Microfluidic biosensor based on an array of hydrogel-entrapped enzymes,”

Here we show that a microfluidic sensor based on an array of hydrogel-entrapped enzymes can be used to simultaneously detect different concentrations of the same analyte (glucose) or multiple analytes (glucose and galactose) in real time. The concentration of paraoxon, an acetylcholine esterase inhibitor, can be quantified using the same approach. The hydrogel micropatch arrays and the microfluidic systems are easy to fabricate, and the hydrogels provide a convenient, biocompatible matrix for the enzymes. Isolation of the micropatches within different microfluidic channels eliminates the possibility of cross talk between enzymes. In this paper, we describe a microfluidic biosensor that uses an array of hydrogel-entrapped enzymes to quantitatively determine the concentration of an analyte and simultaneously detect multiple analytes. The approach relies on the presence of active enzymes within hydrogel micropatches photolithographically defined within microfluidic channels. The enzymes are sufficiently large that they are unable to escape the hydrogel matrix, but the targets are small enough to enter the hydrogel, encounter the enzyme, and be converted into detectable products. By using discrete micropatches contained within multiple channels, and in some cases multiple enzymes within a single hydrogel micropatch, it is possible to detect multiple, structurally similar analytes in parallel and in real time. The apparatus necessary to carry out the assay is straightforward to fabricate. [1][2][3] The performance of biosensors incorporating capture probes is directly linked to the approach used for probe immobilization. In this regard, the following issues are important: (1) the biomaterial must remain active on or within the support, (2) nonspecific adsorption should be minimized, (3) the number of probe receptors should be optimized to provide maximum signal, (4) it should be easy to place the immobilized probes in a desired location, and (5) mass transfer of the target from solution to the probe should be rapid. To address these points, we have focused our recent studies on two families of biomolecular supports that are particularly adaptable to the microfluidic environment: polymeric microbeads 4-6 and monolithic hydrogels. 3,7 For example, we reported that photopolymerized hydrogel micropatches could be used for immobilizing enzymes 3 and bacteria 7 within the channels of microfluidic devices. These relatively large biomaterials are physically entrapped within the photo-cross-linked hydrogel matrix, but analytes are able to diffuse through nanopores within the gel and encounter the probes. Importantly, both enzymes and bacteria retain their activity within the gel, which means that the composite gel/biomaterial can be used as a sensor unit or microbioreactor. Here, we expand upon our earlier findings by demonstrating that an array of hydrogel-entrapped enzymes can be used to simultaneously detect multiple analytes or quantitatively determine the concentration of a single analyte. In addition to our own work, others have shown that hydrogels can be used to immobilize proteins, 8,9 cells, 10-12 and DNA [13][14][15] within microfluidic devices and on planar supports. The size and shape of the gel can be defined by photolithography, 16,17 a mold, 3,10,12 or a robotic spotter. 18 The smallest hydrogel features reported are in the range of tens of micrometers. 16 Unlike array sensors that rely on surface immobilization of DNA or protein monolayers, which are usually designed to bind specific targets, hydrogel micropatches containing enzymes are essentially microbioreactors that consume reactants and generate products. It is important, therefore, to minimize cross-talk between elements of the array. This issue has been addressed by several groups. For example, Arenkov and co-workers fabricated gel pads on hydrophobic surfaces that were covalently linked to enzymes. The hydrophobic surface prevented sample droplets from spreading to nearby gel pads. 8 McDevitt and co-workers developed an * To whom correspondence should be addressed. Voice: 979-845-5629. Fax: 979-845-1399. E-mail: [email protected]. † Present address: Department of Chemistry and Biochemistry, The University of Texas, 1 University Station A5300, Austin, TX 78712-0165. (

An Overview of Recent Strategies in Pathogen Sensing

Pathogenic bacteria are one of the major concerns in food industries and water treatment facilities because of their rapid growth and deleterious effects on human health. The development of fast and accurate detection and identification systems for bacterial strains has long been an important issue to researchers. Although confirmative for the identification of bacteria, conventional methods require time-consuming process involving either the test of characteristic metabolites or cellular reproductive cycles. In this paper, we review recent sensing strategies based on micro- and nano-fabrication technology. These technologies allow for a great improvement of detection limit, therefore, reduce the time required for sample preparation. The paper will be focused on newly developed nano- and micro-scaled biosensors, novel sensing modalities utilizing microfluidic lab-on-a-chip, and array technology for the detection of pathogenic bacteria

Characterization and applications of microfluidic devices based on immobilized biomaterials

Microfluidic biosensors and bioreactors based on immobilized biomaterials are described in this dissertation. Photocrosslinkable hydrogel or polymeric microbeads were used as a supporting matrix for immobilizing E.coli or enzymes in a microfluidic device. This dissertation covers a microfluidic bioreactor based on hydrogel-entrapped E.coli, a microfluidic biosensor based on an array of hydrogel-entrapped enzymes, and a microfluidic bioreactor based on microbead-immobilized enzymes. Hydrogel micropatches containing E.coli were fabricated within a microfluidic channel by in-situ photopolymerization. The cells were viable in the hydrogel micropatch and their membranes could be porated by lysating agents. Entrapment of viable cells within hydrogels, followed by lysis, could provide a convenient means for preparing biocatalysts without the need for enzyme extraction and purification. Our results suggested that hydrogel-entrapped cells, immobilized within microfluidic channels, can act as sensors for small molecules and as bioreactors for carrying out reactions. A microfluidic biosensor based on an array of hydrogel-entrapped enzymes could be used to simultaneously detect different concentrations of the same analyte or multiple analyte in real time. The concentration of an enzyme inhibitor could be quantified using the same basic approach. Isolations of the microchannels within different microfluidic channels could eliminate the possibility of cross talk between enzymes. Finally, we characterized microfluidic bioreactors packed with microbead-immobilized enzymes that can carry out sequential, two-step enzyme-catalyzed reactions under flow conditions. The overall efficiency of the reactors depended on the spatial relationship of the two enzymes immobilized on the beads. Digital simulations confirmed the experimental results

Characterization and applications of microfluidic devices based on immobilized biomaterials

Microfluidic biosensors and bioreactors based on immobilized biomaterials are described in this dissertation. Photocrosslinkable hydrogel or polymeric microbeads were used as a supporting matrix for immobilizing E.coli or enzymes in a microfluidic device. This dissertation covers a microfluidic bioreactor based on hydrogel-entrapped E.coli, a microfluidic biosensor based on an array of hydrogel-entrapped enzymes, and a microfluidic bioreactor based on microbead-immobilized enzymes. Hydrogel micropatches containing E.coli were fabricated within a microfluidic channel by in-situ photopolymerization. The cells were viable in the hydrogel micropatch and their membranes could be porated by lysating agents. Entrapment of viable cells within hydrogels, followed by lysis, could provide a convenient means for preparing biocatalysts without the need for enzyme extraction and purification. Our results suggested that hydrogel-entrapped cells, immobilized within microfluidic channels, can act as sensors for small molecules and as bioreactors for carrying out reactions. A microfluidic biosensor based on an array of hydrogel-entrapped enzymes could be used to simultaneously detect different concentrations of the same analyte or multiple analyte in real time. The concentration of an enzyme inhibitor could be quantified using the same basic approach. Isolations of the microchannels within different microfluidic channels could eliminate the possibility of cross talk between enzymes. Finally, we characterized microfluidic bioreactors packed with microbead-immobilized enzymes that can carry out sequential, two-step enzyme-catalyzed reactions under flow conditions. The overall efficiency of the reactors depended on the spatial relationship of the two enzymes immobilized on the beads. Digital simulations confirmed the experimental results

Detection of a drug and its metabolites from a fingerprint using Raman microscopy

The goal of this research project is to detect a drug and its metabolites from a fingerprint using Raman microscopy. A fingerprint is a residue remained as a result of the evaporation of a sweat transferred from fingers to a surface. Fingerprints are important in forensic science because their unique patterns can be used to identify a person. In addition, some analytical methods can provide the identity of a compound that may be present in the fingerprint. Particularly, if the presence of a drug metabolite in the fingerprint is confirmed by an analytical method, this strongly suggests that the fingerprint owner consumed the drug. Raman microscopy is a non-destructive, imaging method that does not require sample preparation steps. The unique Raman peak frequencies in the Raman spectrum can help identify an unknown compound. But, weak Raman scattering signals makes it difficult to detect low concentrations of a drug metabolite. To resolve this issue, we propose to collect Raman signals using a substrate that shows surface enhanced Raman scattering (SERS). The SERS is a unique phenomenon observed at nanoscaled gold or silver surface. This SERS signal has been known to be 10³-10⁶ times stronger than a normal Raman signal. We examined two types of gold SERS substrates using Raman dye molecules and will present the results of the SERS enhancement factors, reusuability, and short-term stability of the two substrates, and the future plan of this project

Analytical Use of Easily Accessible Optoelectronic Devices: Colorimetric Approaches Focused on Oxygen Quantification

Rapid technological progress in digital color imaging devices, such as charge-coupled devices (CCD), complementary metal-oxide-semiconductor (CMOS) cameras, liquid crystal display (LCD), and digital light projection (DLP) devices, makes these optoelectronic products nearly ubiquitous in our daily lives. For example, relatively high-quality color images can be obtained anytime with mobile phone cameras and wireless webcams (some products as low as several tens of US dollars) and then be transmitted wirelessly. These color imaging devices were originally designed as sensory or perceptual devices that mimic the human eye when responding to visible wavelengths. This chapter introduces the potential use of these color imaging devices as economic analytical instruments. After we briefly review prospective luminophores amenable for colorimetric chemical quantification using color imaging devices, we will describe oxygen quantification as exemplary applications of this approach

Patch It If You Can: Increasing the Efficiency of Patch Generation Using Context

Although program repair is a tremendous aspect of a software system, it can be extremely challenging. An Automated Program Repair (APR) technique has been proposed to solve this problem. Among them, template-based APR shows good performance. One of the key properties of the template-based APR technique for practical use is its efficiency. However, because the existing techniques mainly focus on performance improvement, they do not sufficiently consider the efficiency. In this study, we propose EffiGenC, which efficiently explores the patch ingredient search space to improve the overall efficiency of the template-based APR. EffiGenC defines the context using the concept of extended reaching definition from compiler theory. EffiGenC constructs the search space by collecting the ingredient required for patching in the context. We evaluated EffiGenC on the Defects4j benchmark. EffiGenC decreases the number of candidate patches from 27% to 86% compared to existing techniques. EffiGenC also correctly/plausibly fixes 47/72 bugs. For Future work, we will solve the search space problem that exists in multiline bugs using context