the technology well suited for the development of integrated and disposable assays for use at the point of care.

Generating arbitrary chemical patterns for multipoint dosing of single cells.

Living cells reside within anisotropic microenvironments that orchestrate a broad range of polarized responses through physical and chemical cues. To unravel how localized chemical signals influence complex behaviors, tools must be developed for establishing patterns of chemical gradients that vary over subcellular dimensions. Here, we present a strategy for addressing this critical need in which an arbitrary number of chemically distinct, subcellular dosing streams are created in real time within a microfluidic environment. In this approach, cells are cultured on a thin polymer membrane that serves as a barrier between the cell-culture environment and a reagent chamber containing multiple reagent species flowing in parallel under low Reynolds number conditions. Focal ablation of the membrane creates pores that allow solution to flow from desired regions within this reagent pattern into the cell-culture chamber, resulting in narrow, chemically distinct dosing streams. Unlike previous dosing strategies, this system provides the capacity to tailor arbitrary patterns of reagents on the fly to suit the geometry and orientation of specific cells.

Low-power microfluidic electro-hydraulic pump (EHP)

Low-power electrolysis-based microfluidic pumps utilizing the principle of hydraulics, integrated with microfluidic channels in polydimethylsiloxane (PDMS) substrates, are presented. The electro-hydraulic pumps (EHPs), consisting of electrolytic, hydraulic and fluidic chambers, were investigated using two types of electrodes: stainless steel for larger volumes and annealed gold electrodes for smaller-scale devices. Using a hydraulic fluid chamber and a thin flexible PDMS membrane, this novel prototype successfully separates the reagent fluid from the electrolytic fluid, which is particularly important for biological and chemical applications. The hydraulic advantage of the EHP device arises from the precise control of flow rate by changing the electrolytic pressure generated, independent of the volume of the reagent chamber, mimicking the function of a hydraulic press. Since the reservoirs are pre-filled with reagents and sealed prior to testing, external fluid coupling is minimized. The stainless steel electrode EHPs were manufactured with varying chamber volume ratios (1 : 1 to 1 : 3) as a proof-of-concept, and exhibited flow rates of 1.25 to 30 microl/min with electrolysis-based actuation at 2.5 to 10 V(DC). The miniaturized gold electrode EHPs were manufactured with 3 mm diameters and 1 : 1 chamber volume ratios, and produced flow rates of 1.24 to 7.00 microl/min at 2.5 to 10 V(AC), with a higher maximum sustained pressure of 343 KPa, suggesting greater device robustness using methods compatible with microfabrication. The proposed technology is low-cost, low-power and disposable, with a high level of reproducibility, allowing for ease of fabrication and integration into existing microfluidic lab-on-a-chip and analysis systems.

Abstract C21: Evaluation of an 8-plex cytokine immunoassay on the AVANTRA®, a novel automated biomarker workstation

Abstract Decision Biomarkers, Inc. (DBI) has developed a novel, automated, immunoassay platform to simultaneously evaluate multiple biomarkers in a single reaction vessel. Biomarker panels are used for many research and clinical indications. We have generated data from a multiplex assay measuring a panel of 8 cytokines: IL-1β, IL-2, IL-5, IL-6, Il-8, IL-10, IL-12, and TNFα. The heart of the system is a microarray of the biomarker specific monoclonal antibodies spotted onto a glass slide which has been coated with an ultra-thin, two-dimensional film of nitrocellulose. This microarray is assembled in a plastic biochip (MAX BIOCHIP™): a self-contained device with a series of micro fluidic channels, valves, sensors, and reagent chambers containing dried, biotinylated detection antibodies, streptavidin-conjugated Cy3, and wash buffer. The biochip is processed in the AVANTRA® biomarker workstation following injection of 200 µl of sample (100 µl of plasma diluted in 100 µl of PBS). The instrument initiates, in sequence, the flow of sample, detection antibody, label, and buffer, across the array. The end result is in a fluorescent labeled complex that is then imaged by an internal CCD camera. Spot intensity is correlated with analyte concentration by instrument software. “Master” curves” electronically stored in the instrument are used to calculate analyte concentrations through a 2-point calibration scheme. Each cytokine assay has a 4-log dynamic range and individual analytical sensitivities ranging between 2.7 and 20 pg/ml. Both the intra- and interassay precision results yielded mean CV's consistently &amp;lt;10% Reproducibility was demonstrated by overlapping curves following replicate standard evaluation. The unique surface chemistry, marker specific reagents, and enhanced biochip micro fluidics and instrument automation, all combine to produce a robust and easy to use multiplex immunoassay. Currently in development are a 10-plex angiogenesis panel and a 12-plex cardiac panel that will soon be available. Additionally, assays for numerous other biomarker panels are slated for future development. Custom assay development is also available. In summary, DBI has developed a sensitive and precise, multiplex immunoassay for 8 cytokines that can run on its new, automated platform, the AVANTRA® biomarker workstation. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C20.