Michael Mecklenburg

Microformat imaging ELISA for pesticide determination.

A flat-well microformat competitive enzyme-linked immunosorbent chemiluminescent assay for the detection of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) is described. Thick-film technology was used to pattern a hydrophobic layer 100 μm thick onto glass microscope slides to form an array of 2 × 2 mm(2) squares. These flat wells were able to hold 2 μL of reagents, corresponding to a height of ∼500 μm, with minimal contamination risk. The hydrophobic ink used to pattern the surfaces allowed significantly larger volumes of samples to be applied when compared with surfaces patterned with nonhydrophobic inks. This reduced evaporation effects and permitted greater pipetting accuracy, thereby improving assay reproducibility. A competitive immunoassay was developed based on the ability of free 2,4-D hapten to inhibit binding of anti-2,4-D monoclonal antibodies to 2,4-D-bovine serum albumin conjugate adsorbed onto the glass support. The support was subsequently incubated with alkaline phosphatase (AP) labeled anti-mouse IgG. The amount of AP conjugate bound was determined by quantitating the chemiluminescent emission produced from the enzymatic breakdown of CSPD substrate by AP using a cooled CCD camera. The detection limit of the single-sample microformat assay was 2.7 × 10(-)(11) M, or 6 pg of 2,4-D. The linear ranges of the single-sample and multisample assays were 4.5 × 10(-)(8)-4.5 × 10(-)(11) and 4.5 × 10(-)(7)-1.66 × 10(-)(10) M, respectively. In comparison, the detection limits of a tube-based chemiluminescent assay using standard luminometer and of a colorimetric ELISA were 45 × 10(-)(11) and 9.9 × 10(-)(8) M, respectively. The ability to scale the thick-film-based microformat assay makes it an ideal candidate for the development of affinity arrays and high-throughput assay formats. Prospects for further improvements of this imaging ELISA strategy will be discussed.

Development of an integrated thermal biosensor for the simultaneous determination of multiple analytes

A flow injection thermal microbiosensor was designed for the simultaneous determination of multiple analytes. The biosensor consisted of five thin-film thermistors which were located along a single microchannel. The device was fabricated on a quartz chip by micromachining. The feasibility of employing this system for the detection of two independent enzyme reactions was demonstrated using two different pairs of enzymes, urease–penicillinase and urease–glucose oxidase. The enzymes were immobilized on agarose beads, which were then sequentially packed into distinct regions of the microchannel. Using this method, samples containing urea mixed with penicillin V or with glucose were simultaneously analysed. Linear ranges of up to 20 mmol l–1 urea, 40 mmol l–1 penicillin V and 8 mmol l–1 glucose (saturated with O2) were obtained using a flow rate of 30 µl min–1 and a sample volume of 20 µl. The relative standard deviations for urea and penicillin V assays were 1.13 and 2.42% for the first 100 samples and 1.17 and 2.78% for 200 samples, respectively. The sensor is capable of analysing 25 samples per hour.

Microbiosensor based on an integrated thermopile

A microbiosensor based on an integrated thermopile was designed and fabricated on a quartz chip. The thermopile, which was manufactured by doping boron in polysilicon together with aluminium, provided a potential output of ca. 2 mVK. A silicone rubber membrane was used to form and seal the microchannel. The total column volume was 20 μl. Glucose oxidase and catalase were co-immobilized on spherical CPG beads (controlled pore glass) and in turn charged into the microchannel. Using 1 μl sample volumes, a linear range of 2 to 25 mM glucose was obtained using a flow rate of 105 μlmin. The relative standard deviation for 100 glucose samples (10 mM) was 5%.

A strategy for the broad range detection of compounds with affinity for nucleic acids

Abstract A nucleic acid based optical biosensing strategy for the broad range detection of compounds with affinity for nucleic acids is described. The strategy attempts to use the chemical and structural information contained in the DNA helix as a sensing element per se . Detection is based on measuring changes in the fluorescence signal intensity of ToPro-3 complexed with nucleic acids. ToPro-3 is a cationic, planar aromatic fluorescent nucleic acid dye that binds to DNA by intercalation. The fluorescence of the dye is environment dependent and is greatly enhanced upon intercalation. ToPro-3 has a long excitation wavelength of 642 nm with emission occurring at 661 nm. At these wavelengths, spectral interference from organic compounds is dramatically reduced. The sensor is capable of detecting known intercalating and groove binding compounds over a broad range of binding affinities. Detection limits of 200, 20, 95 and 1 nM were obtained for EtBr, DAPI, PI and TOTO1, respectively. The ability of the assay to detect the known mutagens bisbenzidine, 1,2,4-benzenetriamine and proflavin, as well as of unknown compounds in natural product extracts was also investigated. Furthermore, the use of longer wavelengths allows the implementation of solid-state instrumentation which allows stable DC operation, reduces electronic noise and simplifies miniaturization, all of which increase portability. This scheme provides a flexible strategy for detecting compounds with affinity for DNA that can be used in a wide range of application areas. Approaches for expanding the informational content of the scheme are discussed.

A simple competitive enzyme-linked immunosorbent assay using antigen-β-galactosidase fusions

The fusion of the N-terminal 461 bp of the human interferon-alpha 2 (INF) in frame to the beta-galactosidase gene from Escherichia coli is described. The presence of the expected DNA sequence was shown by restriction mapping and DNA sequencing. A fusion protein was demonstrated in crude extracts of E. coli by Western blots using polyclonal anti-beta-galactosidase and monoclonal anti-IFN antibodies. Using monoclonal antibodies specific for the N-terminal region of IFN-alpha and cell-free extracts from an E. coli strain containing the fusion protein, we set up a simple competitive enzyme-linked immunosorbent assay for human interferon. The test described here was linear down to a lower detection limit of at least 1000 Units, or 5 ng human IFN.

Optimization of a charge coupled device imaging enzyme linked immuno sorbent assay and supports for the simultaneous determination of multiple 2,4-D samples

Abstract A chemiluminescent microformat enzyme linked immuno sorbent assay (ELISA) has been optimized for the simultaneous determination of multiple 2,4-dichlorophenoxyacetic acid (2,4-D) samples. The competitive immunoassay employed a 2,4-DBSA conjugate, anti-2,4-D monoclonal antibodies and alkaline phosphatase (AP) labelled anti-mouse IgG. The bound AP conjugate was determined by quantitating the chemiluminescence emission from the enzymatic decomposition of the luminogenic substrate, CSPD, by AP using a cooled charge coupled device (CCD) camera. The detection limit for the simultaneous determination of multiple samples was 4.3 × 10−10 M corresponding to 96 pg ml−1 or 192 fg well with a coefficient of variation (CV, %) of 12.5%. The linear range of the assay was 4.5 × 10−7–4.5 × 10−10M. The ability of gold coated silicon wafers and glass capillaries to serve as solid phase supports in the imaging ELISA was investigated. The highly reflective gold surfaces improved both the linear range and the sensitivity of the assay, as compared to thick-film patterned surfaces. The capillary supports, on the other hand, lead to a reduction in the linear range and the sensitivity of the assay, as compared to the thick-film patterned surfaces. Initial studies indicate that the capillaries guide the light and may provide a built-in mechanism for collecting the emitted light. Strategies for further development of support materials for imaging-based detectors will be discussed.

Fast determination of whole blood glucose with a calorimetric micro-biosensor

Abstract Fast determination of whole blood glucose without any pretreatment was achieved using a calorimetric FIA micro-biosensor. Glucose oxidase was covalently immobilized, together with catalase, onto controlled pore glass beads and packed into a micro-column. By reducing the sample volume down to 1 μ1, a measurement period of about 40 s and an enlarged linear range of 0.5–20 mM glucose were obtained. The correlation coefficient was 0.98 when the micro-biosensor was compared to the Reflolux-S blood glucose analyser. The relative standard deviation for 100 blood samples was 3.7%.

Differentiation of human serum samples by surface plasmon resonance monitoring of the integral glycoprotein interaction with a lectin panel

Bacterial infection and inflammation result in massive changes in serum glycoproteins. These changes were investigated by the interaction of the saccharide glycoprotein moiety with lectins. A panel of eight lectins (Canavalia ensiformis, Bandeiraea simplicifolia BS-I, Arachis hypogaea, Phytolacca americana, Phaseolus vulgaris, Artocarpus integrifolia, Triticum vulgaris and Pisum sativum) was used to differentiate human serum glycoproteins obtained from patients with various bacterial infections. Lectin functionalised sensing layers were created on gold-coated wafers and lectin-glycoprotein interactions were monitored by surface plasmon resonance. The interaction of the lectin panel with serum glycoproteins produces unique patterns. Principal component analysis (PCA) was used to analyse the patterns. The actual panel of eight lectins enabled discrimination between sera obtained from patients sick with bacterial infection and healthy patients. Extended lectin panels have the potential to distinguish between types of bacterial infection and identify specific disease state. (Less)

Optical surface methods for detection of nucleic acid binding

Abstract Detection of DNA-binding and hybridization using pseudo-Brewster angle reflectometry and ellipsometry is described. DNA was attached to planar silicon surfaces coated with a 150 A layer of nitrocellulose or with a monolayer of aminosilane. Poly-L-lysine was used as a linker between the modified surfaces and the DNA. The course of the reassociation was monitored continuously with the reflectometry method. A reassociation time of approximately one hour was observed for polynucleotides of 0.5–1.0 kb.

Fast determination of antibiotics in whole blood.

There is a need for analytical methods capable of monitoring blood antibiotic levels in real time. Here we present a method for quantifying antibiotic levels in whole blood that does not require any sample pretreatment. The tests employ the enzyme penicillinase to assay for penicillin G, penicillin V and ampicillin using a flow-injected biosensor, the Enzyme Thermistor. Optimal flow rates, sample volumes and pH were determined to be 0.5 mL/min, 100 μL and 7.0, respectively. Analysis of the antibiotics diluted in buffer gave a linear range of 0.17-5.0 mM. Calibration curves prepared using blood spiked with the antibiotics gave a linear range of 0.17-2.0 mM. Linear regression values for all of the calibration curves were 0.998 or higher. Assay cycle time was 5 min. The relative standard deviation value for 100 determinations of a mock blood sample spiked with penicillin G was 6.71%. Despite the elimination of sample pretreatment, no detectable clogging or signal drift was observed. The assay provides a fast, simple, reliable analytical method for determining antibiotic concentrations in blood without the need for any sample pretreatment. This is an important first step towards developing a device capable of real-time monitoring of antibiotic levels in whole blood. The technology has the potential to significantly improve the outcomes of patients undergoing critical care.