A Highly Sensitive Procedure for the Detection of Blood in Stool for Indications of Colon Cancer, Internal Bleeding, Disease and Other Traumas
Malaria is a debilitating, infectious disease characterized by chills, shaking and periodic bouts of intense fever. Each year, there are an estimated 400 million to 600 million cases of malaria and 2.7 million resulting deaths, worldwide. Malaria is found in many locations of the tropical world and in some locations of the subtropics, but there are only four species of the single-celled parasite of the genus Plasmodium that infect humans and cause the disease. Westerners who visit malarious countries are not immune. Several thousand return home from travels each year and are hospitalized with malaria. Expatriates and soldiers who live abroad are at a great risk of contracting malaria. Malaria was the number one cause of hospitalization among American troops deployed to Somalia; the number two cause among troops in Vietnam (after combat injury); and a leading cause among diplomats, missionaries and aid workers. Malaria case numbers are increasing markedly in many third world countries for several reasons including the cessation of malaria vector control programs. Mosquitoes are the vectors – or intermediate carrier of the parasite that causes malaria. The disease is transmitted between humans by the bite of a female mosquito. Vector controls are aimed at reducing the mosquito population and preventing their access to humans. However, financial obstacles limit the success of this effort in many developing countries. Insecticides are expensive and increasingly unsuccessful in killing mosquitoes that are developing resistances to the various chemicals. Antibiotics can be used as a preventative and a treatment. Unfortunately, drug resistance is becoming an alarming problem and contributes to uncontrollable outbreaks. Drug resistance is often connected with a legacy of foolishly overusing or under dosing antimalarial drugs. Expense is, again, an issue.
The present invention overcomes the aforementioned disadvantages by providing methods of producing electrodes comprising stable thiolated surfaces, and methods of using such electrodes to accumulate and detect thiol-binding analytes, especially heme, in a target sample with high degree of sensitivity and selectivity. In particular, applicants have discovered that the thiolated surfaces of the electrodes produced via the present inventions tend to be advantageously stable, i.e. avoid significant degradation, for periods of time as long as several hours to one or more days (or longer) either in the presence or absence of oxygen. Although applicants do not wish to be bound by or to any particular theory of operation, it is believed that the present methods provide electrodes which overcome the relative instability of prior art electrodes in the presence of oxygen by preparing the electrode surface through an electrochemical treatment prior to thiolating the surface. Tests were conducted which comprised aerating a target sample solution comprising heme and introducing an electrode of the present invention thereto. The tests showed that heme was as easily attached to the electrode in such solution as it is in specially de-aerated solutions, suggesting that the bonds formed between dithiol molecules and the electrode substrate according to the present methods do not readily break in the presence of oxygen.
Because of the aforementioned surface stability, the electrodes produced herein can be used advantageously according to the present invention to accumulate and detect amounts of thiol-binding analytes from low concentration analyte solutions with greater accuracy than prior art electrode processes. To ensure sufficient interaction of thiol-binding analyte molecules in relatively low concentration analyte solutions (for example, those having a concentration measured in nanomolar (nM) or even smaller units) with an electrode for the concentration and accurate detection thereof, it is often necessary to allow the electrode to remain in the target analyte solution for a period of time as long as several hours to one or more days. While many prior art electrodes tend to degrade before such necessary interaction times are achieved, the electrodes produced herein tend to be sufficiently stable to remain in solution for periods of time necessary to measure low analyte concentrations with an accuracy not previously achievable using prior art methods. Applicants have recognized, for example, that the electrode of the present invention can be used to detect thiol-binding analytes in solutions comprising an analyte concentration of greater or less than about 100 micromolar (.mu.M). In certain embodiments, the present methods can be used to detect analytes in solutions as low as from about 10 nM to about 100 .mu.M of analytes. Preferably, the present methods are capable of detecting analytes in solution comprising concentrations as low as less than about 10 nM analytes, and even more preferably less than about 1 nM analytes.
Applicants have further recognized that the electrodes having analyte accumulated thereon produced according to the present methods tend to be sufficiently stable to allow the electrode to be transferred from a sample solution to a test solution for use in analyte detection. By concentrating analyte samples onto an electrode and/or transferring the analyte into another solution, the present methods allow for a more sensitive, selective, and accurate detection of low analyte concentrations in sample solutions than is obtainable using prior art electrode methods. In addition, the accumulated-analyte electrodes can be transported in air or aqueous solution from, for example, a field testing site to the laboratory for analysis. This obviates the need to transport entire liquid samples, such as blood samples, which may require refrigeration or other handling and transport considerations, for testing to the laboratory.
According to certain embodiments of the present methods, applicants have also recognized that the production and use of electrodes having a fractal dimension (D.sub.f) of greater than about 2 allows for the detection of analytes in solution with greater sensitivity than prior art methods. As will be recognized by those of skill in the art, the term "fractal dimension" refers to a measurement of fractal geometric dimension. For example, a metal electrode with a flat surface has a D.sub.f =2. As discussed below, certain metal electrodes comprising coiled metal wires (in some cases with surfaces roughened via cyclic voltammetry) produced via the present methods have D.sub.f values of greater than 2. By using electrodes having D.sub.f >2, certain preferred embodiments of the present invention allow for the binding of greater amounts of dithiol compounds, and thus, greater amounts of analyte, to the electrode for the detection of analyte with greater accuracy and sensitivity than prior art methods.
According to one aspect, the present invention provides methods of producing an electrode comprising: providing a substrate capable of binding a dithiol molecule thereto; electrochemically treating the substrate to provide a treated substrate having a fractal dimension of greater than about 2; and contacting the treated substrate with dithiol molecules to produce an electrode having dithiol groups attached thereto and capable of binding an analyte thereto.
According to another aspect, the present invention comprises methods of accumulating an analyte capable of bonding to a dithiol moiety onto an electrode comprising: providing an electrode of the present invention capable of binding the analyte to be detected thereto; and contacting the electrode with a target solution comprising an analyte to bind at least a portion of the analyte to the electrode.