Thin film biosensor for rapid visual detection of nucleic acid targets.

BACKGROUND: We have developed a silicon-based biosensor that generates a visual signal in response to nucleic acid targets. METHODS: In this system, capture oligonucleotide probes are immobilized on the surface of the biosensor. Interaction of the capture probes with a complementary target and a biotinylated detector oligonucleotide allows initiation of formation of an organic thin film on the biosensor. Thin film formation is completed by enzymatic activity of peroxidase conjugated to an anti-biotin antibody. Peroxidase catalyzes deposition of an insoluble product onto the silicon surface, generating a uniform thin film. The increased thickness on the surface alters the perceived color of the biosensor through changes in the interference patterns of reflected light from the surface, causing a color change from gold to purple. RESULTS: The biosensor results may be evaluated by direct visual inspection or quantified by ellipsometry. Results are obtained in 25 min with a detection limit of 5 pmol/L (150 amol/sample). Selectivity of the biosensor is demonstrated by discrimination of single nucleotide mismatches. Multitarget arrays are also analyzed with the thin film biosensor, and the system is capable of detecting targets from human serum and urine. CONCLUSIONS: The biosensor surface is inexpensive to produce, and the assay format is simple and rapid. The thin film biosensor is adaptable to a wide variety of nucleic acid detection applications, including rapid diagnostic testing for infectious disease panels, antibiotic resistance panels, or allelic discrimination of specific genetic markers.

A new optical immunoassay for detection of S-100B protein in whole blood.

BACKGROUND: S-100B is a protein mainly found in astroglial cells and only detected to a low level in blood. Serum levels of S-100B increase in patients with acute brain injuries. The aim of this study was to establish feasibility of a new Optical ImmunoAssay ([OIA], Bio-Star, Inc, Boulder, CO) test for determination of S-100B in blood. METHODS: We have developed a new, rapid, and sensitive OIA test to identify elevated levels of S-100B in whole blood. The OIA test for S-100B combines monoclonal antibodies specific for the B-subunit of S-100 with OIA thin film technology. Each sample was tested for S-100B by the OIA method and a commercially available immunoluminometric assay. Blood samples were drawn serially from 9 patients undergoing coronary artery bypass graft surgery and during the early postoperative period. RESULTS: The OIA test determination of S-100B protein correlated with immunoluminometric assay data (r = 0.8) with a detection limit of 0.25 ng/mL. CONCLUSIONS: The sensitivity and feasibility of this rapid assay may be suitable for rapid evaluation of S-100B in urgent care settings (surgery, intensive care units, or emergency room).

Fixed polarizer ellipsometry for simple and sensitive detection of thin films generated by specific molecular interactions: applications in immunoassays and DNA sequence detection.

Biological thin films may form on a surface by specific molecular interactions. The fixed polarizer ellipsometer (FPE) is a sensitive instrument that detects biological thin films either qualitatively or quantitatively. The design is simple and inexpensive. The assays are formatted on an optical surface, and the FPE detection is based on the phase shift of linearly polarized light after reflection through a thin film. We have constructed mathematical models of the FPE response to reflection through single-layer and two-layer films that agree closely with experimental data. Several biological assays have been measured with the FPE to demonstrate the application of this technology to clinical targets, including ultrasensitive immunoassays for hepatitis B surface antigen (0.1 ng/mL) and alpha-fetoprotein (0.01 ng/ mL) and DNA hybridization (0.5 fmol/microL target probe). A clinical study for detection of group A streptococcus from patient throat swabs demonstrated the qualitative application of the FPE to infectious disease targets. The flexibility and sensitivity of the FPE makes this technology suitable for numerous target analytes and applications.

Cell cycle regulation of induced mutagenesis in yeast.

The Saccharomyces cerevisiae CDC7 gene encodes a protein kinase that functions in three aspects of DNA metabolism: replication, repair, and meiotic recombination. It is likely that these functions overlap and share common elements. The cell cycle dependence of Cdc7 associated DNA repair was examined by UV irradiating a wild type and hypomutable cdc7-7 strain throughout the cell cycle. Both the wild type strain and the cdc7-7 mutant stain delay entry into S phase by 40-60 min when exposed to UV mutagenesis. Cells in G1 are the most sensitive to lethal UV damage while cells in S phase sustain fewer lethal hits. The yield of mutants is greatest for the CDC7 wild type strain when S phase cells are mutagenized. This peak of induced mutagenesis is absent in the cdc7-7 strain. Cdc7 protein may be required for error-prone DNA repair or for translesion error-prone DNA replication and not for the checkpoints in G1 phase. Because Cdc28 protein kinase and Dbf4 protein, a Cdc7 kinase regulator, are also important for induced mutagenesis and the CDC7 promoter is not induced in response to DNA damage, Cdc7 protein kinase may be regulated post-translationally following DNA damage, in the same manner as it is regulated during the cell cycle.

Effect of specimen storage, antibiotics, and feminine hygiene products on the detection of group B Streptococcus by culture and the STREP B OIA test.

Agar culture from vaginal swabs is the routine method for diagnosis of maternal Group B Streptococcus (GBS) colonization. Swab specimens are often transported to a clinical laboratory for processing. In these studies, specimen transport was simulated by inoculating swabs with GBS and storing them at selected temperatures and with or without transport medium. The recovery of viable GBS was assessed by agar culture. GBS antigen was detected immunologically with an Optical ImmunoAssay (OIA) method. Swabs that were stored with transport medium harbored viable but rapidly declining numbers of GBS. In contrast, a strong OIA signal was maintained. Recovery of viable GBS organisms declined more quickly when swabs were stored in the absence of transport medium, whereas detection of GBS antigen remained consistent. Both methods were tested for interference from either antibiotics or feminine hygiene products. These compounds inhibited the detection of GBS by culture but had no detrimental effect on the OIA result.

Molecular genetic studies of the Cdc7 protein kinase and induced mutagenesis in yeast.

The Saccharomyces cerevisiae CDC7 gene encodes a protein kinase that functions in DNA replication, repair, and meiotic recombination. The sequence of several temperature-sensitive (ts) cdc7 mutations was determined and correlated with protein kinase consensus domain structure. The positions of these ts alleles suggests some general principles for predicting ts protein kinase mutations. Pedigree segregation lag analysis demonstrated that all of the mutant proteins are less active or less stable than wild-type Cdc7p. Two new mutations were constructed, one by site-directed and the other by insertional mutagenesis. All of the cdc7 mutants were assayed for induced mutagenesis in response to mutagenic agents at the permissive temperature. Some cdc7 mutants were found to be hypomutable, while others are hypermutable. The differences in mutability are observed most clearly when log phase cells are used. Both hypo- and hypermutability are recessive to wild type. Cdc7p may participate in DNA repair by phosphorylating repair enzymes or by altering chromatin structure to allow accessibility to DNA lesions.

Molecular analysis of hemolytic and phospholipase C activities of Pseudomonas cepacia.

By using a gene-specific fragment from the hemolytic phospholipase C (PLC) gene of Pseudomonas aeruginosa as a probe and data from Southern hybridizations under reduced stringency conditions, we cloned a 4.2-kb restriction fragment from a beta-hemolytic Pseudomonas cepacia strain which expressed hemolytic and PLC activities in Escherichia coli under the control of the lac promoter. It was found, by using a T7 phage promoter-directed expression system, that this DNA fragment carries at least two genes. One gene which shares significant DNA homology with both PLC genes from P. aeruginosa encodes a 72-kDa protein, while the other gene encodes a 22-kDa protein. When both genes on the 4.2-kb fragment were expressed from the T7 promoter in the same cell, hemolytic and PLC activities could be detected in the cell lysate. In contrast, when each individual gene was expressed in different cells or when lysates containing the translated products of each separate gene were mixed, neither hemolytic activity nor PLC activity could be detected. Clinical and environmental isolates of P. cepacia were examined for beta-hemolytic activity, PLC activity, sphingomyelinase activity, and reactivity in Southern hybridizations with a probe from P. cepacia which is specific for the larger gene which encodes the 72-kDa protein. There were considerable differences in the ability of the different strains to express hemolytic and PLC activities, and the results of Southern DNA-DNA hybridizations of the genomic DNAs of these strains revealed considerable differences in the probe-reactive fragments between high- and medium-stringency conditions as well as remarkable variation in size and number of probe-reactive fragments among different strains. Analysis of the genomic DNAs from hemolytic and nonhemolytic variants of an individual strain (PC-69) by agarose gel electrophoresis. Southern hybridization, and transverse alternating pulsed field gel electrophoresis suggests that the conversion of the hemolytic phenotype to the nonhemolytic phenotype is associated with either the loss of a large plasmid (greater than 200 kb) or a large deletion of the chromosome of P. cepacia PC-69.

Molecular comparison of a nonhemolytic and a hemolytic phospholipase C from Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces two secreted phospholipase C (PLC) enzymes. The expression of both PLCs is regulated by Pi. One of the PLCs is hemolytic, and one is nonhemolytic. Low-stringency hybridization studies suggested that the genes encoding these two PLCs shared DNA homology. This information was used to clone plcN, the gene encoding the 77-kilodalton nonhemolytic PLC, PLC-N. A fragment of plcN was used to mutate the chromosomal copy of plcN by the generation of a gene interruption mutation. This mutant produces 55% less total PLC activity than the wild type, confirming the successful cloning of plcN. plcN was sequenced and encodes a protein which is 40% identical to the hemolytic PLC (PLC-H). The majority of the homology lies within the NH2 two-thirds of the proteins, while the remaining third of the amino acid sequence of the two proteins shows very little homology. Both PLCs hydrolyze phosphatidylcholine; however, each enzyme has a distinct substrate specificity. PLC-H hydrolyzes sphingomyelin in addition to phosphatidylcholine, whereas PLC-N is active on phosphatidylserine as well as phosphatidylcholine. These studies suggest structure-function relationships between PLC activity and hemolysis.

Genetic mapping of the structural gene for phospholipase C of Pseudomonas aeruginosa PAO.

An insertion mutation constructed by gene replacement methods was used to map the gene corresponding to the hemolytic phospholipase C (plcS gene) in Pseudomonas aeruginosa PAO1 by R68.45-mediated conjugation. plcS mapped approximately at 67 min on the 75-min chromosomal map (B. W. Holloway, K. O'Hoy, and H. Matsumoto, p. 213-221, in S. J. O'Brien, ed., Genetic Maps 1987, vol. 4, 1987), between the markers pur-67 and pru-375 and considerably distal to the regulatory genes plcA and plcB, which are located at approximately 12 min.

Mutations in the hemolytic-phospholipase C operon result in decreased virulence of Pseudomonas aeruginosa PAO1 grown under phosphate-limiting conditions.

The phospholipase C (PLC) operon of Pseudomonas aeruginosa consists of plcS, which encodes a heat-labile secreted hemolysin, and two in-phase, overlapping genes, plcR1 and plcR2, which may encode Pi-regulatory genes. A 2.8-kilobase-pair deletion mutation in this operon was constructed, and a tetracycline resistance (Tcr) cartridge replaced the deleted sequences. A deletion mutant of strain PAO1 was obtained through recombination between the flanking regions of the mutated cloned PLC operon and the homologous chromosomal regions. The deletion of the chromosomal PLC operon and its replacement by the Tcr cartridge was confirmed by Southern hybridization. The deletion strain, PLC SR, is nonhemolytic. However, it retains PLC activity when measured on a synthetic substrate. A second mutant strain, PLC R, contains a deletion in the plcR genes. This mutant is more hemolytic and produces more enzymatic activity than PAO1. The virulence of both of these mutants was compared with that of PAO1 in the mouse burn model of infection. When mice were infected with cultures grown in a high-Pi medium, there was a 10-fold increase in the 50% lethal dose of the mutants compared with PAO1. In contrast, when the inoculum originated from low-Pi cultures, there was a 200- to 10,000-fold increase in the 50% lethal dose of the mutants over PAO1.

Identification of a new phospholipase C activity by analysis of an insertional mutation in the hemolytic phospholipase C structural gene of Pseudomonas aeruginosa.

The phospholipase C (PLC) gene of Pseudomonas aeruginosa encodes a heat-labile secreted hemolysin which is part of a Pi-regulated operon. The structural gene for PLC, plcS, was mutated in vitro by insertion of a tetracycline resistance gene cartridge. Gene replacement techniques were used to introduce the mutated plcS gene into the P. aeruginosa chromosome in place of the wild-type gene. The precise replacement of wild-type sequences by mutant sequences was confirmed by Southern hybridization. The mutant strain, designated PLC S, is nonhemolytic and lacks a 78-kilodalton protein corresponding to the size of the wild-type PLC. However, there is an additional phospholipase activity present in PLC S capable of hydrolyzing p-nitrophenylphosphorylcholine, a synthetic PLC substrate, and phosphatidylcholine. This enzymatic activity is not a result of a truncated product produced from the mutated plcS gene. The phospholipase activity of PLC S was identified as a nonhemolytic PLC.