Novel Quantitative Methods For Evaluating Preeclampsia Associated Serum Protein And The Preliminary Clinical Application

Preeclampsia is a type of hypertensive disorder with the onset of hypertension and proteinuria after 20 weeks of pregnancy as its main clinical manifestation.The diagnosis of preeclampsia,in the absence of proteinuria,requires the presence of hypertension together with evidence of systemic diseases including injury of nervous system,blood system and digestive system or vitals as heart,liver,kidney and lung.Therefore,preeclampsia is currently a leading cause of maternal and perinatal morbidity and mortality around the world.At present,the administration of preeclampsia is limited to symptomatic treatments such as unplugging,depressurization,etc.Because the pathogenesis of preeclampsia is extremely complicated,and no consent has been reached on the actual mechanism.This may be due to the fact that there is still a lack of reliable individual biomarkers for the precise and specific prediction.Meanwhile,novel technical progress on biosensing and bioanalysis has emulated the traditional serological bioassays in sensitivity,specificity and the speed of response,etc.So,for preeclampsia,it is now in urgent need to develop new quantitative methods of serological detection with integration,automation,efficiency,sensitivity and specialty.This dissertation presents a set of novel electrochemical biosensing methods for evaluating serum biomarkers of preeclampsia.The interaction of various synthesized ligands and the biomarkermolecules are employed to achieve specific and sensitive detection,which has also been preliminarily realized in the bioanalysis of clinical serum samples of preeclampsia.These results may show the potential of the proposed method in promoting the early discovery,early diagnosis and early treatment of preeclampsia.1.Sensitive detection of preeclampsia associated serum protein using peptide nucleic acid-coupled dual cycling reactionsObjective:Proteohormone,which belongs to hormone family,could regulate metabolic activity of various tissue and cell in order to accommodate physiological process.Insulin,a proteohormone secreted by pancreatic β cell,is the center of carbohydrate controlling and fat metabolism and can facilitate the synthesis of glycogen,fat and protein.The aberrant expression of insulin in serum is not only associated with metabolic diseases like diabetes,but also involved in the development of preeclampsia via activation or suppression of coherent signal pathways.Therefore,a new electrochemical biosensor has been designed to detect insulin level with high sensitivity,through the use of peptide nucleic acid-coupled dual cycling reactions as well as nano-composites.Methods:1.The electrochemical biosensor was first constructed.2.Several important experimental aspects of this method were optimized:enzyme concentration,ZrOCl2 solution concentration,incubation time for the binding between insulin and hairpin probe 1,the incubation time for the dual amplification and the incubation time for the immobilization of GNP/DNA/MB nano-composites to DNAs on the electrode surface.3.Square Wave Voltammetry(SWV)was employed to confirm the contribution of the several steps of signal amplification to the finally obtained sensitivity.4.SWV was used to identify standard insulin at different concentrations and obtain the calibration curve.5.A series of control experiments were conducted to verify the specificity.6.Serum samples from normal pregnant women spiked with different concentrations of insulin were investigated.7.Comparison between this method and commercial ELISA kit was used to verify the feasibility.8.A total number of 12 blood samples(including 6 PE and 6 normal)were obtained from primipara women who deliveries from November,2013 to June,2014 in our hospital.The new biosensor was used to detect the expression level of insulin in the blood samples.Results:1.Optimization of experimental conditions:(1)The optimal concentration of work enzyme was 0.25 U/μL.(2)A concentration of 0.25 mM was sufficient for signal amplification by nano-composites.(3)An incubation of 120 min was proper for insulin-probe interaction.(4)80 min was proper for dual DNA amplification.(5)60 min was found to be optimal for sufficient immobilization of GNP/DNA/MB nano-composites with DNAs on the electrode surface.2.The steps of dual amplification and nano-composites both contributed to the sensitivity of the detection.3.A linear relationship between the peak current and the logarithm of insulin concentration could be constituted over the range from 1 to 500 pM,and the linear equation was:y=2.46x+1.71.A detection limit was found to be less than 1 pM,and the average coefficient of variation was within 5%.4.It could be observed that the interference of the control species can be negligible.5.The practical error of concentration between detection by biosensor and spiked sample was within 5%.6.The new method presented higher sensitivity and wider detection range when compared with commercial ELISA kit.7.According to the result detected by new biosensor,on the contrary to the preeclampsia women,the insulin level in the normal women was rather low.Conclusion:Our electrochemical biosensor attains high sensitivity,specificity and a satisfactory analytical performance in detecting complex bio-samples.It may serve as analysis platform with rapid,direct and accurate performance,with a promising prospect in clinical diagnosis in the future.2.Peptide-induced bio-mineralization for detecting preeclampsia related serum proteinObjective:Latest researches have validated that the aberrant expression level of high temperature requirement A1(HtrA1)will lead to dysfunction of trophoblast cells and has an effect on placental implantation,which is closely related to development of preeclampsia.Therefore,rapid,accurate and distinctive methods to detect the level of HtrAl may contribute to prediction and diagnosis of preeclampsia.We have designed a bio-mineralization based method for the sensitive detection of serum HtrAl,using the enzymatic activity of HtrA1 to induce the formation of peptide nanowire and the subsequent peptide templated biomineralization.This design is also inspired by the pathological calcification during the progress of preeclampsia.Methods:1.The electrochemical biosensor was first constructed.2.UV-vis absorption spectrum was performed to characterize the formation of Ag nanoparticles through peptide-initiated bio-mineralization in aqueous solution.3.Atomic force microscopy(AFM)was used to the morphological changes on the electrode surface.4.High-resolution scanning electron microscopy(HRSEM)and high-resolution transmission electron microscopy(HRTEM)were performed to determine the morphology of the Ag nanoparticles on the peptide nanowires.5.Circular dichroism(CD)and fourier transform infrared(FT-IR)spectroscopy were employed to analyze the conformational change of the peptide.6.Electrochemical impedance spectra(EIS)was recorded to show the stepwise treatment and modification on the electrode.7.Several important experimental aspects of this method were optimized:the incubation time of the substrate probe modified electrode with HtrAl,the time needed for the formation of peptide nanowire and the work enzyme concentration.8.Dynamic light scattering(DLS)was employed to study size and size distribution of Ag nanoparticles when confront to different pH.9.MTT was used to analyze the cytotoxicity of Ag nanoparticles.10.Square Wave Voltammetry(SWV)was used to identify standard insulin at different concentrations.11.A series of control experiments have been conducted to verify the specificity.12.A total number of 12 blood samples(including 6 preeclampsia and 6 normal)were obtained from primipara women who deliveries from December,2014 to June,2015 in our hospital.The new biosensor was used to detect the expression level of HtrA1 in the blood samples.13.Comparison between this method and commercial ELISA kit was used to verify the feasibility.Results:1.A characteristic absorption peak of Ag nanoparticle could be observed after the bio-mineralization reaction by UV-vis absorption spectrum.2.It could be observed that the Ag nanoparticles mainly nucleate on specific sites of the peptide nanowire according to AFM,HRSEM and HRTEM.3.The CD spectrum of the peptide alone(Red curve)had a negative peak at 197 nm with a positive peak at 220 nm,while the peptide with Ag nanoparticles had two negative peaks at 220 nm and 197 nm.On FT-IR,the peptide without the formation of Ag nanoparticles showed the evident bands of Amide A around 3500 cm-1,and Amide I between 1600-1700 cm-1.4.After each step of modification on electrode,the obtained signals of EIS were changed.5.Optimization of experimental conditions:(1)An incubation of 2 h was sufficient for the interaction between the electrode and HtrA1.(2)100 min was proper for the formation of peptide nanowire.(3)The optimal concentration of work enzyme was 0.3 mg/mL.6.The intermediate pH relevant to biosensing application could produce relatively small and uniformly sized Ag nanoparticles.7.Only greatly excessive amount of Ag nanoparticles would generate cytotoxicity.8.A linear relationship between the peak current and the logarithm of HtrAl concentration could be constituted over the range from 50 to 2800 pg/mL,and the linear equation was:y=115x-137.A detection limit was as low as 21.7 pg/mL,and the average coefficient of variation was within 5%.9.It could be observed that the interference from the control species can be negligible.10.According to the result detected by new biosensor,the HtrA1 level in the preeclampsia women was higher than that in the normal women.11.The new method presented preferable analytical performance when compared with commercial ELISA kit.Conclusion:The new designed biosensor shows satisfactory analytical performance,with low background signal,high stability and highly sensitivity that may of potential value for the clinical analysis of preeclampsia in the future.3.Detection of preeclampsia associated serum protein using bi-functional peptide nucleic acid probeObjective:Preeclampsia can often induce prethrombotic state.The disturbances of blood coagulation,namely the imbalance of coagulation and anticoagulation are among the pathogenetic factors of preeclampsia.So it is important to monitor hypercoagulable state and coagulation indicators in women before,during and after pregnancy-Increased generation of thrombin in vivo can directly indicate the prethrombotic state,so monitoring of its concentration and activity may contribute to the timely discovery of hypercoagulable state,as well as facilitating the prognosis of preeclampsia.We have designed a method for detecting thrombin,using a peptide nucleic acid(PNA)aptamer of thrombin.By modifying the sequence of this aptamer,a bi-functional probe is obtained,which can either bind with thrombin,or induce DNA cleavage cycling to generate amplified signal readout.Methods:1.The electrochemical biosensor was first constructed.2.Electrochemical impedance spectra(EIS)was recorded to show the stepwise treatment and modification on the electrode.3.Several important experimental aspects of this method were optimized:the incubation time of the electrode with peptide nucleic acid(PNA),the time needed for cycle invasion and digestion on the interface as well as the nuclease S1 concentration.4.Square Wave Voltammetry(SWV)was used to identify standard thrombin at different concentrations.5.A series of control experiments were conducted to verify the specificity.6.Three blood samples were obtained from normal primipara women who deliveries during June,2015 in our hospital.Serum samples spiked with different concentrations of thrombin were investigated to validate the applicability.Results:1.After each step of modification on electrode,the obtained signals of EIS were changed.2.Optimization of experimental conditions:(1)An incubation of 80 min was sufficient for the interaction between the electrode and PNA.(2)120 min was proper for cycle invasion and digestion on the interface.(3)The optimal concentration of nuclease S1 was 5 U/mL.3.A linear relationship between the peak current and the logarithm of HtrAl concentration could be constituted over the range from 63 pM-40 nM,and the linear equation was:y = 2.11 x+1.94.A detection limit was as low as 27.4 pM,and the average coefficient of variation was within 5%.4.It could be observed that the interference of the control species could be negligible.5.The practical error of concentration between detection by biosensor and spiked sample was within 5%.Conclusion:In this design,a bi-functional PNA probe is developed and the controlled functional switching of it has also been achieved to enable the sensitive detection of thrombin in biological and clinical serum samples.Since this method has no special requirement for the sequence of the aptamer,it is potentially generalizable,for the assay of various biomarkers with rapid,satisfactory sensitivity,indicating a great potential for the clinical analysis in the future.