|
Harmful algal blooms （HABs）, one of global marine environmental problems, has serious impacts on the marine ecosystem balance and lead to enormous economic losses, which make it a pressing topic in marine science. The qualitative and quantitative detection for HABs species is the basis in HABs study. Besides the traditional microscopic method, more and more new techniques, such as flow cytometer and microscope （FlowCAM）, chemotaxonomy, spectrofluorimetry for algal classes, oligonucleotide probes and immunoassays, have been developed and played important roles in this field.In this study, electrochemiluminescence - molecular probe （ECL-MP）, a new method for harmful algal detection, was established based on the sandwich hybridization integrated with nuclease protection assay （NPA-SH）, which was improved by the electrochemiluminescence （ECL）.Firstly, an ECL analyzer was designed and built according to the principle of ECL and some related studies. Then performance optimization tests were made to it, including the values of voltage and current, the concentration of tripropylamine （TPrA） and the pH of phosphate buffer （PBS）. It was proved by facts that this analyzer was stable and highly sensitive, with a detection range of Ru（bpy）3Cl2·6H2O from 0.4 pmol to 4 nmol in the optimal reaction conditions with voltage of 1.0 V, current of 1.0 mA, TPrA concentration of 1.5 mol·L -1 and pH of 7.4.Secondly, Prorocentrum minimum （Pavillard） Schiller was detected by ECL-MP. The NPA probes for P. minimum were labeled with Ru（bpy）32+ and biotin （B-NPA-Ru）, so that they could be bound with streptavidin-coupled magnetic beads and generate electrochemiluminescence in the ECL analyzer. Then the B-NPA-Ru probes hybridized with complementary sequence of rRNA, forming DNA/RNA hybrids. The unbound and imperfectly matched probes were digested by S1 nuclease which could degrade single-stranded nucleic acids into 5’phosphoryl mono or oligonucleotides, leaving intact double-stranded nucleic acids. The B-NPA-Ru probes were released after the denaturation of DNA/RNA hybrids and injected into the ECL analyzer attaching to the magnetic beads. The Ru（bpy）32+ labelled on the B-NPA-Ru probes interacted with TPrA and generated ECL, when the potential reached a certain value. The ECL-MP calibration curve of P. minimum was established through analyzing the relations between the ECL intensity （count per second, CPS） and cell numbers. When samples from individual and mixed cultures as well as field collections were detected by ECL-MP and microscopy, there was no significant difference with 95% confidence level （t-test） between the two methods, which indicated that the ECL-MP had good specificity, reliability and accuracy in the P. minimum detection.The ECL-MP took advantage of the specificity of NPA-SH on the one hand, converting easily degraded rRNA into complementary stable DNA probes stoichiometrically so that the P. minimum can be analyzed qualitatively and quantitatively; on the other hand, the ECL-MP developed its high sensitivity and rapid analysis with ECL. The detection range of P. minimum cells was from 6.25×102 to 4×104, which was more sensitive compared with the NPA-SH range from 1.1×104 to 3.7×105. Meanwhile, the analysis time was reduced to 4.5 hours compared to 7.5 hours in NPA-SH. Therefore, if species-specific NPA probes for different kinds of harmful algae are designed, the daily monitoring of HABs can be performed with ECL-MP, and it is a new way for pre-warns and mechanism research of HABs.
|