Mass-producible microporous silicon membranes for specific leukocyte subset isolation, immunophenotyping, and personalized immunomodulatory drug screening in vitro.

Widespread commercial and clinical adaptation of biomedical microfluidic technology has been limited in large part due to the lack of mass producibility of polydimethylsiloxane (PDMS) and glass-based devices commonly as reported in the literature. Here, we present a batch-fabricated, robust, and mass-producible immunophenotyping microfluidic device using silicon micromachining processes. Our Si and glass-based microfluidic device, named the silicon microfluidic immunophenotyping assay (SiMIPA), consists of a highly porous (∼40%) silicon membrane that can selectively separate microparticles below a certain size threshold. The device is capable of isolating and stimulating specific leukocyte populations, and allows for measuring their secretion of cell signaling proteins by means of a no-wash homogeneous chemiluminescence-based immunoassay. The high manufacturing throughput (∼170 devices per wafer) makes a large quantity of SiMIPA chips readily available for clinically relevant applications, which normally require large dataset acquisitions for statistical accuracy. With 30 SiMIPA chips, we performed in vitro immunomodulatory drug screening on isolated leukocyte subsets, yielding 5 data points at 6 drug concentrations. Furthermore, the excellent structural integrity of the device allowed for samples and reagents to be loaded using a micropipette, greatly simplifying the experimental protocol.

Belt-Mounted Micro-Gas-Chromatograph Prototype for Determining Personal Exposures to Volatile-Organic-Compound Mixture Components.

We describe a belt-mountable prototype instrument containing a gas chromatographic microsystem (µGC) and demonstrate its capability for near-real-time recognition and quantification of volatile organic compounds (VOCs) in moderately complex mixtures at concentrations encountered in industrial workplace environments. The µGC comprises three discrete, Si/Pyrex microfabricated chips: a dual-adsorbent micropreconcentrator-focuser for VOC capture and injection; a wall-coated microcolumn with thin-metal heaters and temperature sensors for temperature-programmed separations; and an array of four microchemiresistors with thiolate-monolayer-protected-Au-nanoparticle interface films for detection and recognition-discrimination. The battery-powered µGC prototype (20 × 15 × 9 cm, ∼2.1 kg sans battery) has on-board microcontrollers and can autonomously analyze the components of a given VOC mixture several times per hour. Calibration curves bracketing the Threshold Limit Value (TLV) of each VOC yielded detection limits of 16-600 parts-per-billion for air samples of 5-10 mL, well below respective TLVs. A 2:1 injection split improved the resolution of early eluting compounds by up to 63%. Responses and response patterns were stable for 5 days. Use of retention-time windows facilitated the chemometric recognition and discrimination of the components of a 21-VOC mixture sampled and analyzed in 3.5 min. Results from a "mock" field test, in which personal exposures to time-varying concentrations of a mixture of five VOCs were measured autonomously, agreed closely with those from a reference GC. Thus, reliable, near-real-time determinations of worker exposures to multiple VOCs with this wearable µGC prototype appear feasible.

Efficient molecular evolution to generate enantioselective enzymes using a dual-channel microfluidic droplet screening platform.

Directed evolution has long been a key strategy to generate enzymes with desired properties like high selectivity, but experimental barriers and analytical costs of screening enormous mutant libraries have limited such efforts. Here, we describe an ultrahigh-throughput dual-channel microfluidic droplet screening system that can be used to screen up to ~107 enzyme variants per day. As an example case, we use the system to engineer the enantioselectivity of an esterase to preferentially produce desired enantiomers of profens, an important class of anti-inflammatory drugs. Using two types of screening working modes over the course of five rounds of directed evolution, we identify (from among 5 million mutants) a variant with 700-fold improved enantioselectivity for the desired (S)-profens. We thus demonstrate that this screening platform can be used to rapidly generate enzymes with desired enzymatic properties like enantiospecificity, chemospecificity, and regiospecificity.

AC Electroosmosis-Enhanced Nanoplasmofluidic Detection of Ultralow-Concentration Cytokine.

Label-free, nanoparticle-based plasmonic optical biosensing, combined with device miniaturization and microarray integration, has emerged as a promising approach for rapid, multiplexed biomolecular analysis. However, limited sensitivity prevents the wide use of such integrated label-free nanoplasmonic biosensors in clinical and life science applications where low-abundance biomolecule detection is needed. Here, we present a nanoplasmofluidic device integrated with microelectrodes for rapid, label-free analysis of a low-abundance cell signaling protein, detected by AC electroosmosis-enhanced localized surface plasmon resonance (ACE-LSPR) biofunctional nanoparticle imaging. The ACE-LSPR device is constructed using both bottom-up and top-down sensor fabrication methods, allowing the seamless integration of antibody-conjugated gold nanorod (AuNR) biosensor arrays with microelectrodes on the same microfluidic platform. Applying an AC voltage to microelectrodes while scanning the scattering light intensity variation of the AuNR biosensors results in significantly enhanced biosensing performance. The AC electroosmosis (ACEO) based enhancement of the biosensor performance enables rapid (5-15 min) quantification of IL-1ß, a pro-inflammatory cytokine biomarker, with a sensitivity down to 158.5 fg/mL (9.1 fM) for spiked samples in PBS and 1 pg/mL (58 fM) for diluted human serum. Together with the optimized detection sensitivity and speed, our study presents the first critical step toward the application of nanoplasmonic biosensing technology to immune status monitoring guided by low-abundance cytokine measurement.

Fully Automated Portable Comprehensive 2-Dimensional Gas Chromatography Device.

We developed a fully automated portable 2-dimensional (2-D) gas chromatography (GC x GC) device, which had a dimension of 60 cm × 50 cm × 10 cm and weight less than 5 kg. The device incorporated a micropreconcentrator/injector, commercial columns, micro-Deans switches, microthermal injectors, microphotoionization detectors, data acquisition cards, and power supplies, as well as computer control and user interface. It employed multiple channels (4 channels) in the second dimension (2D) to increase the 2D separation time (up to 32 s) and hence 2D peak capacity. In addition, a nondestructive flow-through vapor detector was installed at the end of the 1D column to monitor the eluent from 1D and assist in reconstructing 1D elution peaks. With the information obtained jointly from the 1D and 2D detectors, 1D elution peaks could be reconstructed with significantly improved 1D resolution. In this Article, we first discuss the details of the system operating principle and the algorithm to reconstruct 1D elution peaks, followed by the description and characterization of each component. Finally, 2-D separation of 50 analytes, including alkane (C6-C12), alkene, alcohol, aldehyde, ketone, cycloalkane, and aromatic hydrocarbon, in 14 min is demonstrated, showing the peak capacity of 430-530 and the peak capacity production of 40-80/min.

Low-Power Miniaturized Helium Dielectric Barrier Discharge Photoionization Detectors for Highly Sensitive Vapor Detection.

This paper presents the design, fabrication, and characterization of a microhelium dielectric barrier discharge photoionization detector (µHDBD-PID) on chip with dimensions of only ∼15 mm × âˆ¼10 mm × âˆ¼0.7 mm and weight of only ∼0.25 g. It offers low power consumption (<400 mW), low helium consumption (5.8 mL/min), rapid response (up to ∼60 ms at a flow rate of 1.5 mL/min), quick warm-up time (∼5 min), an excellent detection limit (a few picograms), a large linear dynamic range (>4 orders of magnitude), and maintenance-free operation. Furthermore, the µHDBD-PID can be driven with a miniaturized (∼5 cm × âˆ¼2.5 cm × âˆ¼2.5 cm), light (22 g), and low cost (∼$2) power supply with only 1.5 VDC input. The dependence of the µHDBD-PID performance on bias voltage, auxiliary helium flow rate, carrier gas flow rate, and temperature was also systematically investigated. Finally, the µHDBD-PID was employed to detect permanent gases and a sublist of the EPA 8260 standard reagents that include 51 analytes. The µHDBD-PID developed here can have a broad range of applications in portable and microgas chromatography systems for in situ, real-time, and sensitive gas analysis.

Multiplexed Nanoplasmonic Temporal Profiling of T-Cell Response under Immunomodulatory Agent Exposure.

Immunomodulatory drugs-agents regulating the immune response-are commonly used for treating immune system disorders and minimizing graft versus host disease in persons receiving organ transplants. At the cellular level, immunosuppressant drugs are used to inhibit pro-inflammatory or tissue-damaging responses of cells. However, few studies have so far precisely characterized the cellular-level effect of immunomodulatory treatment. The primary challenge arises due to the rapid and transient nature of T-cell immune responses to such treatment. T-cell responses involve a highly interactive network of different types of cytokines, which makes precise monitoring of drug-modulated T-cell response difficult. Here, we present a nanoplasmonic biosensing approach to quantitatively characterize cytokine secretion behaviors of T cells with a fine time-resolution (every 10 min) that are altered by an immunosuppressive drug used in the treatment of T-cell-mediated diseases. With a microfluidic platform integrating antibody-conjugated gold nanorod (AuNR) arrays, the technique enables simultaneous multi-time-point measurements of pro-inflammatory (IL-2, IFN-γ, and TNF-α) and anti-inflammatory (IL-10) cytokines secreted by T cells. The integrated nanoplasmonic biosensors achieve precise measurements with low operating sample volume (1 µL), short assay time (∼30 min), heightened sensitivity (∼20-30 pg/mL), and negligible sensor crosstalk. Data obtained from the multicytokine secretion profiles with high practicality resulting from all of these sensing capabilities provide a comprehensive picture of the time-varying cellular functional state during pharmacologic immunosuppression. The capability to monitor cellular functional response demonstrated in this study has great potential to ultimately permit personalized immunomodulatory treatment.

In situ calibration of micro-photoionization detectors in a multi-dimensional micro-gas chromatography system.

A photoionization detector (PID) is widely used as a gas chromatography (GC) detector. By virtue of its non-destructive nature, multiple PIDs can be used in multi-dimensional GC. However, different PIDs have different responsivities towards the same chemical compound with the same concentration or mass due to different aging conditions of the PID lamps and windows. Here, we carried out a systematic study regarding the response of 5 Krypton µPIDs in a 1 × 4-channel 2-dimensional µGC system to 7 different volatile organic compounds (VOCs) with the ionization potential ranging from 8.45 eV to 10.08 eV and the concentration ranging from ∼1 ng to ∼2000 ng. We used one of the PIDs as the reference detector and calculated the calibration factor for each of the remaining 4 PIDs against the first PID, which we found is quite uniform regardless of the analyte, its concentration, or chromatographic peak width. Based on the above observation, we were able to quantitatively reconstruct the coeluted peaks in the first dimension using the signal obtained with a PID array in the second dimension. Our work will enable rapid and in situ calibration of PIDs in a GC system using a single analyte at a single concentration. It will also lead to the development of multi-channel multi-dimensional GC where multiple PIDs are employed.

Flow-through microfluidic photoionization detectors for rapid and highly sensitive vapor detection.

A photoionization detector (PID) is well known for its high sensitivity, large dynamic range, and non-destructive vapor detection capability. However, due to its tardy response, which results from the relatively large ionization chamber and dead volume, the application of the PID in gas chromatography (GC) has been limited. Here, we developed a rapid, flow-through, and highly sensitive microfluidic PID that was microfabricated directly on a conductive silicon wafer. The microfluidic PID has a significantly reduced ionization chamber volume of only 1.3 µL, nearly 10 times smaller than that of state-of-the-art PIDs and over 100 times smaller than that of commercial PIDs. Moreover, it has virtually zero dead volume due to its flow-through design. Consequently, the response time of the microfluidic PID can be considerably shortened, ultimately limited by its residence time (7.8 ms for 10 mL min(-1) and 78 ms for 1 mL min(-1)). Experimentally, the response of the microfluidic PID was measured to be the same as that of the standard flame ionization detector with peak full-widths-at-half-maximum of 0.25 s and 0.085 s for flow rates of 2.3 mL min(-1) and 10 mL min(-1), respectively. Our studies further show that the microfluidic PID was able to detect analytes down to the picogram level (at 3σ of noise) and had a linear dynamic range of six orders of magnitude. Finally, because of the very short distance between the electrodes, low voltage (<10 VDC, over 10 times lower than that in a regular PID) can be used for microfluidic PID operation. This work will open a door to broad applications of PIDs in gas analyzers, in particular, micro-GC and multi-dimensional GC.

Multiplex serum cytokine immunoassay using nanoplasmonic biosensor microarrays.

Precise monitoring of the rapidly changing immune status during the course of a disease requires multiplex analysis of cytokines from frequently sampled human blood. However, the current lack of rapid, multiplex, and low volume assays makes immune monitoring for clinical decision-making (e.g., critically ill patients) impractical. Without such assays, immune monitoring is even virtually impossible for infants and neonates with infectious diseases and/or immune mediated disorders as access to their blood in large quantities is prohibited. Localized surface plasmon resonance (LSPR)-based microfluidic optical biosensing is a promising approach to fill this technical gap as it could potentially permit real-time refractometric detection of biomolecular binding on a metallic nanoparticle surface and sensor miniaturization, both leading to rapid and sample-sparing analyte analysis. Despite this promise, practical implementation of such a microfluidic assay for cytokine biomarker detection in serum samples has not been established primarily due to the limited sensitivity of LSPR biosensing. Here, we developed a high-throughput, label-free, multiarrayed LSPR optical biosensor device with 480 nanoplasmonic sensing spots in microfluidic channel arrays and demonstrated parallel multiplex immunoassays of six cytokines in a complex serum matrix on a single device chip while overcoming technical limitations. The device was fabricated using easy-to-implement, one-step microfluidic patterning and antibody conjugation of gold nanorods (AuNRs). When scanning the scattering light intensity across the microarrays of AuNR ensembles with dark-field imaging optics, our LSPR biosensing technique allowed for high-sensitivity quantitative cytokine measurements at concentrations down to 5-20 pg/mL from a 1 µL serum sample. Using the nanoplasmonic biosensor microarray device, we demonstrated the ability to monitor the inflammatory responses of infants following cardiopulmonary bypass (CPB) surgery through tracking the time-course variations of their serum cytokines. The whole parallel on-chip assays, which involved the loading, incubation, and washing of samples and reagents, and 10-fold replicated multianalyte detection for each sample using the entire biosensor arrays, were completed within 40 min.

Directed self-assembly of nanogold using a chemically modified nanopatterned surface.

Electron-beam lithography (EBL) was used to define an aminosilane nanopatterned surface in order to electrostatically self-assemble gold nanoparticles (Au NPs). The chemically modified nanopatterned surfaces were immersed into a Au NP solution to allow the Au NPs to self-assemble. Equilibrium self-assembly was achieved in only 20 min. The number of Au NPs that self-assembled on an aminosilane dot was controlled by manipulating the diameters of both the Au NPs and the dots. Adding salt to the Au NP solution enabled the Au NPs to self-assemble in greater numbers on the same sized dot. However, the preparation of the Au NP solution containing salt was sensitive to spikes in the salt concentration. These spikes led to aggregation of the Au NPs and non-specific deposition of Au NPs on the substrate. The Au NP patterned surfaces were immersed in a sodium hydroxide solution in order to lift-off the patterned Au NPs, but no lift-off was observed without adequate physical agitation. The van der Waals forces are too strong to allow for lift-off despite the absence of electrostatic forces.

Molded transparent photopolymers and phase shift optics for fabricating three dimensional nanostructures.

This paper introduces approaches that combine micro/nanomolding, or nanoimprinting, techniques with proximity optical phase mask lithographic methods to form three dimensional (3D) nanostructures in thick, transparent layers of photopolymers. The results demonstrate three strategies of this type, where molded relief structures in these photopolymers represent (i) fine (<1 microm) features that serve as the phase masks for their own exposure, (ii) coarse features (>1 microm) that are used with phase masks to provide access to large structure dimensions, and (iii) fine structures that are used together phase masks to achieve large, multilevel phase modulations. Several examples are provided, together with optical modeling of the fabrication process and the transmission properties of certain of the fabricated structures. These approaches provide capabilities in 3D fabrication that complement those of other techniques, with potential applications in photonics, microfluidics, drug delivery and other areas.