Larry J. Kricka

Liquid Transport In Micron And Submicron Channels

There has been a growth of interest in fluid transport in very small structures. The basis for this interest derives from the application of micromachining technology to problems in fluidics. Several aspects of this problem are reviewed and discussed including some of our recent research on this topic. The problems discussed may be separated into those dealing with biological systems and those that explore the applicability of the macroscopic Navier-Stokes equations to very small planar channels. In the work conducted at the University of Pennsylvania, an experimental investigation of fluid flow in extremely small channels was conducted. Three devices have been constructed with channels of rectangular cross-section ranging in area from 7200 to 80 square microns. It was found that in the relatively large flow channels that the experimental observations were in rough agreement with the predictions from the Navier-Stokes equations. However, in the smallest of the chan-nels, there was a significant deviation from the Navier-Stokes predictions.

[29] Enhanced chemiluminescent reactions catalyzed by horseradish peroxidase

Abstract Enhancement of light emission from the horseradish peroxidase-catalyzed oxidation of diacyl hydrazides on addition of 6-hydroxybenzothiazole or phenol derivatives forms the basis of rapid, specific, and sensitive chemiluminescent assays for peroxidase. The advantages and wide applicability of the technique have been demonstrated in a range of ligand-binding assays. Careful selection of chemiluminescent reagents, enhancer, their relative proportions and reaction conditions, and more detailed knowledge of the mechanism of enhancement should enable further improvements in sensitivity and the intensity or constancy of light emission.

Clinical applications of chemiluminescence

Abstract This article reviews the clinical applications of chemiluminescence in routine testing and surveys the diverse applications of chemiluminescence in clinical research. In routine clinical testing, chemiluminescent labels (acridinium ester, acridinium sulfonamide) and detection reactions for peroxidase and alkaline phosphatase enzyme labels (luminol and adamantyl 1,2-dioxetane-based reactions, respectively) are widely used in immunoassay and nucleic acid probe assays (e.g. hybridization protection assay, Hybrid Capture® assay). In clinical research the sensitivity, dynamic range and diversity of chemiluminescent assays has led to a vast range of applications, notably in protein and nucleic acid blotting, microarray-based assays, monitoring reactive oxygen species, and as detection reactions for substances separated by HPLC, capillary electrophoresis (CE), and flow-injection analysis.

Microchips, microarrays, biochips and nanochips: personal laboratories for the 21st century

Micro miniaturization of analytical procedures is having significant impact on diagnostic testing, and will enable highly complex clinical testing to be miniaturized and permit testing to move from the central laboratory into non-laboratory settings. The diverse range of micro analytical devices includes microchips, gene chips, bioelectronic chips. They have been applied to several clinically important assays (e.g., PCR, immunoassay). The main advantages of the new devices are integration of multiple steps in complex analytical procedures, diversity of application, sub-microliter consumption of reagents and sample, and portability. These devices form the basis of new and smaller analyzers (e.g., capillary electrophoresis) and may ultimately be used in even smaller devices useful in decentralized testing (lab-on-a-chip, personal laboratories). The impact of microchips on healthcare costs could be significant via timely intervention and monitoring, combined with improved treatments (e.g., microchip-based pharmacogenomic tests). Empowerment of health consumers to perform self-testing is limited, but microchips could accelerate this process and so produce a level of self-awareness of biochemical and genetic information hitherto unimaginable. The next level of miniaturization is the nanochip (nanometer-sized features) and the technological foundation for these futuristic devices is discernable in nanotubes and self-assembling molecular structures.

Enhanced chemiluminescent method for the detection of DNA dot-hybridization assays

A simple enhanced chemiluminescent procedure for the quantitation of DNA hybridization to dot blots is described. The method utilizes DNA probes labeled with biotin, which are detected using a biotinylated streptavidin-horseradish peroxidase complex. The peroxidase enzyme then takes part in an enhanced chemiluminescent reaction with luminol, peroxide, and an enhancer. The method can be used to give quantitative results using a photomultiplier tube or qualitative results by recording the light emission on instant photographic film.

Stains, labels and detection strategies for nucleic acids assays

Selected developments and trends in stains, labels and strategies for detecting and measuring nucleic acids (DNA, RNA) and related molecules [e.g. oligo(deoxy)nucleotides, nucleic acid fragments and polymerase chain reaction products] are surveyed based on the literature in the final decade of the 20th century (1991-2000). During this period, important families of cyanine dyes were developed for sensitive detection of double-stranded DNA, single-stranded DNA, and oligo(deoxy)nucleotides in gels and in solution, and families of energy transfer primers were produced for DNA sequencing applications. The continuing quest for improved labels for hybridization assays has produced a series of candidate labels including genes encoding enzymes, microparticles (e.g. quantum dots, nanocrystals, phosphors), and new examples of the fluorophore (e.g. cyanine dyes) and enzyme class of labels (e.g. firefly luciferase mutants). Label detection technologies for use in northern and southern blotting assays have focused on luminescent methods, particularly enhanced chemiluminescence for peroxidase labels and adamantyl 1,2-dioxetanes for alkaline phosphatase labels. Sets of labels have been selected to meet the demands of multicolour assays (e.g. four-colour sequencing and single nucleotide primer extension assays). Non-separation assay formats have emerged based on fluorescence polarization, fluorescence energy transfer (TaqManTM, molecular beacons) and channelling principles. Microanalytical devices (microchips), high-throughput simultaneous test arrays (microarrays, gene chips), capillary electrophoretic analysis and dipstick devices have presented new challenges and requirements for nucleic acid detection, and fluorescent methods currently dominate in many of these applications.

Clinical and biochemical applications of luciferases and luciferins

Recent advances in the analytical applications of bacterial and firefly luciferases and firefly luciferin are reviewed. Luciferases have been used in soluble and immobilized/co-immobilized forms in assays for a variety of enzymes, substrates, and cofactors. The firefly luciferase reaction forms the basis of rapid microbiological tests which have found application in susceptibility testing, detection of bacteriuria, activated sludge analysis, and food testing. Rapid microbiological assays are also possible using bacteriophages containing the lux genes from Virbrio harveyi. Both the firefly and the bacterial luciferase reaction have been applied in immunoassay and DNA probe assays and the firefly luciferin phosphate substrate for alkaline phosphatase labels has proven particularly successful.

Advantages of firefly luciferase as a reporter gene: application to the interleukin-2 gene promoter.

The chloramphenicol acetyltransferase (CAT) gene is widely used in recombinant constructs employed to study promoter and enhancer control of gene expression. However, CAT-based assays require a laborious, multi-step procedure for quantitation of promoter activity. We have applied the recently described firefly luciferase (LUC) reporter gene to the study of the interleukin-2 (IL2) promoter and have further defined the properties of this reporter gene system. We find that IL2-LUC constructs have multiple advantages over IL2-CAT constructs. The LUC assay is highly sensitive and requires 1/10 the cells used in the CAT system. A final quantitative measure of promoter activity can be obtained within 25 h following transfection with IL2-LUC, compared to 108-160 h with IL2-CAT. Light emission significantly (fourfold) above background is detectable 3 h after induction in a direct assay of extracts from transfected cells. We have described the variability of the assay, the minimum number of transfected cells required to detect light, the stability of luciferase in cell extracts, the effect of Triton X-100 on the assay, and a rapid cell lysis procedure. The luciferase system is a simple, rapid, and sensitive method for the study of promoter activity in transfected cells, particularly for weakly expressed genes such as IL2 which give low activity in the CAT assay.

Fabrication of plastic microchips by hot embossing

Plastic microchips with microchannels (100 microm wide, 40 microm deep) of varying designs have been fabricated in polymethylmethacrylate by a hot embossing process using an electroform tool produced starting with silicon chip masters. Hot-embossed chips were capped with a polymethylmethacrylate top using a proprietary solvent bonding process. Holes were drilled through the top of the chip to allow access to the channels. The chips were tested with fluid and shown to fill easily. The seal between the top of the chip and the hot embossed base was effective, and there was no leakage from the channels when fluid was pumped through the microchannels. The chips were also tested with a semen sample and the plastic chip performed identically to the previous silicon-glass and glass versions of the chip. This microfabrication technique offers a viable and potentially high-volume low cost production method for fabricating transparent microchips for analytical applications.

Enhancement of the horseradish peroxidase-catalyzed chemiluminescent oxidation of cyclic diacyl hydrazides by 6-hydroxybenzothiazoles

6-Hydroxybenzothiazole, 2-cyano-6-hydroxybenzothiazole, and 2-(6-hydroxy-2-benzothiazolyl)thiazole-4-carboxylic acid (dehydroluciferin) dramatically enhance light emission from the horseradish peroxidase conjugate catalyzed oxidation of luminol, isoluminol, N-(6-aminobutyl)-N-ethyl isoluminol, and 7-dimethylaminonaphthalene-1,2-dicarboxylic acid hydrazide by either peroxide or perborate. Light emission is enhanced by up to 1000-fold, which is an improvement over the enhancement previously observed using firefly luciferin (4,5-dihydro-2-(6-hydroxy-2-benzothiazolyl)thiazole-4-carboxylic acid). Enhancement is influenced by enhancer concentration and pH. Spectral scans of light emitted in enhanced and unenhanced reactions are similar, suggesting that aminophthalate products, and not the enhancers, are the emitters.