Microfluidic Array Research Articles

Microfluidic Flow Cell Array for Controlled Cell Deposition in Engineered Musculoskeletal Tissues.

Musculoskeletal tissues contain critical gradients in extracellular matrix (ECM) composition and cell types that allow for proper mechanical function of tissues and integration between adjacent tissues. However, properly controlling these patterns in engineered tissues is difficult and tissue engineering (TE) is presently in need of methods to generate integration zones for tissue anchoring, transition zones between tissues, and controlled ECM gradients for proper mechanical function. In this study, we present a novel method of using a microfluidic flow cell array (MFCA) to precisely control cell deposition onto TE constructs to produce tunable cell patterns on engineered constructs. In this study, we characterized MFCA cell deposition to efficiently and reliably deposit cells in controllable patterns and densities. We developed methods for deposition of human adipose-derived stem cells and human osteoblasts using a 12-channel pilot printhead. We mimicked key gradients and transitions by creating two-cell and three-cell-type transitions characteristic of the integration zones of musculoskeletal tissues. Overall, we demonstrate the ability to precisely and reproducibly control cell deposition on engineered constructs using this method and control cell population gradients. We establish the production of multicell transitions and multicell interfaces utilizing MFCA cell deposition, to demonstrate the potential of the method to create an extensive variety of engineered musculoskeletal tissues. Furthermore, customization of the printhead design can accommodate various structures, sizes, shapes, and number of flow cell channels to meet specific requirements for a broad range of musculoskeletal tissues.

(Invited) Electrochemical Measurement Approaches for Planetary Exploration

2018 marks the 10-year anniversary of the landing of the NASA Phoenix spacecraft on the ice-rich northern plains of Mars. The Phoenix Lander carried the Wet Chemistry Laboratory (WCL), a component instrument of the Microscopy, Electrochemistry, and Conductivity Analyzer (MECA) and was used to perform a comprehensive electrochemical characterization of martian soil in an aqueous solution. Designed to characterize the habitability of the martian surface, WCL contained an array of ion selective electrodes, a conductivity cell, a platinum electrode to measure redox potential, electrodes to perform perform cyclic voltammetry, chronopotentiometry and anodic stripping voltammetry, as well as a set of chemical reagents used to perform a sulfate titration and sample acidification. The measurements made with WCL resulted in a number of major discoveries, including the detection of percent levels of perchlorate on the martian surface, and represent the first, and to date, the only time electrochemical methods have been used for planetary exploration beyond Earth. NASA is now embarking on a new era of astrobiology exploration as plans are being formulated for missions to Ocean Worlds of the outer solar system, including Europa, an ice covered Jovian moon. Based on lessons learned during the development of WCL and its operation on the surface of Mars, we will discuss new design approaches and strategies that will enable the implementation of autonomous microfluidic electrochemical sensor arrays during upcoming missions to these yet to be explored solar system environments.

Integrated Bacterial Identification and Antimicrobial Susceptibility Testing for Polymicrobial Infections Using Digital PCR and Digital High-Resolution Melt in a Microfluidic Array Platform.

In diagnosing bacterial infection, rapid bacterial identification (ID) and antimicrobial susceptibility testing (AST) are critical to clinicians in order to provide an effective treatment in a timely manner. The gold standard, culture-based approach provides both ID and antimicrobial susceptibility but requires several days of turnaround time. Especially in polymicrobial infections, where there are more than one organisms interacting collectively that can complicate the treatment. Here, we demonstrate a rapid bacterial diagnostic approach that is capable of bacterial ID/AST in heterogeneous samples within less than 4 hours by using digital PCR (dPCR) and digital high-resolution melt via microfluidic devices. By utilizing dPCR, we are able to quantify amount of nucleic acid, which correlates to phenotypic responses of {\bf individual pathogens in a mixed sample and also shorten the required time of antibiotic exposure. In addition, we employ a machine learning algorithm to automatically identify bacterial species based on melt profiles of 16S rRNA gene.

Note: Multi-sheet light enables optical interference lithography

We propose and demonstrate a modified spatial filter-based single-shot lithography technique for fabricating an array of microfluidic channels. This is achieved by illuminating the photopolymer specimen with a multiple light sheet (MLS) pattern. Modified spatial filtering is employed in a cylindrical lens system to generate the MLS pattern. The transmission window [the difference (α - β) angle] of the spatial filter determines the characteristics of the pattern and the fabricated microfluidic channel array. After exposing to a negative photoresist (DPHPA monomer with rose bengal as the photoinitiator), this gives rise to an array of micro-fluidic channels (post development process). We studied the effect of micro-channel geometry (channel width, inter-channel separation, and aspect ratio) for varying exposure times that show near-linear dependence. The results show that the fabricated array has 7 prominent channels with an individual channel width and inter-channel separation of approximately 5 μm and 12 μm, respectively. The proposed technique enables selective plane patterning and reduces the overall cost for large-scale production.

Automated 3D-Printed Microfluidic Array for Rapid Nanomaterial-Enhanced Detection of Multiple Proteins.

We report here the fabrication and validation of a novel 3D-printed, automated immunoarray to detect multiple proteins with ultralow detection limits. This low cost, miniature immunoarray employs electrochemiluminescent (ECL) detection measured with a CCD camera and employs touch-screen control of a micropump to facilitate automated use. The miniaturized array features prefilled reservoirs to deliver sample and reagents to a paper-thin pyrolytic graphite microwell detection chip to complete sandwich immunoassays. The detection chip achieves high sensitivity by using single-wall carbon nanotube-antibody conjugates in the microwells and employing massively labeled antibody-decorated RuBPY-silica nanoparticles to generate ECL. The total cost of an array is $0.65, and an eight-protein assay can be done in duplicate for $0.14 per protein with limits of detection (LOD) as low as 78-110 fg mL-1 in diluted serum. The electronic control system costs $210 in components. Utility of the automated immunoarray was demonstrated by detecting an eight-protein prostate cancer biomarker panel in human serum samples in 25 min. The system is well suited to future clinical and point-of-care diagnostic testing and could be used in resource-limited environments.

Autonomous Magnetic Microrobots by Navigating Gates for Multiple Biomolecules Delivery.

The precise delivery of biofunctionalized matters is of great interest from the fundamental and applied viewpoints. In spite of significant progress achieved during the last decade, a parallel and automated isolation and manipulation of rare analyte, and their simultaneous on-chip separation and trapping, still remain challenging. Here, a universal micromagnet junction for self-navigating gates of microrobotic particles to deliver the biomolecules to specific sites using a remote magnetic field is described. In the proposed concept, the nonmagnetic gap between the lithographically defined donor and acceptor micromagnets creates a crucial energy barrier to restrict particle gating. It is shown that by carefully designing the geometry of the junctions, it becomes possible to deliver multiple protein-functionalized carriers in high resolution, as well as MCF-7 and THP-1 cells from the mixture, with high fidelity and trap them in individual apartments. Integration of such junctions with magnetophoretic circuitry elements could lead to novel platforms without retrieving for the synchronous digital manipulation of particles/biomolecules in microfluidic multiplex arrays for next-generation biochips.

Fully 3D printed integrated reactor array for point-of-care molecular diagnostics

Molecular diagnostics that involve nucleic acid amplification tests (NAATs) are crucial for prevention and treatment of infectious diseases. In this study, we developed a simple, inexpensive, disposable, fully 3D printed microfluidic reactor array that is capable of carrying out extraction, concentration and isothermal amplification of nucleic acids in variety of body fluids. The method allows rapid molecular diagnostic tests for infectious diseases at point of care. A simple leak-proof polymerization strategy was developed to integrate flow-through nucleic acid isolation membranes into microfluidic devices, yielding a multifunctional diagnostic platform. Static coating technology was adopted to improve the biocompatibility of our 3D printed device. We demonstrated the suitability of our device for both end-point colorimetric qualitative detection and real-time fluorescence quantitative detection. We applied our diagnostic device to detection of Plasmodium falciparum in plasma samples and Neisseria meningitides in cerebrospinal fluid (CSF) samples by loop-mediated, isothermal amplification (LAMP) within 50 min. The detection limits were 100 fg for P. falciparum and 50 colony-forming unit (CFU) for N. meningitidis per reaction, which are comparable to that of benchtop instruments. This rapid and inexpensive 3D printed device has great potential for point-of-care molecular diagnosis of infectious disease in resource-limited settings.

Deterministic Capture of Individual Circulating Tumor Cells Using a Flow-Restricted Microfluidic Trap Array.

Circulating tumor cells (CTCs) are regarded as a strong biomarker which includes clinically valuable information. However, CTCs are very rare and require precise separation and detection for effective clinical applications. Furthermore, downstream analysis has become necessary to identify the distinct sub-population of CTCs that causes metastasis. Here, we report a flow-restricted microfluidic trap array capable of deterministic single-cell capture of CTCs. The extent of flow restriction, correlating with the device geometry, was then optimized using a highly invasive breast cancer cell line (LM2 MDA-MB-231) to achieve 97% capture efficiency with a single-cell capture rate of 99%. Single-cell capture of CTCs from mice with full-blown metastasis was also demonstrated. The single-CTC capturing ability of the flow-restricted trap array not only showed cell enumerating ability but also high prospects for application in future automated downstream analysis.

Neuregulin 1 discovered as a cleavage target for the HCV NS3/4A protease by a microfluidic membrane protein array

The hepatitis C virus (HCV) non-structural protein 3 (NS3) is essential for HCV maturation. The NS3/4A protease is a target for several HCV treatments and is a well-known target for HCV drug discovery. The protein is membrane associated and thus probably interacts with other membrane proteins. However, the vast majority of known NS3 host partners are soluble proteins rather than membrane proteins, most likely due to lack of appropriate platforms for their discovery. Utilization of an integrated microfluidics platform enables analysis of membrane proteins in their native form. We screened over 2800 membrane proteins for interaction with NS3 and 90 previously unknown interactions were identified. Of these, several proteins were selected for validation by co-immunoprecipitation and for NS3 proteolytic activity. Bearing in mind the considerable number of interactions formed, together with the popularity of NS3/4A protease as a drug target, it was striking to note its lack of proteolytic activity. Only a single protein, Neuregulin1, was observed to be cleaved, adding to the 3 known NS3/4A cleavage targets. Neuregulin1 participates in neural proliferation. Recent studies have shown its involvement in HCV infection and hepatocellular carcinoma. We showed that NS3/4A triggers an increase in neuregulin1 mRNA levels in HCV infected cells. Despite this increase, its protein concentration is decreased due to proteolytic cleavage. Additionally, its EGF-like domain levels were increased, possibly explaining the ErbB2 and EGFR upregulation in HCV infected cells. The newly discovered protein interactions may provide insights into HCV infection mechanisms and potentially provide new therapeutic targets against HCV.

Microfluidic Droplet Array as Optical Irises Actuated via Electrowetting

Initiated by a task in tunable microoptics, but not limited to this application, a microfluidic droplet array in an upright standing module with 3 × 3 subcells and droplet actuation via electrowetting is presented. Each subcell is filled with a single (of course transparent) water droplet, serving as a movable iris, surrounded by opaque blackened decane. Each subcell measures 1 × 1 mm2 and incorporates 2 × 2 quadratically arranged positions for the droplet. All 3 × 3 droplets are actuated synchronously by electrowetting on dielectric (EWOD). The droplet speed is up to 12 mm/s at 130 V (Vrms) with response times of about 40 ms. Minimum operating voltage is 30 V. Horizontal and vertical movement of the droplets is demonstrated. Furthermore, a minor modification of the subcells allows us to exploit the flattening of each droplet. Hence, the opaque decane fluid sample can cover each water droplet and render each subcell opaque, resulting in switchable irises of constant opening diameter. The concept does not require any mechanically moving parts or external pumps.

Up-regulated Pro-inflammatory MicroRNAs (miRNAs) in Alzheimer's disease (AD) and Age-Related Macular Degeneration (AMD).

Alzheimer's disease (AD) of the brain neocortex and age-related macular degeneration (AMD) of the retina are two complex neurodegenerative disorders, which (i) involve the progressive dysregulation and deterioration of multiple neurobiological signaling pathways, (ii) exhibit the temporal accumulation of pro-inflammatory lesions including the amyloid beta (Aβ) peptide-containing senile plaques of AD and the drusen of AMD, and (iii) culminate in an insidious inflammatory neurodegeneration ending, respectively, in neural cell atrophy and death and progressive loss of cognition and central visual function. Recent independent research studies have indicated that AD and AMD share common, pathological signaling defects and disease mechanisms at the molecular genetic level. Using high-integrity total RNA samples pooled from AD brain and AMD retina, microfluidic hybridization miRNA arrays, and bioinformatics, the current study was undertaken to quantify microRNA (miRNA) speciation and complexity common to both AD and AMD. These small non-coding (sncRNAs) are known to post-transcriptionally regulate multiple neurobiological pathways and an abundance of research information has already been generated on the roles of these miRNAs in pathological situations involving inflammatory neuropathology and neural cell decline. Here, for the first time, we report the sequence and abundance of a septet of sncRNAs including miRNA-7, miRNA-9-1, miRNA-23a/miRNA-27a, miRNA-34a, miRNA-125b-1, miRNA-146a, and miRNA-155 that are significantly increased in abundance and common to both AD-affected superior temporal lobe neocortex (Brodmann A22) and the AMD-affected macular region of the retina. Bioinformatics, miRNA-mRNA complementarity, next-gen RNA sequencing, and feature alignment analysis further indicate that these 7 up-regulated miRNAs have the potential to interact with and down-regulate~9460 target messenger RNAs (mRNAs; about 3.5% of the genome) involved in the synchronization of amyloid production and clearance, phagocytosis, innate-immune, pro-inflammatory, and neurotrophic signaling and/or synaptogenesis in diseased tissues.

Rapid and label-free identification of single leukemia cells from blood in a high-density microfluidic trapping array by fluorescence lifetime imaging microscopy.

The rapid screening and isolation of single leukemia cells from blood has become critical for early leukemia detection and tumor heterogeneity interrogation. However, due to the size overlap between leukemia cells and the more abundant white blood cells (WBCs), the isolation and identification of leukemia cells individually from peripheral blood is extremely challenging and often requires immunolabeling or cytogenetic assays. Here we present a rapid and label-free single leukemia cell identification platform that combines: (1) high-throughput size-based separation of hemocytes via a single-cell trapping array, and (2) leukemia cell identification through phasor approach and fluorescence lifetime imaging microscopy (phasor-FLIM), to quantify changes between free/bound nicotinamide adenine dinucleotide (NADH) as an indirect measurement of metabolic alteration in living cells. The microfluidic trapping array designed with 1600 highly-packed addressable single-cell traps can simultaneously filter out red blood cells (RBCs) and trap WBCs/leukemia cells, and is compatible with low-magnification imaging and fast-speed fluorescence screening. The trapped single leukemia cells, e.g., THP-1, Jurkat and K562 cells, are distinguished from WBCs in the phasor-FLIM lifetime map, as they exhibit significant shift towards shorter fluorescence lifetime and a higher ratio of free/bound NADH compared to WBCs, because of their glycolysis-dominant metabolism for rapid proliferation. Based on a multiparametric scheme comparing the eight parameter-spectra of the phasor-FLIM signatures, spiked leukemia cells are quantitatively distinguished from normal WBCs with an area-under-the-curve (AUC) value of 1.00. Different leukemia cell lines are also quantitatively distinguished from each other with AUC values higher than 0.95, demonstrating high sensitivity and specificity for single cell analysis. The presented platform is the first to enable high-density size-based single-cell trapping simultaneously with RBC filtering and rapid label-free individual-leukemia-cell screening through non-invasive metabolic imaging. Compared to conventional biomolecular diagnostics techniques, phasor-FLIM based single-cell screening is label-free, cell-friendly, robust, and has the potential to screen blood in clinical volumes through parallelization.

Accuracy of serum-specific IgE test with microfluidic array enzyme-linked immunosorbent assay for diagnosing inhalant allergen sensitization in asthma and/or rhinitis allergy patients in Jakarta, Indonesia.

BackgroundAsthma and allergic rhinitis are a global health burden. Inhalant allergens worsen the symptoms and clinical manifestations of asthma and allergic rhinitis. Skin prick test is the gold standard for diagnosing allergen sensitization but is associated with some limitations. In contrast, in vitro serum-specific immunoglobulin E (SSIgE) test is convenient and is not associated with an anaphylactic risk.ObjectiveThe present study compared the accuracy of the SSIgE test by using microfluidic array enzyme-linked immunosorbent assay (ELISA) with that of the skin prick test for diagnosing inhalant allergen sensitization in patients with asthma and/or allergic rhinitis.MethodsThis diagnostic study included patients with asthma and/or allergic rhinitis. Of these, 100 patients underwent the SSIgE test for diagnosing sensitization to house dust mites (Dermatophagoides pteronyssinus, Dermatophagoides farinae, and Blomia tropicalis), dog dander, cat dander, and cockroach allergen. All the patients also underwent the skin prick test for diagnosing allergen sensitization. The sensitivity, specificity, predictive value, and likelihood ratio (LR) of the SSIgE test were evaluated for each allergen.ResultsSensitivity of the SSIgE test for diagnosing house dust mite sensitization was 48%–77%, with the highest sensitivity (77%) observed for diagnosing D. farinae sensitization. Specificity of the SSIgE test for diagnosing house dust mite sensitization was 64%–95%, with the highest specificity (95%) observed for diagnosing B. tropicalis sensitization. Although the SSIgE test showed high specificity and LR+ for diagnosing cockroach allergen sensitization, it showed low sensitivity (12%). Moreover, the SSIgE test showed high specificity (89%) but low sensitivity (3%) for diagnosing dog dander sensitization and high specificity (88%) but low sensitivity (10%) for diagnosing cat dander sensitization.ConclusionThe SSIgE test using microfluidic array ELISA shows moderate accuracy for diagnosing house dust mite sensitization and low accuracy for diagnosing cockroach allergen and dog and cat dander sensitization.

A novel and accurate microfluidic assay of CD62L in bladder cancer serum samples.

We report a low-cost, sensitive, bead-based electrochemical immunoarray for soluble L-selectin (or CD62L protein), a potential biomarker for staging bladder cancer. We used a semi-automated modular microfluidic array with online antigen capture on superparamagnetic beads, which were subsequently delivered to a detection chamber housing multiple sensors. The assay was designed to accurately detect CD62L in diluted serum with a limit of detection (LOD) of 0.25 ng mL-1 and a dynamic range of 0.25-100 ng mL-1. The microfluidic array gave significantly better accuracy and higher sensitivity than a standard ELISA kit, which was shown to be subject to significant systematic error at high and low concentration ranges. 31 serum samples from patients with varying grades of bladder cancer and cancer-free controls were analyzed by the immunoarray and ELISA, and the CD62L levels correlated. This work establishes a new accurate assay for determining CD62L levels and highlights the potential of this protein as a biomarker for detecting locoregional progression of bladder cancer.

Construction of Tumor Tissue Array on An Open-Access Microfluidic Chip

Abstract An open-access microfluidic chip which enabled automatic cell distribution and complex multi-step operations was developed. The microfluidic chip featured a key structure in which a nanoporous membrane was sandwiched by a cell culture chamber array layer and a corresponding media reservoir array layer. The microfluidic approach took advantage of the characteristics of nanoporous membrane. On one side, this membrane permitted the flow of air but not liquid, thus acting as a flow-stop valve to enable automatic cell distribution. On the other side, it allowed diffusion-based media exchange and thus, mimicked the endothelial layer. In synergy with a liquid transferring platform, the open-access microfluidic system enabled complex multi-step operations involving medium exchange, drug treatment, and cell viability testing. By using this microfluidic protocol, a 10 × 10 tissue arrays was constructed in 90 s, followed by schedule-dependent drug testing. Morphological and immunohistochemical assays results indicated that the resultant tumor tissue was faithful to that in vivo. Drug testing assays showed that the microfluidic tissue array promised multi-step cell assays under biomimetic microenvironment, thus providing an advantageous tool for cell research.

A microfluidic chip capable of generating and trapping emulsion droplets for digital loop-mediated isothermal amplification analysis.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that rapidly amplifies specific DNA molecules at high yield. In this study, a microfluidic droplet array chip was designed to execute the digital LAMP process. The novel device was capable of 1) creating emulsion droplets, 2) sorting them into a 30 × 8 droplet array, and 3) executing LAMP across the 240 trapped and separated droplets (with a volume of 0.22 nL) after only 40 min of reaction at 56 °C. Nucleic acids were accurately quantified across a dynamic range of 50 to 2.5 × 103 DNA copies per μL, and the limit of detection was a single DNA molecule. This is the first time that an arrayed emulsion droplet microfluidic device has been used for digital LAMP analysis. When compared to microwell digital nucleic acid amplification assays, this droplet array-based digital LAMP assay eliminates the constraint on the size of the digitized target, which was determined by the dimension of the microwells for its counterparts. Moreover, the capacity for hydrodynamic droplet trapping allows the chip to operate in a one-droplet-to-one-trap manner. This microfluidic chip may therefore become a promising device for digital LAMP-based diagnostics in the near future.

High-throughput microfluidic single-cell trapping arrays for biomolecular and imaging analysis.

Single-cell analysis is of critical importance in revealing population heterogeneity, identifying minority sub-populations of interest, as well as discovering unique characteristics of individual cells. Microfluidic platforms work at the scale comparable to cell diameter and is suitable for single-cell manipulation. Here we present a microfluidic trapping array which is able to rapidly and deterministically trap single-cells in highly-packed microwells. This chapter first describes the design and fabrication protocols of the trapping array, and then presents its two representative applications: single-cell mRNA probing when integrated with a dielectrophoretic nanotweezer (DENT), and live-cell real-time imaging when combined with fluorescence lifetime imaging microscopy (FLIM). As the single-cell trapping efficiency is determined by the channel design instead of the flow rate, this trapping array can be coupled with different microfluidic sample processing units with different flow rates for various single-cell analyses.

Colloidal transport within nematic liquid crystals with arrays of obstacles.

We have investigated the gravity-driven transport of spherical colloids suspended in the nematic liquid crystal 4-cyano-4'-pentylbiphenyl (5CB) within microfluidic arrays of cylindrical obstacles arranged in a square lattice. Homeotropic anchoring at the surfaces of the obstacles created periodic director-field patterns that strongly influenced the motion of the colloids, whose surfaces had planar anchoring. When the gravitational force was oriented parallel to a principal axis of the lattice, the particles moved along channels between columns of obstacles and displayed pronounced modulations in their velocity. Quantitative analysis indicates that this modulation resulted from a combination of a spatially varying effective drag viscosity and elastic interactions engendered by the periodic director field. The interactions differed qualitatively from a sum of pair-wise interactions between the colloids and isolated obstacles, reflecting the distinct nematic environment created by confinement within the array. As the angle α between the gravitational force and principal axis of the lattice was varied, the velocity did not follow the force but instead locked into a discrete set of directions commensurate with the lattice. The transitions between these directions occurred at values of α that were different from those observed when the spheres were in an isotropic liquid, indicating the ability of the liquid crystal forces to tune the lateral displacement behavior in such devices.

Rapid detection of multiple respiratory viruses based on microfluidic isothermal amplification and a real-time colorimetric method.

Respiratory viruses are major threats causing development of acute respiratory tract infections, which are common causes of illness and death throughout the world. Here, an integrated microsystem based on real-time colorimetry was developed for diagnosing multiple respiratory viruses. The microsystem employed magnetic beads for nucleic acid extraction and an eight-channel microfluidic array chip integrated with a loop-mediated isothermal amplification system for point-of-care screening of respiratory viruses. The overall detection process (including sample collection, nucleic acid extraction, sample loading, real-time detection, and signal output) could be completed within 1 h. Our results show that the developed method could specifically recognize influenza A virus subtypes (H1N1, H3N2, H5N1, and H7N9), influenza B virus, and human adenoviruses. The results obtained with 109 clinical samples indicate that the developed method has high specificity (100%, confidence interval 94.9-100.0) and sensitivity (96%, confidence interval 78.1-99.9). The integration of magnetic bead-based pre-treatment techniques and microfluidic isothermal amplification provides an effective solution for rapidly detecting etiological agents of respiratory diseases. The strategy of using a closed chip system and real-time colorimetry reduced aerosol contamination and ensured the accuracy of the results. The developed method provides an effective alternative for rapid point-of-care screening for viruses that cause respiratory disease syndromes and further aids in accurate and timely detection to control and prevent the spread of respiratory diseases caused by such pathogens.

Automated 4-Sample Protein Immunoassays using 3D-Printed Microfluidics.

Low cost, miniaturized assay platforms that work with small sample volumes, high sensitivity and rapid detection will have high value in future biomolecular diagnostics. Herein we report an automated, 3D printed electrochemiluminescent (ECL) immunoarray integrated with a nanostructured pyrolytic graphite sheet (PGS) microwell chip configured to detect 2 proteins simultaneously from complex liquid samples with high sensitivity and selectivity. Assays are done in 18 min at cost of < $1.00 using 1-2 microliters of sample. 3D printed microfluidic array design integrates reagent and sample chambers with rapid ECL detection. A commercial programmable syringe pump used with a preset program allows pump to pause and resume reagent delivery as required for completion of the sandwich immunoassays. Nanostructured surfaces feature antibody-decorated single wall carbon nanotube forests on PGS chip microwells, and sensitivity is amplified via massively labeled RuBPY-silica nanoparticles for detection. Prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA) were measured simultaneously from human serum on the immunoarray with detection limits 150 fg mL-1 for PSA and 230 fg mL-1 for PSMA, with dynamic ranges up to 5 ng mL-1. Validation of the immunoarray by measuring these proteins in human serum showed good correlation with single protein ELISA. These 3D printed platforms can be easily adapted to multiple applications and configurable CAD files for the immunoarray can be downloaded from our lab's website.