Methods for using redox liposome biosensors

Details Methods for using redox liposome biosensors Methods for using redox liposome biosensors

2000-06-30

2002-05-14

Stucker, Jeffrey (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C435S005000, C435S006120, C435S007100, C435S007200, C435S007210, C435S007310, C435S007320, C435S007370, C436S501000, C436S518000, C436S149000, C436S151000, C436S801000, C436S806000, C436S904000, C204S403060

Reexamination Certificate

active

06387614

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides novel methods and compositions for preparing and using electrochemical sensors to signal the presence of biological molecules.
BACKGROUND OF THE INVENTION
Bacterial toxins are a primary cause of a variety of human diseases. For instance, some strains of
Escherichia coli
residing in the intestines of man and many other animals are capable of secreting various enterotoxigenic toxins. Indeed, these toxigenic strains of
E. coli
are generally considered as a cause of many diarrheal diseases (Scotland et al., Lancet i:90 [1980]).
E. coli
is the organism most commonly isolated in clinical microbiology laboratories, as it is usually present as normal flora in the intestines of humans and other animals. However, it is an important cause of intestinal, as well as extraintestinal infections. For example, in a 1984 survey of nosocomial infections in the United States,
E. coli
was associated with 30.7% of the urinary tract infections, 11.5% of the surgical wound infections, 6.4% of the lower respiratory tract infections, 10.5% of the primary bacteremia cases, 7.0% of the cutaneous infections, and 7.4% of the other infections (Farmer and Kelly, “Enterobacteriaceae,” in
Manual of Clinical Microbiology,
Balows et al.(eds), American Society for Microbiology, [1991], p. 365). Surveillance reports from England, Wales and Ireland for 1986 indicated that
E. coli
was responsible for 5,473 cases of bacteremia (including blood, bone marrow, spleen and heart specimens); of these, 568 were fatal. For spinal fluid specimens, there were 58 cases, with 10 fatalities (Farmer and Kelly, supra, at p. 366). There are no similar data for United States, as these are not reportable diseases in this country.
Studies in various countries have identified certain serotypes (based on both the O and H antigens) that are associated with the four major groups of
E. coli
recognized as enteric pathogens. Table 1 lists common serotypes included within these groups. The first group includes the classical enteropathogenic serotypes (“EPEC”); the next group includes those that produce heat-labile or heat-stable enterotoxins (“ETEC”); the third group includes the enteroinvasive strains (“EIEC”) that mimic Shigella strains in their ability to invade and multiply within intestinal epithelial cells; and the fourth group includes strains and serotypes that cause hemorrhagic colitis or produce Shiga-like toxins (or verotoxins) (“VTEC” or “EHEC” [enterohemmorrhagic
E. coli
]).
TABLE 1
Pathogenic
E. coli
Serotypes
Group
Associated Serotypes
Enterotoxigenic
O6:H16; O8:NM; O8:H9; O11:H27; O15:H11;
(ETEC)
O20:NM; O25:NM; O25:H42; O27:H7; O27:H20;
O63:H12; O78:H11; O78:H12; O85:H7; O114:H21;
O115:H21; O126:H9; O128ac:H7; O128ac:H12;
O128ac:H21; O148:H28; O149:H4; O159:H4;
O159:H20; O166:H27; and O167:H5
Enteropathogenic
O26:NM; O26:H11; O55:NM; O55:H6; O86:NM;
(EPEC)
O86:H2; O86:H34; O111ab:NM; O111ab:H2;
O111ab:H12; O111ab:H21; O114:H2; O119:H6;
O125ac:H21; O127:NM; O127:H6; O127:H9;
O127:H21; O128ab:H2; O142:H6; and O158:H23
Enteroinvasive
O28ac:NM; O29:NM; O112ac:NM; O115:NM;
(EIEC)
O124:NM; O124:H7; O124:H30; O135:NM;
O136:NM; O143:NM; O144:NM; O152:NM;
O164:NM; and O167:NM
Verotoxin-
O1:NM; O2:H5; O2:H7; O4:NM; O4:H10; O5:NM;
Producing
O5:H16; O6:H1; O18:NM; O18:H7; O25:NM;
(VTEC))
O26:NM; O26:H11; O26:H32; O38:H21; O39:H4;
O45:H2; O50:H7; O55:H7; O55:H10; O82:H8;
O84:H2; O91:NM; O91:H21; O103:H2; O111:NM;
O111:H8; O111:H30; O111:H34; O113:H7;
O113:H21; O114:H48; O115:H10; O117:H4;
O118:H12; O118:H30; O121:NM; O121:H19;
O125:NM; O125:H8; O126:NM; O126:H8; O128:NM;
O128:H2; O128:H8; O128:H12; O128:H25;
O145:NM; O125:H25; O146:H21; O153:H25;
O157:NM; O157:H7; O163:H19; O165:NM;
O165:19; and O165:H25
Detection of these toxins commonly involves the use of biological (i.e., animal) assays and immunoassays (for review, See, Jay (ed.),
Modern Food Microbiology,
Chapman and Hall, New York [1996]). Bioassays typically involve whole- or part-animal test, which are expensive and require a few days to complete. Imunological assays, on the other hand, couple antibody binding with optical signaling and amplification. The assay time can be reduced to one day or a few hours, depending on the type of method and toxin involved. Currently, various immunoassays are available for bacterial toxins include gel diffusion, reverse passive latex agglutination (RPLA) and enzyme-linked immunosorbent assay (ELISA) (See e.g., Pimbly and Patel, J. Appl. Microbiol. (Suppl.), 84:S98 [1998]). However, these techniques do not give results in a real-time detection fashion. In addition, RPLA can be affected by non-specific interference from materials which physically interfere the formation of a tight button (Mpamugo et al., J. Med. Microbiol., 43:442 [1995]), while ELISA is subject to enzymatic interference from samples containing peroxidase (Park et al., Appl. Environ. Microbiol., 60:677 [1994]). Poor antibody stability limits the use of these methods in field applications. An alternative approach for toxin detection involves the use of the polymerase chain reaction (PCR) to detect the bacterial gene(s) responsible for the production and release of toxins. While highly specific and sensitive, results from PCR may be inconclusive, as the gene may be present, but the toxin may be absent when the organisms are killed. Furthermore, inhibitors of DNA polymerases found in some samples may interfere with the detection of toxin.
Clearly, there is a need for fast, reliable, specific and sensitive methods for the detection of bacterial toxins for use in outbreak investigations, clinical diagnostics and quality monitoring in the food and feed industries. Recent progress in toxin detection methods has primarily focused on supramolecular assemblies (e.g., LB [Langmuir-Blodgett] monolayers and lipid bilayer membranes) coupled with specific cell surface receptors (See e.g., Charych et al., Chem. Biol., 3:113 [1996]). It has been shown that by engineering lipid membranes with desirable optical ‘reporting’ ability, colorimetric detection of cholera toxin (CT) can be achieved. The colorimetric sensor consists of self-assembly of the amphiphilic diacetylenic lipids and cell surface receptor GM1 in forms of LB monolayers and bilayer vesicles. Nonetheless, in spite of improvements in optical sensors, a still greater degree of sensitivity is needed in the art.
SUMMARY OF THE INVENTION
The present invention provides novel methods and compositions for preparing and using electrochemical sensors to signal the presence of biological molecules. It is not intended that the present invention be limited to any particular biological molecule. For example, it is contemplated that the present invention will find use in the detection and/or identification of microorganisms, prions, microbial toxins, antigens and/or antibodies, antigen and antibody complexes, and other suitable compounds or compositions of interest.
In some embodiments, the present invention is directed to novel biosensors for amperometric detection of
E. coli
heat-labile enterotoxin, in particular, enterotoxin LT. In preferred embodiments, the novel biosensor couples a redox supramolecular assembly with a sol-gel thin film electrode. In particularly preferred embodiments, the sensor utilizes an open platform to host biosensory elements, thereby allowing fast access of the target molecules to the redox vesicles for detection. Compared to other designs involving sol-gel encapsulation of enzymatic centers for biosensing, the diffusion process is greatly improved and molecular access is less restricted in the present invention. The measured apparent diffusion coefficients for lipid ferrocene are about 2-3 orders of magnitude higher than those for redox-doped polymer/gels. The response time and sensitivity can therefore be improved by the enhancement in mass transport.
The present invention also provides methods for using the amperometric biosensor. As described in the Exampl

Affiliated with

Also associated with

Rating

Methods for using redox liposome biosensors does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Methods for using redox liposome biosensors, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods for using redox liposome biosensors will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2886845