Myra T. Koesdjojo

On-chip aptamer-based sandwich assay for thrombin detection employing magnetic beads and quantum dots.

In this paper, we report the development of an on-chip aptamer-based fluorescence assay for protein detection and quantification based on sandwich ELISA principles. Thrombin was selected as a model analyte to validate the assay design, which involves two DNA thrombin aptamers recognizing two different epitopes of the protein. Aptamer-functionalized magnetic beads were utilized to capture the target analyte, while a second aptamer, functionalized with quantum dots, was employed for on-chip detection. The binding of thrombin to the two aptamers via sandwich assay was monitored by fluorescence microscopy. The sandwich assay was performed on disposable microfluidic devices, fabricated on double-sided tapes and polymeric materials using a laser cutting approach. The approach enabled rapid thrombin detection with high specificity. Experimental conditions, such as reagent consumption and incubation time, were optimized in the microchip platform for the lowest limit of detection, highest specificity, and shortest assay time. The analytical performance of the microchip based assay was compared to that in the well plate format (generally utilized for ELISA-based methodologies). The results show that microfluidic chip proved to be a rapid and efficient system for aptamer-based thrombin assays, requiring only minimal (microliter) reagent use. This work demonstrated the successful application of on-chip aptamer-based sandwich assays for detection of target proteins of biomedical importance.

Fabrication of a microfluidic system for capillary electrophoresis using a two-stage embossing technique and solvent welding on poly(methyl methacrylate) with water as a sacrificial layer.

Methods for fabricating poly(methyl methacrylate) microchips using a novel two-stage embossing technique and solvent welding to form microchannels in microfluidic devices are presented. The hot embossing method involves a two-stage process to create the final microchip design. In its simplest form, a mold made of aluminum is fabricated using CNC machining to create the desired microchannel design. In this work, two polymer substrates with different glass transition temperatures (Tg), polyetherimide (PEI) and poly(methyl methacrylate) (PMMA), were used to make the reusable secondary master and the final chip. First, the aluminum mold was used to emboss the PEI, a polymeric substrate with Tg approximately 216 degrees C. The embossed PEI was then used as a secondary mold for embossing PMMA, a polymeric substrate with a lower Tg ( approximately 105 degrees C). The resulting PMMA substrate possessed the same features as those of the aluminum mold. Successful feature transfer from the aluminum mold to the PMMA substrate was verified by profilometry. Bonding of the embossed layer and a blank PMMA layer to generate the microchip was achieved by solvent welding. The embossed piece was first filled with water that formed a solid sacrificial layer when frozen. The ice layer prevented channel deformation when the welding solvent (dichloroethane) was applied between the two chips during bonding. Electrophoretic separations of fluorescent dyes, rhodamine B (Rh B) and fluorescein (FL), were performed on PMMA microchips to demonstrate the feasibility of the fabrication process for microreplication of useful devices for separations. The PMMA micro-chip was tested under an electric field strength of 705 V cm-1. Separations of the test mixture of Rh B and FL generated 55 500 and 66 300 theoretical plates/meter, respectively.

Technique for Microfabrication of Polymeric-Based Microchips from an SU-8 Master with Temperature-Assisted Vaporized Organic Solvent Bonding

Novel means of fabricating polymeric microfluidic devices are presented. An SU-8 master is applied in two-stage embossing, followed by vaporized organic solvent bonding. The primary master is created by standard photolithography; the inexpensive SU-8 primary master is used in a two-stage process to generate microfeatures in hard polymers. A vaporized solvent bonding technique that readily produces complete microfluidic chips, without the need of a sacrificial layer to prevent channel deformation, was used to form complete multilayer microfluidic devices. This technique provides a more direct method to generate hard polymer microfluidic chips than classical techniques and therefore is highly amenable to rapid prototyping. The technique lends itself readily to many polymers, facilitating device production for a variety of applications, even permitting hybrid polymer chips, and provides a rapid, cost-effective, simple, and versatile approach to the production of polymer-based microdevices. The fabrication technique was tested to build microchips to perform several analyses, including chromatographic separations and a quantitative indicator assay. High separation efficiencies of 10,000-45,000 plates/m were obtained using the fabricated liquid chromatography (LC) microchip. The fabrication method was also tested in building a passive micromixer that contained high-density microfeatures and required three polymer layers. A glycine assay using o-phthaldialdehyde (OPA) was performed in the micromixer. With glycine concentrations ranging from 0.0 to 2.6 microM, a linear calibration plot was obtained with a detection limit of 0.032 microM.

Low-cost, high-speed identification of counterfeit antimalarial drugs on paper

With the emergence of artesunate antimalarial counterfeiting in Southeast Asia and sub-Saharan Africa, we present the production of a rapid, inexpensive and simple colorimetric-based testing kit for the detection of counterfeit artesunate in order to preserve life and prevent the development of multi-drug resistant malaria. The kit works based on paper microfluidics which offer several advantages over conventional microfluidics, and has great potential to generate inexpensive, easy-to-use, rapid and disposable diagnostic devices. Here, we have developed a colorimetric assay that is specific to artesunate and turns yellow upon addition of the sample. The test can be done within minutes, and allows for a semi-quantitative analysis of the artesunate tablets by comparing the developed yellow color on the paper test to a color-coded key chart that comes with the kit. A more accurate and precise analysis is done by utilizing a color analyzer on an iPhone camera that measures the color intensity of the developed color on the paper chip. A digital image of the chip was taken and analyzed by measuring the average gray intensity of the color developed on the paper circle. A plot of the artesunate concentration versus the average gray scale intensity was generated. Results show that the intensity of the yellow color developed on the paper test was consistent and proportional to the amount of artesunate present in the sample. With artesunate concentrations ranging from 0.0 to 20mg/mL, a linear calibration plot was obtained with a detection limit of 0.98 mg/mL.

Cost-efficient fabrication techniques for microchips and interconnects enabled by polycaprolactone

In this paper we present a novel fabrication technique that utilizes polycaprolactone (PCL) as a bonding medium due to its low melting temperature property. PCL is biodegradable polyester with a melting point of 60??C, and a glass transition temperature of??60??C [1?10]. It is used as a rapid bonding medium in the fabrication process that readily produces complete microfluidic chips. The microchannels are produced via laser ablation micromachining and thermal embossing, followed by bonding with PCL. The PCL is uniformly coated on a piece of polymer sheet to produce a thin film on its surface. A complete microfluidic channel is formed by enclosing the open channel with the PCL-coated polymer piece. This fabrication technique lends itself readily to various polymers, such as (poly)methylmethacrylate (PMMA), polycarbonate (PC), polyetherimide (PEI) and poly(ethylene terephthalate) (PETE), facilitating device production for a variety of application, even permitting hybrid polymer chips. The bonding was performed rapidly at 60??C. This approach provides a more direct method to generate hard polymer microfluidic chips than classical techniques and is therefore highly amendable to rapid prototyping. This work also explores the use of PCL as an alternative approach to making simple, cost-effective universal adhesive for bonding interconnects. Bonding is performed at 60??C, by placing the adhesive layer in between an interconnect port and a microchip. This method allows for connections to be made easily and quickly.

18 Molecularly imprinted polymers as sorbents for separations and extractions

Abstract Molecularly imprinted polymers (MIPs) are materials that mimic biological receptors with synthetic recognition sites that exhibit pre-determined selectivity toward analyte(s) of interest. MIPs have become increasingly popular in recent years, and have been applied as selective sorbents for extractions and chromatography and in other areas where high specificity is required. This chapter presents a summary of factors of importance in the synthesis and use of MIPs, describes various different synthesis approaches, and reviews recent advances in the field of molecularly imprinted materials.

Impact of Parylene-A Encapsulation on ZnO Nanobridge Sensors and Sensitivity Enhancement via Continuous Ultraviolet Illumination

The impact of parylene-A encapsulation and the effect of continuous ultraviolet (UV) exposure on ZnO nanobridge sensor response are investigated. ZnO nanowire (NW) devices are fabricated using a novel method that involves selective growth of ZnO nanobridges between lithographically defined pads of carbonized photoresist (C-PR). We find that a thin coating of parylene-A effectively attenuates the response of NW devices to O2, H2O vapor, and UV illumination. The accessibility of the amine group on parylene-A for chemical functionalization is verified by transforming the amine groups on the surface of the parylene-A coating into aromatic imine groups, followed by UV–Vis absorption. Our results suggest that, in addition to modulating environmental sensitivity and providing protection of the ZnO NWs for liquid- and vapor-phase sensing, the parylene-A encapsulation may also serve as an activation layer for further specific functionalization targeting selective sensing. We also found that the sensitivity and response time of ZnO nanobridge devices to O2 are dramatically improved by continuously exposing the nanobridge devices to UV illumination. Finally, we show that the C-PR directed growth method can also be used to isolate free-standing NW carpet.

Rapid Synthesis of a Long Double-Stranded Oligonucleotide from a Single-Stranded Nucleotide Using Magnetic Beads and an Oligo Library.

Chemical synthesis of oligonucleotides is a widely used tool in the field of biochemistry. Several methods for gene synthesis have been introduced in the growing area of genomics. In this paper, a novel method of constructing dsDNA is proposed. Short (28-mer) oligo fragments from a library were assembled through successive annealing and ligation processes, followed by PCR. First, two oligo fragments annealed to form a dsDNA molecule. The double-stranded oligo was immobilized onto magnetic beads (solid support) via streptavidin-biotin binding. Next, single-stranded oligo fragments were added successively through ligation to form the complete DNA molecule. The synthesized DNA was amplified through PCR and gel electrophoresis was used to characterize the product. Sanger sequencing showed that more than 97% of the nucleotides matched the expected sequence. Extending the length of the DNA molecule by adding single-stranded oligonucleotides from a basis set (library) via ligation enables a more convenient and rapid mechanism for the design and synthesis of oligonucleotides on the go. Coupled with an automated dispensing system and libraries of short oligo fragments, this novel DNA synthesis method would offer an efficient and cost-effective method for producing dsDNA.

Development of photoactivable nanofiber membranes for efficient metal ion extraction

A novel spiropyran nanofiber was developed an applied to the extraction of heavy metal ions from drinking water. The objective of this work was to capitalize on the high surface area achievable via nanotechnology to develop a high efficiency, cost effective system to enable analysis for a range of applications including ground, lake and river water monitoring. Assessment and remediation of heavy metal contamination is of utmost importance in industrial, environmental, and human water consumption applications. We have developed technology for the removal of heavy metal ions from aqueous solutions without the need for ancillary reagents in the metal binding-and-release process, as are common in more conventional methods. As an alternative, we have employed photoactivable compounds, triggered by illumination with light of appropriate wavelengths, to control binding and release events. This technology has the potential to reduce the high consumption of ancillary reagents and decrease waste stream volume, not to mention the capacity to reduce the high recurring costs borne by users.

pH/ion sensitive nanoprobes with optical tweezers

We present fluorescence-based pH/ion nanosensors, positioned and manipulated using holographic optical tweezers, with simultaneous fluorescence read-out, within a microfluidic device and within a biological cell.