Analytica Chimica Acta (v.457, #1)

Preface by William E Lee (1-2).

Chemiluminescence multichannel immunosensor for biodetection by Erwin Yacoub-George; Leonhard Meixner; Waltraud Scheithauer; Andreas Koppi; Stephan Drost; Hans Wolf; Christine Danapel; Klaus A Feller (3-12).
An improved portable detector for biological compounds, the chemiluminescence multichannel immunosensor (CL-MADAG), has been developed and characterised. The device is based on a capillary ELISA technique in combination with a miniaturised fluidics system and uses chemiluminescence as the detection principle. The fluidics system construction allows three chemiluminescence immunoassays to be performed simultaneously within three fused silica capillaries (FSC). The CL-MADAG was characterised in a series of experiments with staphylococcal enterotoxin B (SEB) as a model toxin, the bacterial phage virus M13 as a virus simulant, and a pathogenic strain of Escherichia coli as simulant for bacteria. It was shown that the CL-MADAG can assay liquid samples for these substances within 24 min. The detection limits were 5 ng/ml for SEB, 105  cfu/ml for E. coli O157:H7 and 107  pfu/ml for M13.
Keywords: Biosensor; Chemiluminescence; Immunoassay; Multianalyte detection;

Selection of phage displayed peptides for the detection of 2,4,6-trinitrotoluene in seawater by Ellen R. Goldman; Mehran P. Pazirandeh; Paul T. Charles; Eric D. Balighian; George P. Anderson (13-19).
We have selected for phage displayed peptides that showed specific binding to a 2,4,6-trinitrotoluene (TNT) derivative, 2,4,6-trinitrobenzene (TNB) in environmentally relevant conditions, and have integrated the selected phage into a continuous flow immunosensor platform for the detection of TNT. A library of 12 random amino acid peptides (12-mers) displayed on phage was panned against TNB coupled to the protein bovine serum albumin (BSA) in a solution of artificial seawater. Eight phage clones, seven of which share an identical amino acid sequence, bound selectively to TNB–BSA in artificial seawater as judged by enzyme-linked immunosorbent assay (ELISA). Addition of TNT, inhibited binding of the phage. Whole phage were labeled with the dye cyanine 5 (Cy5), and incorporated into a flow sensor platform. Labeled phage were loaded onto a TNB–affi-gel packed column, and a reproducible signal, at least five times greater than background, was observed on repeat injections of 10 mg/l TNT dissolved in seawater. This study presents one of the first examples of phage selection in a non-physiological medium, and the first demonstration that dye-labeled phage can be integrated into a continuous flow sensor.
Keywords: Phage display; Biosensor; Explosive detection;

Staphylococcal enterotoxin B (SEB) was labeled with tetramethylrhodamine isothiocyanate (TRITC) and used as a probe for a competitive immunoassay. Labeling conditions such as solution pH and time were varied to observe the effect on the fluorescent product. It was found that solution pH of the labeling reaction had little effect on the fluorescence signal of the resulting products. However, labeling at pH 7.0 produced a probe that had a higher affinity for the antibody used in this study than the probes produced at pH 8.0 and 9.0. The fluorescent probes were used to perform a competitive assay for SEB in model skim milk samples. Detection limit was approximately 300 fg of SEB. Quantitation was achieved by curve fitting of fluorescent signals for bound/free probe versus log[SEB] with logarithmic functions. Accuracy in the model skim milk samples was acceptable for 3 and 5 nM SEB, but decreased considerably for a concentration of less than 1 nM SEB. The error was attributed to deviation in linearity in the standard curve at lower concentrations. Reproducibility for the analysis of both standard solutions used for the calibration curves and the model skim milk samples was excellent, with standard deviations of approximately 10% from data collected over a 3-week period. No cross-reactivity was found when the assay was tested with a 700 nM sample of staphylococcal enterotoxin A. Although competitive immunoassays are usually used for small molecules, such as therapeutic drugs, the results demonstrate that relatively large molecules (SEB, 27 kDa) can also be assayed with the technique.
Keywords: Staphylococcal enterotoxin B; SEB; Capillary electrophoresis; Immunoassay; Fluorescence;

Short single-stranded DNA (ssDNA) oligonucleotides can be grown on the surface of fused silica by automated nucleic acid synthesis. The immobilized ssDNA can be deposited at a desired average density. The density of ssDNA provides a controlled parameter that in combination with temperature, ionic strength and pH, can be used to define the selectivity of hybridization. Furthermore, the density of ssDNA can be used to control the affinity of complementary DNA so that it associates with the nucleic acids on the surface rather than areas that are not coated with ssDNA. The characteristic melt temperature observed for immobilized double-stranded DNA (dsDNA) 20mer shifts by up to 10 °C when a single base pair mismatch is present in the center of a target oligonucleotide. Optimization of quantitative analysis of such single base pair mismatches requires use of select experimental conditions to maximize the formation of the fully matched target duplex while minimizing the formation of the mismatched duplex. Results based on fiber optic biosensors that are used to study binding of fluorescein-labeled complementary DNA demonstrate that it is possible to achieve a selectivity coefficient of fully matched to single base pair mismatch of approximately 85–1, while maintaining >55% of the maximum possible signal that can be obtained from the fully matched target duplex.
Keywords: DNA; Hybridization; Fiber optic; Fluorescence; Selectivity; Biosensor;

Detection, identification and characterisation of biological material, for example micro-organisms, toxins and viruses, occupy an important position in industrial, medical and environmental analysis. Bioanalytes may be investigated by one of numerous analytical techniques available, however, the recognition principle often utilises either antibodies, an alternative protein recognition principle, or nucleic acid probes. Common techniques include ELISA, surface plasmon resonance and other evanescent wave detectors, flow cytometry, immunofluoresence microscopy, and PCR-based assays. Even though antibodies are the most successfully employed recognition component for identifying bioanalytes, they have a number of intrinsic negative aspects to their application. Up until now however, a suitable alternative has not been identified. Carbohydrate recognition is a mechanism by which a high proportion of biological molecules first come into contact with each other in the process of cellular adhesion. This article will discuss the possibility of using this principle as a biosensor recognition element, and will review the current situation.
Keywords: Carbohydrate recognition; Adhesion; Bioanalyte detection;

We report on the development of reagentless fluorescence-based sensing films utilizing hydrolytic enzymes co-entrapped with polymers that are labelled with pH sensitive fluorophores. Aqueous solutions of a hydrophilic enzyme (urease) or a lipophilic enzyme (lipase) containing fluorescein or carboxy-seminaphtharhodafluor-1 (SNARF-1), either free or conjugated to a dextran polymer backbone, were mixed with hydrolyzed alkoxysilane solutions and cast onto planar surfaces to form thin, biologically active sol–gel derived films (ca. 500 nm thick). The films also contained various additives, such as methyltrimethoxysilane, dimethyldimethoxysilane, polyethylene glycol or polyvinyl alcohol, to optimize the activity of the entrapped enzymes. The photostability, leaching, pK a and pH response of the entrapped probes were characterized, as was the performance of the entrapped enzymes, and an optimal set of processing conditions was obtained for each different sensing film. In general, the results indicated that SNARF-labelled dextran was the most useful pH sensitive dye owing to insensitivity to leaching and photobleaching. Furthermore, it was observed that the pK a and pH response of this probe was insensitive to preparation conditions. The performance of the co-entrapped enzymes was highly dependent on the type and level of additive, but in all cases, it was possible to obtain active enzymes with good performance characteristics. Reagentless sensing films for urea and glyceryl tributyrate (GTB) are demonstrated based on the detection of enzyme-mediated pH changes from films coated onto planar substrates.
Keywords: Fluorescence; Protein; Biosensor; Ormosil; Sol–gel;

Here we report a strand-specific fluorescent homogeneous assay format for rapid polymerase chain reaction (PCR). A number of similar assays are commonly used for research applications and are an ideal solution for a closed tube quantitative PCR. These assays use fluorescent resonant energy transfer (FRET) between donor and acceptor fluorescent moieties as the reporting mechanism. However, for different reasons these assays do not report amplification when very rapid cycling times are used. This is because current assays, such as TaqMan®, are limited, in terms of assay speed, by the 5′–3′ exonuclease activity of Taq DNA polymerase. Other assays based on hybridisation require either a complex de-conformational event to occur, or require more than one probe to report amplification. Reducing the complexity of the experiment reduces costs in terms of design, optimisation and manufacture. Here, we describe ResonSense® chemistries that use a simple linear fluorescent-labelled probe and a DNA minor-groove binding dye as either donor or acceptor moieties in a homogeneous assay format on the LightCycler®. This assay format will provide for rapid analysis of samples and so it is particularly well suited to point-of-use testing.
Keywords: Rapid PCR; ResonSense®; Angler®; LightCycler®; Fluorescence; SYBR®Gold;

Mass spectrometric identification of toxins of biological origin by James R Hancock; P.A D’Agostino (71-82).
The chemical/biological (CB) threat spectrum encompasses a wide range of potential agents including chemical warfare agents, biological warfare agents and toxins of biological origin that fall between these two main agent categories. These proteinaceous and non-proteinaceous toxins, commonly referred to as mid-spectrum agents, range in molecular mass from a few hundred to more than a hundred thousand daltons. The large number of potential candidates as well as the structural diversity of possible mid-spectrum agents makes identification of these compounds a challenge. The NATO defense community has recognized these challenges and has a working group that is developing identification protocols and evaluating methods through a series of international analytical exercises. Identification strategies rely heavily on recent advances that have been made in both mass spectrometry (MS) and liquid chromatography (LC), with LC–MS typically being employed as the primary method for separation/identification. While this paper focuses on the application of these and related instrumental analytical techniques for the identification of mid-spectrum agents, the approach described could be applied in the fields of toxicology, forensic science and environmental analysis. Areas for future research have been identified and application of developed mid-spectrum identification methods to the ongoing biological and toxin weapons convention (BTWC) are anticipated.
Keywords: Biological and toxin weapons convention; Liquid chromatography; Mass spectrometry;

Fingerprinting bacterial strains using ion mobility spectrometry by R.T. Vinopal; J.R. Jadamec; P. deFur; A.L. Demars; S. Jakubielski; C. Green; C.P. Anderson; J.E. Dugas; R.F. DeBono (83-95).
Ion mobility spectrometry (IMS) is currently in widespread use for the detection and identification of narcotic and explosive compounds without prior sample clean-up or concentration steps. IMS analysis is rapid, less than a minute, and sensitive, with detection limits in the nanogram to picogram range, depending on the target analyte. Our studies indicate that this technique has potential for detection of specific components of bacterial cells and for identification and differentiation of bacterial strains and species within a minute, and with no specialized test kits or reagents required. When microgram quantities of whole bacterial cells are thermally desorbed, complex positive or negative ion patterns (plasmagrams) are obtained. These plasmagrams differ reproducibly for different strains and species and for different conditions of growth, and can be used for the classification and differentiation of specific strains and species of bacteria, including pathogens. Methods for improved ion peak detection, most notably sequential sample desorption at stepped increases in temperature (programmed temperature ramping), are described.
Keywords: Ion mobility spectrometry; Microbiology; Pathogens; Bacterial fingerprinting;

Cholera toxin-induced modulation of gene expression: elucidation via cDNA microarray for rational cell-based sensor design by Hsingchi J. Lin; P.T. Charles; Joanne D. Andreadis; A.M. Churilla; David A. Stenger; J.J. Pancrazio (97-108).
Cell-based biosensors utilize functional changes in cellular response to identify the biological threats in a physiological relevant manner. Cell-based sensors have been used for a wide array of applications including toxicological assessment and drug-screening. In this paper, we utilize DNA arrays to identify differential gene expression events induced by toxin exposure for the purpose of developing a reporter gene assay system compatible with insertion into a cell-based sensor platform. HT29, an intestine epithelial cell line, was used as a cell model to study the cholera toxin (CT)-induced host cell modulation using DNA array analysis. A false positive model was generated from analysis of housekeeping genes in untreated control experiments to characterize our system and to minimize the number of false positives in the data. Threshold probability scores (−3.72), which gives <0.02% false positives for up/down regulation from the false positive model, were used to identify 73 and 25 known genes/expression tag sequences (ESTs) that were up- and down-regulated, respectively, in cells exposed 23 nM of CT. Using quantitative multiplex PCR assay, the gene expression levels for several genes shown to be modulated according to the microarray experiments, such as apolipoprotein D (Apol D), E-cadherin, and cyclin A2, were confirmed. The differential expression of genes encoding cytochrome P450, glutathione transferase (GST), and MGAT2 were noteworthy and consistent with previous studies. Our study provides an approach to analyze cDNA microarray data with defined false positive rates. The utility of cDNA microarray information for the design of cell-based sensor using a reporter gene approach is discussed.
Keywords: cDNA microarray; False positive; Cholera; Epithelial; Gene expression; Enterotoxin; Lymphoma;

Rapid and specific detection of bacteria using bioluminescence by D.J. Squirrell; R.L. Price; M.J. Murphy (109-114).
This paper describes rapid methods for the detection of very low numbers of bacteria. Specificity was obtained by the use of antibodies in immunomagnetic separation, a bacteriophage to allow targeted cell lysis, or a combination of both. The amplified endpoint assay used cell-derived adenylate kinase to convert added adenosine diphosphate (ADP) to adenosine triphosphate (ATP) which could then act as a substrate for the firefly bioluminescence reaction. Specific and non-specific assays were evaluated using Escherichia coli O157 as the test organism. Limits of detection of around 102  cells ml−1 could be obtained with an upper end cut-off of around 107  cells ml−1. Depending on the level of specificity offered, the assays took from 5 min to just under 1 h.
Keywords: Bacteria; Detection; Bioluminescence; Adenylate kinase; Bacteriophage; Immunomagnetic separation;

Development of a thermostable firefly luciferase by L.C Tisi; P.J White; D.J Squirrell; M.J Murphy; C.R Lowe; J.A.H Murray (115-123).
Firefly luciferase forms the basis of a wide range of analytical techniques. However, the enzyme is unstable and rapidly loses activity even at room temperature. This leads to losses in sensitivity and precision in analytical applications and also severely limits the fieldability of devices incorporating luciferase-based technologies. A number of point mutations have previously been identified that significantly increase the thermostability of the enzyme. We show here that when such mutations are combined they can have an additive effect on the stabilisation of the enzyme. As such, we have constructed a luciferase mutant containing four point mutations, relative to the wildtype enzyme, resulting in remarkably greater thermostability.
Keywords: Firefly luciferase; Microbiological detection; Thermostability; Protein engineering.;

Biological aerosol detection in real time is an urgent civilian and military requirement. Such detection capability will be useful in environmental monitoring, for example, in gathering information in perceived hazardous areas such as housing developments downwind of sewage treatment plants. To be truly functional, the instrument has to operate continuously, 24 h a day and 7 days a week with minimal maintenance and few false alarms. A novel concept is proposed. The system employs a rapid front-end warning/alarming mechanism based on optical technologies that provides useful information for protection decision makers. This is connected to a sample collector that feeds a slower back-end liquid chemistry system that provides analytical results to the medical personnel to assist in prophylaxis and therapy decisions. Experience gained from measuring fluorescence signals of single bacterial spores under flow cytometry (FCM) using UV excitation at 340–360 nm, was applied to concept testing of a prototype instrument, built to do the same for aerosols. This machine was capable of resolving particle size as well as fluorescence intensity of each particle under laboratory and field conditions; it was called the fluorescent aerodynamic particle sizer (FLAPS). This paper describes practical aspects of measuring biological aerosols when the results must be compared to reference samplers that provide culturable or “live” data. Treatment of particle size and fluorescence information is discussed with respect to FLAPS and reference data fidelity. Along with an objective method to evaluate FLAPS data correlation to reference data, an approach for determining limit of detection in the field is discussed. In addressing the back-end detector chemistry, we have prioritized a number of important biological characteristics that must be given to a clinician to help in prophylaxis and therapy decisions. A series of biochemical measurements are proposed to define the threat of a sample and different solutions are given to implement these tests. We predict that the future for biological detection looks promising for fluorescence in situ hybridization (FISH) techniques in identifying microorganisms. A conceptual instrument based on merging FCM and microchip-based analysis is described.
Keywords: Fluorescent aerodynamic particle sizer (FLAPS); Fluorescence in situ hybridization; Flow cytometry (FCM); Immunoassay; Levan; Antigen masking;