Small-Volume Detection of Plasmodium falciparum CSP Gene Using a 50-(mu)m-Diameter Cavity with Self-Contained Electrochemistry

摘要

An electrochemical enzyme-linked immobilized DNA-hybridization assay for the detection of Plasmodium falciparum has been developed. The target molecule was a segment of the repeat sequence of the gene coding for the circumsporozoite (CSP) protein from the AF54087 gene. This analyte offers the possibility of specifically detecting P. falciparum. The assay involves attachment of a biotinylated primary DNA probe via its 5'-amine-terminus to the streptavidin-coated surface of microwells in a 96-well plate. The primary DNA probe (1~(0)P, which was of two different sequences we call 1~(0)P_(a) and 1~(0)P_(b)) was used to capture the target (T, which was of two different sequences, T_(1) sequence 481-590 and T_(2) sequence 472-590 of AF54087 gene for the CSP gene) by hybridization to a complementary sequence on the target. On 1~(0)P_(a), 47 bases were complementary to T_(1) and T_(2) at 543-590, while on 1~(0)P_(b), 35 bases were complementary to T_(1) and T_(2) at 555-590. A secondary DNA probe that contained 36 bases with alkaline phosphatase (2~(0)P-AP) label on the 3' end was hybridized to a complementary base sequence on the 5' end of the target. p-Aminophenol, which is enzymatically generated by the immobilized AP from p-aminophenyl phosphate (PAPP), is detected using electrochemistry. The peak current of cyclic voltammograms from a PAPP solution incubated inside the microwells modified with the complete assembly of the assay components gives a linear relationship with the concentration of the target (2-50 ng/mL, where P_(1) (P_(1a) and P_(1b)) and P_(2)-AP concentrations are 50 ng/mL). A detection limit of 1.4 ng/mL (or 46 pM) of the DNA target was obtained. The signals of the assays were not significantly affected when performed in the presence of human hepatocytes, pig liver, or chicken serum indicating the viability of this assay in real clinical samples.