| With the speeding up of the globalization process and the deterioration of climate and environment, the large-scale outbreak of infectious disease is becoming more and more frequently. In case of the large-scale event of highly pathogenic infectious disease, such as severe acute respiratory syndromes (SARS), a large number of suspected patients need to be diagnosed as soon as possible, for the sake of accurate assessment of the epidemic situation to make effective controlling measures. For such a large workload, the most economic and effective way is to employ a rapid, accurate and automated molecular diagnostic method. Application of a rapid detection of specific DNA sequence in a patient’s sample will take a shorter time for a patient to be confirmed. The realization of the automated nucleic acid detection system has greatly promoted the clinical applications of the nucleic acid analysis technology, and the relate systems are receiving more and more attention.The realization of the automated nucleic acid analysis system is involved in a number of technical problems, one of the most critical techniques is micro-volume liquid handling technology since its accuracy and repeatability will directly affect the reliability of the testing results. A variety of micro-volume pipetting technologies have been developed, however, few of them can be used in practical applications. The pipetting technology based on the principle of air displacement is still a preferable solution for liquid handling in nucleic acid analysis system.In this paper, we first studied the principle of air displacement pipetting technology, and analyzed various influence factors of micro-volume liquid handling process. Accordingly, a dynamic calibration model based on the characteristics of liquid was put forward. Meanwhile, the liquid level detection technology and the realtime pipetting monitoring technology were studied, then an automated liquid handling platform based on a multi-channel air displacement pipettor had been developed. Base on the above study, a high throughput, automated nucleic acid analysis system was realized, and its application for the processing and analysis of clinical samples was checked. The main contents are as follows:1. Study on the air displacement pipetting technologyThe working principle of the air displacement pipetting technology was studied, and a testing platform for pipetting was constructed according to the standards including ISO-8655 and JJG-646-2006. Factors (e.g., environmental factors, equipment factors, operating factors and fluid characteristics) influencing the performances of pipetting were analyzed, and some corresponding countermeasures were put forward according to the origin of those factors. To further improve the performance of the micro-volume pipetting process, a dynamic calibration model based on fluid characteristics (density and viscosity) was developed. A 20μL pipettor was used to verify the effectiveness of the dynamic calibration model. After calibration, the pipetting errors of anhydrous ethanol and blood were reduced by 3.3% and 0.93% on average.2. Study on the automated liquid handling technologyFirstly, two assistive technologies used for automated liquid handling operation were studied. One is the capacitance-based liquid level detection technology, another is the realtime pressure monitoring technology. Since the measuring capacitance was 4.6 pF in the air and at least 6.1 pF in the liquid, liquid level can be detected by capturing the change of capacitance when the pipettor passing into the air-liquid interface. Real-time pipetting monitoring model was developed based on the operation of pure water, and a variety of abnormal operations (e.g., suction, bubble, jams, etc.) can be discriminated and recorded. Secondly, an 8-channel micro-volume pipetting module was developed based on the principle of air displacement. And an automated liquid handling platform was also developed consisting of the 8-channel micro-volume pipetting module, mechanical gripper module and a robotic arm based positioning system. Performance testing was carried out on this platform. The operation range of the 8-channel pipettor was 1～300μL. The pipetting errors of 20μL,100μL and 200μL were 0.61%, 0.75% and 0.51% respectively, and the corresponding repeatabilities were 0.62%,0.53% and 0.56%(CV%). The repositioning precisions of the robotic arm based positioning system were 30.71μm,27.10μm and 23.60μm. The testing results indicated that the overall platform had an excellent ability for liquid handling.3. Development of the automated nucleic acid analysis systemAccording to the requirement of the nucleic acid analysis experiment, the target system was divided into three parts, including sample processing, nucleic acid amplification and signal detection. The whole system was constructed based on the automated liquid handling platform, and the experimental requirement of nucleic acid detection was realized by integrating some functional modules. The self-developed functional modules include multi-functional nucleic acid extraction module, high throughput magnetic separation module and temperature oscillation module. Among them, DNA purification of 96 samples can be finished within 2 hours with the help of the multi-functional nucleic acid extraction module. High throughput DNA extraction based on magnetic nanoparticles can be carried out on the high-throughput magnetic separation module, and this module also provides 4 working location for SBS (Society for Biomolecular Screening) labware. The temperature uniformity of the temperature oscillation module is ±1℃, and provides 5 class amplitudes of oscillation. As a whole, the automated system employs a uniform electric control scheme, and provides a programmable interface for on-line design of the experimental process. As a result, the experimental procedure can be carried out from sample to final report automatically.4. Performance testing of the automated nucleic acid analysis system and its applications in clinicalTo test the performance of the automated system, two BSA gradient dilution separately prepared by the automated system and manual process were measured by Bradford method. The linearities of both standard curves are 0.984 and 0.974 respectively, and the repeatabilities were 2.00% and 2.35% (CV%) on average. Secondly, as many as 20 pieces of the 96 well plates were carried out to prepare the potassium dichromate gradient dilution, the repeatability from automation, manual process with single channel pipettor and 8-channel pipettor were 1.94%, 2.40% and 4.54% respectively. The results indicated that the pipetting accuracy and repeatability of the automation system is superior to manual operation. Then the sample processing capability of the presented system was verified as follows:HBV plasmid sample of certain concentration was respectively purified with the automated system and manual process, the extraction efficiency were 95.01% and 94.7%; High throughput nucleic acid extraction of E.coli samples was carried out with this automation system, and the result showed that there was no cross contamination happening in this system. Also, clinical blood samples were treated with our system, and the purity of the product is 1.72～1.91 (A260/A280), the average concentration was 25.92 ng/μL. Finally,32 clinical HBV serum samples were subjected to the HBV-DNA quantitative analysis with our system. The result showed that the magnitude levels of the viral load were between 105～109 copies/mL, and all of them were greater than 103 copies/mL. And the results indicated that all of those 32 patients were HBV positive, and the virus was very active in the body. |
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