Ⅰ:Construction And Application Of Leaky Surface Acoustic Wave Biosensor Ⅱ:Identification Of Two Rare Pathogenic Microorganisms

Objective1. A high-sensitive and high-specific sensing technology for the detection of circulating tumor cells (CTCs) and proteins was developed based on a label-free leaky surface acoustic wave (LSAW) biosensor.2. Isolation and identification of Burkholderia thailandensis (B. thailandensis) strain and capnophilic Escherichia coli strain from clinical specimens. Besides, the complete genome of B. thailandensis strain was sequenced to investigate the genetic characteristics.MethodsConstruction and application of leaky surface acoustic wave biosensor1. The LSAW biosensor was designed as double two-port resonators style, and fabricated using LiTaO3 crystal as piezoelectric substrate. Then, a 2 X 3 model of LSAW biosensor array was constructed. The detection circuit and biosensor monitor system (BSMS) version 2.0 software were developed. On the basis of above, the detection system of resonant LSAW biosensor was constructed.2. The binding specificity of aptamer and MCF-7 cells was investigated by the flow cytometry analysis. The aptamer was assembled to the surface of LSAW biosensor by mercapto group. The concordance of aptamer immobilization and the optimum concentration of aptamer immobilization were determined based on the phase shift (Δ Phase) of LSAW biosensor.3. We constructed a high-sensitive and rapid approach for the detection of CTCs by a label-free LSAW aptasensor. The linear range, specificity, reproducibility, and stability of LSAW aptasensor were observed.4. The LSAW immunosensor was first immobilized with protein A from Staphylococcus aureus and monoclonal anti-Cyclosporin A (CsA) antibody. A series of experiments were performed to evaluate the concordance of antibody immobilization, the effect of antibody concentration on ΔPhase, and the effect of pH value on ΔPhase.5. We constructed a high-sensitive and label-free approach for the detection of CsA by LSAW immunosensor. The linear range, specificity, and reproducibility of LSAW immunosensor were observed. In order to explore the feasibility of the detection of clinical samples, the CsA concentrations in clinical whole-blood samples were detected by LSAW immunosensor and enzyme multiplied immunoassay technique. The consistency and comparability between LSAW immunosensor and enzyme multiplied immunoassay technique were evaluated by the Bland-Altman difference analysis.Identification of two rare pathogenic microorganisms1. The morphological method, VITEK 2 Compact system, API 20NE system, and 16S rRNA sequence were used to identify the B. thailandensis strain.2. The complete genome of B. thailandensis strain was sequenced to investigate the genetic characteristics. The structure variation, species evolution, and unique gene of B. thailandensis strain were analyzed by the comparative genomics.3. The capnophilic Escherichia coli strain was incubated at 35℃ in air and 35℃ in air containing 5% CO2 to observe morphological characteristics. The VITEK 2 Compact system, API 20E system, and 16S rRNA sequence were used to identify the capnophilic Escherichia coli strain. The antimicrobial drug susceptibility profile was determined.ResultsConstruction and application of leaky surface acoustic wave biosensor1. The double two-port resonant LSAW biosensor was fabricated using simple side polished 36° rotated, y-cut and x-propagation LiTaO3 crystal as piezoelectric substrate. The LSAW biosensor consisted of input reflector array, input interdigital transducer, biological reaction area, output interdigital transducer, and output reflector array. The basic resonance frequency of LSAW biosensor is up to 100 MHz. The APhase of LSAW biosensor was less than 0.1 degree in gas phase and liquid phase.2. Using ΔPhase as the detection signal, the detection circuit of LSAW biosensor was self-designed. The BSMS version 2.0 software was developed by the LabVIEW. The detection system of resonant LSAW biosensor was constructed by combining the LSAW biosensor, NI-PXI system, BSMS version 2.0 software, computer, and power supply.3. The concordance of aptamer immobilization was good. It was a good way of aptamer immobilization by mercapto group. The LSAW biosensor displayed an optimum sensitivity of the response at aptamer concentration of 2.0 μM.4. The average Δ Phase of LSAW aptasensor was linear to the logarithm of concentration of MCF-7 cells in the range from 1×102 cells mL-+1 to 1×107 cells mL-1. The linear regression equation was calculated as ΔPhase= 0.465 1g C-0.112, where C was the concentration of MCF-7 cells (cells mL-1).5. The ΔPhase of LSAW aptasensor could be observed in the presence of MCF-7 cells, whereas the ΔPhase of Roams cells or K562 cells were approximately the same as the ΔPhase of blank solution. The LSAW aptasensor exhibited excellent specificity. After ten regenerating cycles, the sensing activity of LSAW aptasensor has possessed 72.7% of the original. The coefficient of variation was 9.79%, i.e., less than 10%, which indicated a feasible regeneration of LSAW aptasensor. The LSAW aptasensor was dried and stored at 4℃。 After 4 weeks, the APhase of LSAW aptasensor was about 89.51% compared with its initial response.6. The concordance of monoclonal antibody immobilization was good. It was a good way of monoclonal antibody immobilization by Staphylococcal protein A. The LSAW immunosensor displayed an optimum sensitivity of the response at antibody concentration of 2.0 μM and pH value of 7.4.7. The ΔPhase of LSAW immunosensor was linear to the CsA concentration from 1 ng/mL to 1000 ng/mL. The linear regression equation was calculated as ΔPhase= 0.0039X +0.929, where X was the concentration of CsA. The APhase of LSAW immunosensor could be observed in the presence of CsA, whereas the APhase of FK506, valproic acid, digoxin, carbamazepine, and theophylline were approximately the same as the APhase of blank solution. The LSAW immunosensor exhibited excellent specificity. The piranha (1:3 mixture of 30% hydrogen peroxide and 98% sulfuric acid) was used to regenerate the immunosensor. After ten regenerating cycles, the binding activity of LSAW immunosensor had retained 73.53% of the original. The coefficient of variation was 9.33%, i.e., less than 10%.8. The result of Bland-Altman difference analysis revealed that the consistency and comparability between LSAW immunosensor and enzyme multiplied immunoassay technique were good.Identification of two rare pathogenic microorganisms1. The first strain of B. thailandensis was identified by morphological method, VITEK 2 Compact system, API 20NE system, and 16S rRNA sequence in China.2. The complete genome of B. thailandensis strain was sequenced to determine the genetic characteristics. The structure variation, virulence factor (VirB/VirD4 type IV secretion system, HSI-I, and Mycobactin), species evolution, and unique gene (BPMGL000390, BPMGL000391, BPMGL001875, BPMGL001949, and BPMGL002594) were determined by the comparative genomics.3. The first strain of capnophilic Escherichia coli was identified by morphological method, VITEK 2 Compact system, API 20E system, and 16S rRNA sequence in China.Conclusion:1. The double two-port resonant LSAW biosensor and 2X3 model of array were fabricated. The detection circuit and BSMS version 2.0 software were developed. Then, the detection system of resonant LSAW biosensor was constructed.2. A high-sensitive and rapid approach for the detection of MCF-7 cells was constructed by a label-free LSAW aptasensor. This technique combined the sensitivity of LSAW biosensor with the specificity of aptamer, and provided a new method for identifying the tumor metastasis and investigating the mechanism.3. A high-sensitive and label-free approach for the detection of CsA was constructed by a novel LSAW immunosensor. This technique combined the sensitivity of LSAW biosensor with the specificity of antigen-antibody reaction, and provided a new immunoassay for diagnosing the clinical disease and monitoring the drug concentration.4. The first strain of B. thailandensis was isolated and identified in China. The complete genome of B. thailandensis strain was analyzed to determine the genetic variation, virulence, and biological characteristics.5. The first strain of capnophilic Escherichia coli was isolated and identified in China.