Experimental Studies Of Multichannel Biosensor Chip Using Enzyme-linked Immunoassay As Technology For Detection Of Acute Promyelocytic Leukemia MRNA

Acute promyelocytic leukemia (APL) accounts for approximately 10% to15% of all cases of acute myeloid leukemia (AML). 95% of APL patients have PML/RARαfusion gene which is APL specific signal of the molecular biology. The detection of PML/RARαfusion gene not only assists the diagnosis of APL, but the quantitation of the fusion gene also has the important significance to the detection of minimal residual disease (MRD). Because the traditional morphology and routine PCR technology can't quantitate, we designed a novel method to detect APL PML/RARαmRNA in this study. Based on electrochemical DNA biosensor and multi-channel biosensor chip, the multi-channel electrochemical chip was developed for detection of PML/RARαmRNA in APL. Electrochemical DNA biosensor is a novel biosensor which developed rapidly in recent years, with the advantages of fast detection, simplity, sensitivity, low price, and so on. The multi-channel biosensor chip is also a new technology which is coming to the forefront of life science in the recent years. There are 16 wells on the multi-channel biosensor chip, and every tunnel is a detecting head, including a working electrode, reference electrode and auxiliary electrode. Therefore, each tunnel could be a independent sensor. Combining the functions of both, the multichannel electrochemical chip can implement that once operation and multiple detection, which is of fast, high efficient and simultaneous treatment, which is of wide prospect.Deoxyribonucleic acid, or DNA, is a nucleic acid molecule that contains the genetic instructions used in the development and functioning of all known living organisms. Many deoxynucleotide residues which are linked together with 3', 5'-phosphodiester bond in turn make up the long strands, named DNA . Bulk of DNA, as a long-chain molecule, were discovered to be a double helix. Under high temperature, the DNA duplex is melting into the single-strand DNA(ssDNA) which can rehybridize into double strands(dsDNA) thus to interfere the target DNA hybridization between and DNA probe. Ribonucleic Acid, or RNA, is a long chain molecule which forms by the condensation of ribonucleotide through phosphate ester linkage. RNA is general a long-chain molecule not a double helix, which is different with DNA. Accordingly, RNA could hybridize fully with DNA probe withou denaturation and renaturation. Therefore, we take for APL PML/RARαmRNA as the testing targets.Two novel DNA electrochemical probes (hairpin probe and Sandwich-type probe) were respectively designed and developed the multichannel electrochemical chips for detection of PML/RARαmRNA in acute promyelocytic leukemia (APL) in this article. The stability and the specificity of these multichannel electrochemical chips were also been investigated.Therein, Sandwich-type DNA electrochemical probe was designed according to APL PML/RARαmRNA fragment in this study. And then, based on the electrochemical enzyme immunoassay, the relationship between amperometric current and the concentration of synthetic target mRNA complementary strand is linear in the range of 0.1 pM to 200 pM with the detection limit of 1.2×10-14 M. At the same time, the method that demonstrates its simple and good specificity to distinguish the target sequences, single-base mismatched sequences, PML sequences, RARαsequences and non-complementary sequences. It is possible to qualitatively and quantitatively detect PML/RARαmRNA in APL.Besides, hairpin DNA electrochemical probe (HP) was designed according to APL PML/RARαmRNA fragment. And then, based on the electrochemical enzyme immunoassay, the hairpin probe modified multichannel electrochemica chip was developed for detection of PML/RARαfusion gene in APL in this study. The results indicate that, under the optimal conditions, the relationship between amperometric current and the concentration of synthetic target mRNA complementary strand is linear in the range of 0.5 pM to 300 pM with the detection limit of 5.0×10-14 M. Otherwise, the biosensor shows a good specificity to distinguish the complementary sequences and the different mismatched sequences, even the different kinds of the single-base mutation sequences (A-base mismatch, G-base mismatch and C-base mismatch). The obvious difference of results after hybridization demonstrated this multichannel electrochemical chip could detect PML/RARαmRNA qualitatively and quantitatively.