This project will advance the development of a point-of-care (POC) molecular diagnostic device for the rapid and inexpensive detection of infectious disease-causing pathogens in nontraditional heath care settings. An existing, fully integrated microfluidic Chemistry and Reagent Device (CARD(tm)) will be optimized for detection of pathogens in sexually transmitted infections (STIs) and urinary tract infections (UTIs). The CARD(tm) is capable of automatically isolating and lysing infectious agents, then extracting and purifying DNA and/or RNA and PCR amplifying and detecting target sequences (to date, whole blood, plasma, saliva and vaginal swabs have been successfully processed). To improve the ease-of-use of the existing CARD(tm), this project will further integrate the use of stable liposomes (at least three year real time stability has been demonstrated) to increase the signal intensity and simultaneously transduce the biological signal into an electronic one. This will be accomplished by conjugating oliogonucleotide probes that are complementary to portions of the amplified target sequences, to the exterior surface of liposomes, which, in turn, will encapsulate electroactive compounds. The resulting liposome-target complexes will be subsequently magnetically immobilized within the microfluidic channels of the CARD(tm) by use of magnetic beads conjugated to other oligonucleotide probes that are complementary to another region of the target sequences. Upon lysis of the immobilized liposomes, the released electroactive compounds will be allowed to flow onto the surface of an interdigitated ultramicroelectrode array where a continuous and reversible redox reaction will generate electric current that will be directly proportional to the concentration of the analyte. This project will therefore allow sophisticated molecular diagnostics to be performed by individuals of varying degrees of training (including unskilled), since once the sample is introduced all preparative, analytical and readout functions are automatically performed.
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