Pathogen Detection Using Mesoporous Silicon Smart Bandages

 L.A. DeLouise

University of Rochester Medical School Department of Dermatology and The Center for Future Health, 697 Elmwood AveRochester NY 14642

At the University of Rochester Center for Future Health we are exploring new technologies that will help shape the future of consumer health care. Thrusts are focused on utilizing non-invasive imaging methods to help consumers detect early signs of disease including neurological disorders and infection.  The smart bandage activity is part of a consortium of pathogen detection projects focused on developing inexpensive and easy to use affinity based biosensor technologies that can provide early detection of pathogenic organisms in for example food or a wound.  The smart bandage is highly interdisciplinary project requiring expertise that spans the boundaries between material science, optics, chemistry, molecular and microbiology. The sensor is comprised of mesoporous silicon (PSi) which is a unique photonic material that can be fabricated into a variety of structures exhibiting characteristic optical properties. The smart bandage leverages the photoluminescence (PL) and highly reflecting properties of PSi microcavities to transduce the biosensor signal. The operating principle of the device is based on detecting an index of refraction change caused by the direct and selective binding of target molecule to an immobilized probe.  

This talk will begin with an overview of the material and photonic aspects of porous silicon and the microcavity structure.  The methods and challenges to covalently immobilize a biomacromolecular probe inside the porous 3D microstructure will be introduced. Critical parameters include the porosity and pore size relative to molecular weight and shape of the protein probe.  Well-defined controlled solutions employing know binding partners are currently employed in the early phases of developing this technology. The enterohemorrhagic stain of E. coli bacteria O157:H7 serves as our model system.  A 7KDa recombinantly engineered translocated intimin receptor (Tir) protein serves as the probe.  A 35KDa adhesin protein, Intimin, isolated from the bacterial outer membrane serves as the target. We will demonstrate how this model system has enabled us to mature our device fabrication procedures, operational protocols and methods to quantify device selectivity and sensitivity.

 http://www.futurehealth.rochester.edu/research/pathogen.html

Using Inverse Gas Chromatography (IGC) to Investigate
Batch-to-Batch Related Problems

Dan Burnett and Frank Thielmann

Surface Measurement Systems

Batch-to-batch variations of active materials pose significant problems for a wide range of industrial sectors; including pharmaceutical, food, flavors, personal care, catalysis, and polymers.  The choice of techniques available for the analysis, characterization and differentiation of chemically identical products can be limited by the nature of the materials and the sensitivity to small physicochemical differences between them.  Inverse gas chromatography (IGC), a highly sensitive and versatile gas phase technique first developed over 40 years ago to study surface and bulk properties of particulate and fibrous materials, has recently been demonstrated to offer vast potential for solving batch-to-batch problems and investigating subtle surface modifications for a wide variety of materials.  IGC is a versatile and highly sensitive technique that is uniquely capable of differentiating subtle changes by introducing a wide range of molecular probes (vapor or gas) either at very low concentrations (infinite dilution regime), or at higher concentrations (finite dilution regime) to provide a comprehensive understanding of both surface and bulk properties of a material.  IGC is particularly sensitive if measurements are carried out in the low concentration or infinite dilution regime (the linear or Henry portion of the isotherm).  At infinite dilution conditions, very few probe molecules are available for an interaction with the surface of the material under investigation and will therefore only interact with the highest energy sites on the surface.  For this reason even the smallest differences between similar materials or batches of the same material that have been processed under slightly different conditions can be measured by varying probe concentration, temperature, flow rate, or background relative humidity. 

This talk will summarize a number of examples from the literature as well as our own work in which IGC has been utilized to address important batch-to-batch issues and study surface modified materials.  For example, we have used IGC to correlate dissolution rates of a quick release painkiller made under different processing conditions to changes in surface chemistry measured at infinite dilution.  Additionally, finite dilution IGC experiments have been used study hair samples modified with different chemical treatments.[i]  IGC has also been used to investigate the effects of different chemical treatments on a range of carbon black samples.[ii]  Finally, in the catalysis sector, IGC has been used to study the effect of grinding on the surface energy properties of a-alumina samples.[iii]  In addition to the above examples, other IGC applications and case studies will be introduced. 

 Implementation of FDA’s Electronic Records and Electronic Signatures requirements (21 CFR Part 11) in the Analytical Laboratory

 Mr. John Osler

Analytical Development Celltech Pharmaceuticals Rochester, New York

Celltech Pharmaceuticals is a manufacturer of prescription and OTC products and as such is required to implement the Code of Federal Regulations (CFR) and related Good Manufacturing Practices put in place by the Food and Drug Administration (FDA).

In 1997, new requirements on the use of electronic records and electronic signatures were introduced by the FDA and codified as 21 CFR Part 11 – Electronic Records; Electronic Signatures.

Celltech has implemented several laboratory improvements based on the ‘Part 11’ requirements, including an enhanced Chromatographic Data System (Agilent’s ChemStoreâ product) and an electronic data archiving scheme (NuGenesis’ ARCHIVEâ product). 

The presentation will give a background on the FDA’s Electronic Records and Signatures requirements, their impact on the Analytical laboratory, and provide practical examples of meeting those requirements gathered pre and post implementation.

Exploring Proteomics Using Practice and Simulation

 Paul A. Craig

Department of Chemistry Rochester Institute of Technology

Proteomics is the study of the full complement of proteins expressed in a particular organism, tissue or cell line.  Two-dimensional gel electrophoresis (2DE) has been a core technology for proteomics for over two decades, based on its ability to separate and display thousands of proteins in a single experiment.  In 2DE, proteins are separated in the first dimension by their isoelectric points (the pH at which their net charge is zero) and in the second dimension by their molecular weights.  We are studying 2DE from two directions – experimental practice in the lab and simulation of the experiments on computers. 

Students in the chemistry lab are exploring protein expression in Pseudomonas putida, an environmental microbe that has extraordinary abilities to metabolize environmental contaminants.   Their efforts in optimizing conditions for growth, protein extraction, and separation conditions will be described.  The entire genome of this organism has recently been sequenced, which will contribute to our efforts both in the lab and in silico.

Computer science students are developing a 2DE simulation which can accept input in three different file formats: FASTA, Genbank and Protein Data Bank.  Both dimensions of 2DE are included in the simulation and individual protein spots in the final output are linked to data about that protein and to external databases.

Detailed Polymer Characterization Using Gel Permeation Chromatography
and Hyphenated Technologies

X. Michael Liu¹, E. Peter Maziarz¹, William J. Simonsick², David J. Heiler¹ 

1.      Bausch & Lomb, Research, Development and Engineering, Rochester, NY 14609
2.  DuPont Marshall R&D Laboratory, Philadelphia, PA 19146

 Email: x.michael.liu@bausch.com

A more complete understanding of new polymeric materials used for making implant devices becomes increasingly important in the medical device industry.  Often such detailed information requires utilization of a combination of analytical techniques.  In this study, we characterize a silicone material, polyester, and a polymer blend using gel permeation chromatography (GPC) hyphenated with electrospray ionization (ESI), matrix assisted laser desorption ionization (MALDI) mass spectrometry (MS) techniques.  Here we obtain comprehensive compositional information of the polymers such as repeat units, end group chemistry, and identification /quantitation of impurities in the low molecular weight region. 

GPC with laser light scattering, differential viscometry, and differential refractive index detection (Triple Detection) is also used to obtain information on a trace amount of high molecular weight impurities that are undetected by both GPC-ESI and MALDI MS techniques.  GPC-Triple Detection directly measures absolute molecular weight distributions and determines molecular conformation (e.g. branching, cyclic, linear) of the materials. Furthermore the radius of gyration and intrinsic viscosity of the polymer materials are also determined as a function of molecular weight distributions.  Finally we present and compare average molecular weight values of the polymeric materials obtained by conventional GPC, GPC-Triple Detection, GPC-ESI, and GPC-MALDI MS techniques.

Optimizing Method Parameters and Types of Samples used in
Large Volume Injection

 ^{Lee Marotta PerkinElmer Instruments, 761 Bridgeport Avenue, Shelton, CT}

Analytical scientists are continuously being asked to improve detection limits.   This seminar will discuss the applications for Large Volume Injection and the optimization of the necessary parameters.

 First, we will review the advantages of Large Volume Injection or Solvent Purge injections.  There are analytical advantages for injecting samples into a cold injection port, and for preventing solvents from reaching the column and detector.  We will discuss the benefits of replacing classical splitless injections with solvent purge injections. 

We will discuss what sample types lend themselves to solvent purge injection.  Typically, there needs to be a boiling point difference in 6 carbons between the 1st analyte and the solvent which is being purged.  Experiments and parameters on how to utilize this technique when only a three carbon difference exists will be presented.  For example, we are able to maintain recovery of nonane while purging hexane from the injector port. 

Method parameters and method development techniques will be presented.  Many questions will be answered, such as:  What purge temperature should be used?    Which solvents are more appropriate?  What are the optimum solvent purge times? How can I minimize or eliminate any solvent from entering system?   Large injection – how large is large?  Which liners should be used?  Which columns can optimize this technique? 

Characterization of Polymorphs by Inverse Gas Chromatography 

Frank Thielmann¹⁾, David Butler¹⁾, Lesley Mackin²⁾

¹⁾ Surface Measurement Systems UK, 3 Warple Mews, Warple Way, London W3 0RF

²⁾ Pharmacia-Pfizer, Chicago, USA

The batch-to-batch variation of active materials poses a significant problem to the pharmaceutical industry. One common origin of such variation is in the presence of two or more polymorphs, often chemically identical, but with different physical material properties. The choice of techniques available for the analysis and characterization of such polymorphs can be limited by the nature of the materials and the sensitivity to small physicochemical differences between them. One applicable technique is Inverse Gas Chromatography (IGC). Different polymorphs will expose different crystal surfaces and different functional groups at the surface. IGC measures these differences in the surface properties to provide a sensitive tool to compare and identify polymorphs.

Two polymorphs of Xemilofiban (Pharmacia), were studied by IGC to measure their dispersive surface energies and specific polar interactions with a number of solvents. Clear differences were observed between the two polymorphs. Specific interactions could reflect differences in the surface chemistry.

Milled samples of Polymorph A were shown to contain a proportion of amorphous material, also seen in sorption studies, with a glass transition at approx. 55% RH at 30^oC. Humidity treatment, to recrystallize this material resulted in a sample identical to the original Polymorph A, suggesting that a sample of polymorph A remained in that polymorphic form after milling and exposure to high humidities. The results further suggested that underlying sample morphology rather than thermodynamic stability drives the recrystallization.

Acknowledgements

The authors would like to thanks Pharmacia-Pfizer, Chicago, USA for supplying the material for this study and Dr Lesley Mackin for her support.

Determination of Thermodynamic Parameters of Carbon-based Materials by Inverse Gas Chromatography

 Frank Thielmann¹⁾, Goeff Fowler²⁾

 ¹⁾ Surface Measurement Systems Ltd., 3 Warple Mews, Warple Way, London W3 0RF, United Kingdom

²⁾ Department of Chemical Engineering and Chemical Technology, Imperial College of Science, Technology and Medicine, London SW7 2BY, United Kingdom

The determination of surface thermodynamic parameters of carbon-based materials is of high interest for the prediction of their macroscopic properties. The surface energy and the acid-base properties are often-used parameters since they reflect the energetic situation on the surface for both non-polar and specific molecular interactions.

One of the most powerful methods for the determination of these properties is inverse gas chromatography (IGC). IGC involves the sorption of a known adsorbate (vapor) onto an unknown adsorbent stationary phase (solid sample). This approach inverts the conventional relationship between mobile and stationary phases found in analytical gas-solid chromatography.

Thermodynamic studies are usually carried out at infinite dilution since these parameters reflect exclusively the interaction between the clean solid surface and the probe molecule under these conditions.  Therefore, IGC has been successfully applied for characterization of carbon black and related materials. Recently the correlation of acid-base parameters with surface groups identified by other techniques became of particular interest.

New experimental results will be shown to illustrate the potential of IGC at infinite dilution for the study of adsorption site energetics on different carbon-based materials at low temperatures.

Last modified:10/28/03
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