Studies On Liquid Phase And Solid-Liquid Phase Detection Of Bacterial Endotoxin Based On Nucleic Acid Aptamer

Infectious disease is one of the world's top ten killer diseases, especially in developing countries. Children and trauma patients have the higher mortality rate in these diseases. In the infectious disease, the highest mortality comes from sepsis, endotoxemia and septic shock by gram-negative bacteria. Gram-negative bacteria (GNB) infection, bacteria and/or LPS or endotoxin may enter the blood circulation and activate the release of inflammatory mediators in many inflammatory cells, causing inflammation and multiple organ failure seriously.LPS is the main component of GNB outer membrane, built by three parts named lipid A, core oligosaccharide and O-specific polysaccharide side chain, respectively. LPS plays a major role in maintaining the permeability and fluidity of the GNB outer membrane. It can also induce the apoptosis of immune cells directly or indirectly, inhibit immune function, and increase the incidence of sepsis. Thus the detection of LPS is important in many fields of medical science. There are several methods for endotoxin detection at present, such as limulus amebocyte lysate (LAL) test, dynamic turbidity method, and recombinant factor C-related test methods. The most common method is the LAL test, which is sensitive and has higher specificity. However, the LAL test also has many disadvantages, such as extremely sensitive to LPS contamination, high demand for the disinfection of the utensils and the results ofLAL test are sometimes vulnerably affected by certain metal ions, antibiotics, amino acids, sugars, proteins and other plasma. So the application of LAL test is limited in some cases.Recent studies suggested that DNA and RNA not only store and transfer genetic information, but also have the unique structures to react with other molecules. So an artificial random oligonucleotide library can be designed on this theory, and the process of screening highly specific binding fragment from random sequence library is known as "systematic evolution of ligands by exponential enrichment" (SELEX). These molecules were called "nucleic acid aptamer" or "aptamer". The function of aptamer is similar with antibody, and it can combine the targert molecule with high affinity and specificity. The dissociation constant of aptamer can reach pictogram level.The aim of this study is to develop a new detection method for endotoxin which is high stable, specific, sensitive and not affected by external factors. It can also lay foundation for the research and development of endotoxin detection kit or bio-sensors, and raise the level of bacterial endoxin detection in China.Methods:1 Selection of buffer solution. Fresh water for injections (WFI, pH= 5. 61, endotoxin content<0.03 EU·ml^-1), the phosphate buffer (PBS, 0.1mol/L, pH= 7.3, prepared with sterile purified water) and trihydroxymethyl aminomethane (Tris, 0.02mol/L, pH= 7.4) buffer were used in the selection of media environment experiments of endotoxin and aptamer's incubation.2 Optimization of incubation time. PBS was used for the optimization of incubation time as an incubation medium. The incubation time is determined for 30 minutes,1 hour,2 hours,2 hours and 4 hours separately.3 Optimal concentration of aptamers. We selected 5μmol/L,10μmol/L,20μmol/L,40μmol/L for the optimized aptamers of the experiments concentration.4 The comparison of specificity of endotoxion combining with different aptamers. The selectivity of aptamers is an important factor for the detection method. The randomly selected aptamers, which were replaced by AptamerⅠAptamerⅢand AptamerⅡreacted with LPS to determine whether LPS had non-specific binding with other aptamer.5 Detection of bacterial endotoxin using solid-liquid phase assays based on aptamer. Considering all the optimized results of various testing conditions, the experiment was divided into 0.0312 EU/ml,0.0625 EU/ml,0.125 EU/ml,0.25 EU/ml,0.5 EU/ml,1 EU/ml,2 EU/ml and 4 EU/ml endotoxin group separately for the solid phase/liquid phase assays with aptamers.6 Detection of bacterial endotoxin using liquid phase assay based on aptamer. The magnetic nanoparticles activated by hydroxyl were combined with 5'-amino-terminal modified aptamer. The LPS molecules were captured by these aptamer. Then, the magnetic separation technology was applied to remove impurities interference. Another aptamer with a suitable fluorescent marker was combined to another point of LPS for detecting the amount of LPS.7 Detection of liquid separation of magnetic beads by the LAL test. The special method was used to elute the conjugates which were attracted to the beads (including the bacterial endotoxin and aptamer) and the LAL test was used to detect the eluted solution for the validity of endotoxin.8 The stability test of solid phase/liquid phase assays. The reaction solution of solid phase /liquid phase assays was incubated at 37℃.9 Statistical analysis. SPSS 13.0 version was used for statistical analysis. The bivariate correlation analysis (Pearson's) was used for the solid phase/liquid phase assays of endotoxin; the univariate analysis of variance and factorial design analysis of variance for the choice of buffer solution, the optimization of incubation time, optimization of aptamers dose and the comparative tests for the selectivity of aptamers during the endotoxin detection.The data were presented as mean±standard deviation (x±s). The difference of experimental groups was analyzed by LSD pairwise comparison.Results:1 Selection of buffer solution. Endotoxin was incubated with aptamers in the WFI, PBS and Tris buffer. The affinity of endotoxin with aptamers was strong in all those buffers. The different fluorescence signal was not statistically significant(P = 0.536) in three types of buffers. The phosphate buffer and three Tris buffers containing Mg2+, Ca2+, Na+and K+had no interference on the binding of endotoxin with aptamers. Therefore, these buffers are all suitable for the use of detection.2 Optimization of incubation time. At the beginning of incubation, fluorescence intensity gradually increased with time and the amount of endotoxin binded with aptamers gradually increased after incubating for 2 hours and the fluorescence intensity was stable relatively. At this concentration, the fluorescence intensity reached the saturation state. At the same time, the combination of endotoxin with aptamers had completed basically. The results between 2 hours and 4 hours were not different. Therefore, the 2 hours was chosen for the incubation time of aptamers and endotoxin. 3 Optimal concentrations of the aptamers. Endotoxin have binded to the fixed aptamersⅡafter reacting with aptamerⅠat different concentrations. The fluorescence intensity increased gradually from the concentration at 5μmol L and the slope from 5μmol to 10μmol/L was obvious. The statistical analysis of multiple measurements showed that the difference between the two dose groups was statistically significant (P<0.001). The fluorescence values were stable from 10μmol L to 80μmol/L and the differences had not statistical significance (P> 0.5). The 10μmol/L of aptamerⅠandⅡwas selected as the incubation concentration.4 The comparison of specificity of endotoxion combining with different aptamers. When an unrelated aptamer was used to replace LPS-specific aptamers to react with endotoxin, no significant fluorescence was observed, which indicated that the endotoxin-specific Aptamers (ⅠandⅡ) can combine specifically with LPS. The intensity of fluorescence was dependent on the concentration of endotoxin, indicating that this aptamer detection for endotoxin had high selectivity.5 Detection of bacterial endotoxin using solid-liquid phase assays based on aptamer.The result showed that fluorescence intensity increased with the increasement of LPS concentration and there was a linear relationship between them. The result of bivariate correlation analysis showed that both LPS concentration and fluorescence intensity were significantly correlated (Pearson's:r= 0.897, P= 0.003). From the optimization of the test conditions, we found the fluorescence intensity in the solution increased with the dose of endotoxin. The strong affinity between aptamer and endotoxin was used to detect LPS with a satisfactory effect.6 Detection of bacterial endotoxin using liquid phase assay based on aptamer.. Fluorescence intensity increased linearly with the increasement of endotoxin. Tt was significantly related to the LPS concentration. The result of bivariate correlation analysis showed that both LPS and fluorescence intensity was significantly correlated (Pearson's:r= 0.867, P= 0.005).7 Detection of liquid separation of magnetic beads using the LAL test. The lower dose had no obvious positive results and the other groups showed different degrees of gelation except for the 0.0312 EU/ml group. The binding to beads on the material was eluted with the specific methods and confirmed by gel bead separation medium containing the components of endotoxin. The magnetic beads/aptamer/endotoxin binding experiments was effective.8 The stability test of solid phase/liquid phase assays. The results of 6-hour group,12-hour group and 24-hour group showed that no obvious decay of the fluorescence signal, indicating that the two detection methods for bacterial endotoxin had strong stability.Conclusions:1 The sandwich structure of two different aptamers with endotoxin is feasible for detection of endotoxin. It has a wide linear range of detection, with specificity and high sensitivity. The detective signal is stable and reproducible and is not interfered by other unrelated aptamers.2 The liquid phase detection method with bead separation technology can significantly eliminate non-specific absorption, improve the efficiency of detection further.The sensitivity of liquid phase assay is higher than that of the solid-liquid phase assay.3 The method of endotoxin detection based on the endotoxin-specific aptamers showed strong stability for solid phase-liquid phase assay and liquid phase assay. The fluorescence signal can maintain for 24 hours without decaying.