Developing Electrochemical Biosensors For Clinical Detection Of Essential Proteins Of Pre-eclampsia

Pre-eclampsia (PE), a gestational idiopathic disease, is the leading cause of pregnant and prenatal morbidity and mortality around the world. The pathogenesis of PE is currently a hot topic in obstetrical research, arousing heated debate, as well as considerable controversies. Up to date, no consent has been reached on the actual mechanism of PE pathogenesis. Inevitably, the current administration of PE mainly involves symptomatic treatment such as unplugging, depressurization, etc, always with poor effect and prognosis. Moreover, although recent biological research has greatly augmented our understanding of PE pathogenesis, such progress rarely results in significant improvement in clinical practice, due to the lack of reliable disease marker. On the other hand, as biomedical research is migrating from static qualitative investigation to real-time quantitative analysis, high standard protocol for the detection of clinical indicators such as disease-associated protein biomarker, should be established. So the development of state-of-the-art protein analytical technology with unrivaled sensitivity and specificity, and capable of dynamic in vivo protein analysis, will greatly promote biomedical research as well as clinical early diagnosis. In this dissertation, novel electrochemical protein biosensing methods are established based on recent progress in the field of chemical biology, surface chemistry and nonmaterial research. Various aspects of the designed protein biosensors, such as labeling of the protein, molecular recognition, surface modification, quality of signal readout, have been studied and streamlined to provide biosensing performance suitable for clinical detection of several potential biomarker proteins of PE.Part Ⅰ:Detection of CREB Phosphorylation via Zr (Ⅳ) Ion Mediated Signal AmplificationObjective:Protein phosphorylation is a fundamental regulatory mechanism involved in virtually all biological processes. Phosphorylation directly activates/de-activates the function of many essential bio-regulatory proteins, such as CREB (cAMP response factor binding protein) that is indispensible for bio-activities such as apoptosis and immunoresponse. Recent studies have established the imitate relationship between phosphorylation of CREB and pathogenesis of PE. In this section, a novel electrochemical biosensing method to detect phosphorylation of CREB has been developed and preliminarily applied in the detection of clinical sampleMethods:Phosphorylation level of CREB is quantified via the recognition of phosphorylation sites on the target protein with Zr (IV) ion, as well as by employing gold nanoparticle/DNA/methylene blue (GNP/DNA/MB) nano-composite label. A biosensing surface layer is first constructed on the working electrode using high affinity DNA probe of CREB, this electrode surface layer can capture CREB molecules from the sample solution, subsequently, the phosphate group of the captured CREB is recognized by Zr (IV) ion, which then serve to mark the phosphorylated CREB with the nano-composite label. This method has achieved highly sensitive detection with a limit of detection as low as 0.25 nM for the assay of phosphorylated CREB. The feasibility of the method has also been tested in clinical samples retrieved from normal pregnant women, as well as pregnant women with mild PE, severe PE.Results:1. Optimization of experimental conditions(1). The optimal concentration of PKA is 500 unit/mL.(2). An incubation of 120 min is sufficient for the biosensing layer to capture CREB in the sample solution.(3). Effective signal amplification can be accomplished with as low as 0.2 mM Zr (Ⅳ) ion.(4). The optimal incubation time for GNP/DNA/MB nano-composite label to bind with the electrode is 60 min.2. A saturation binding curve can be obtained for the phosphorylated CREB, from which a calibration curve can be derived for the determination CREB phosphorylation in biologically complex sample.3. CREB phosphorylation in the placenta of pregnant women, detected using our method, shows parallel with the severity of PE.Conclusion:This method attains a satisfactory analytical performance in detecting clinical samples, showing a promising prospect in clinical analysis in the future.Part Ⅱ:Detection of STAT 3 Bioactivity via Peptide-controlled Reversible Protein/DNA InteractionObjective:Signal transducer and activator of transcription 3 (STAT 3) is a house-keeping protein for biological processes such as proliferation, differentiation and apoptosis of cell, as well as angiogenesis. Latest researches have validated the close correlation between STAT 3 activity and the pathogenesis of many diseases, such as cancer and PE. In this section, an electrochemical method to detect the activity of STAT 3 is developed based on the reversible STAT 3/DNA interaction that can be controlled by specific binding peptide. The method has also been tested by the detection of clinical sample.Methods:This design is inspired by the differentiated interaction between STAT 3 and its two ligands:phosphorylated peptide and DNA. This peculiar interaction is employed for the detection of bio-active STAT 3 using graphene-based nano-composite catalytic label. At first, a biosensing layer is fabricated on the surface of working electrode using high affinity DNA probe of STAT 3, the probe is also phosphate-ended. The probe can then capture the dimer of active STAT 3 from the sample solution. DNase I can subsequently cleave all surplus probes, leaving only the STAT 3-bound probe molecules, which then release the captured STAT 3, in response to the phosphorylated peptide-triggered dissociation of STAT 3 dimer. These probes are then quantified by attaching onto them the graphene-based catalytic label using Zr (IV)/phosphate interaction. Through the signal amplification with the catalytic label, high sensitivity with a LOD of 0.085 nM is attained. The designed method is tested by quantifying the activity level of STAT 3 in placenta sample collected from the normal, mild PE and severe PE pregnant women having delivered at the obstetric department of the first affiliated hospital of Nanjing Medical University.Results:1. Optimization of experimental conditions:(1). Interaction between STAT 3 and the surface-immobilized probe needs 120 min to complete.(2). The optimal cleavage time of DNase I is 30 min.(3). To dissociate STAT 3 from the electrode, the interaction between the electrode and the phosphorylated peptide should be allowed to proceed for 60 min.(4).60 min is sufficient for the labeling of the electrode with the graphene-based catalytic label.2. In the designed method, the detected level of activity increases with the concentration of STAT 3, before a plateau is reached.3. The parallel between STAT 3 activity and the severity of PE is also observed in placenta sample from delivered women.Conclusion:It is observed that the designed method has satisfactory sensitivity, specificity and reproducibility in detecting STAT 3 bio-activity. Such results indicate a great potential for the clinical analysis of active proteins using our method.Part III:Sensitive and Low Background Detection of Corin via Supramolecular RecognitionObjective:Protease cleavage is an essential type of protein post translational modification. The protease digestion by serine protease, a member of type II trans-membrane protease, regulates processes such as maturation of hormone, apoptosis, regulation of blood pressure and blood coagulation. Latest studies have identified a serine protease, corin, to be strongly correlated with the pathogenesis of hypertension, heart-failure and cancer. Moreover, corin has also been found to be involved with the progress of PE. So in this section, corin, a potential biomarker of PE, is detected by using a designed supramolecular recognition to induce amplified signal readout. This sensitive method has been verified in the analysis of corin activity in serum samples from pregnant women with PE.Methods:In this design, the probe for the target protein is a bi-functional peptide consisting of both the substrate of corin and the binding site of cucurbituril. On a surface biosensing layer formed by the peptide probe, the absence of corin cleavage allows all the probes to bind with the primer of rolling circle amplification (RCA) via the cucurbituril supramolecular recognition of both the probe and the primer. The primer then initiates extensive RCA on the electrode surface. In the presence of corin, on the other hand, corin cleavage removes large number of cleaved fragments that contains the cucurbituril binding site, from the electrode surface, resulting in greatly reduced binding of RCA primer and subsequent low level of RCA reaction. Since the RCA product can effectively block the electron transfer between the electrode and the solution, the contrast between the signal readout in the absence/presence of corin is greatly enhanced, improving the sensitivity of this method to a practical level for clinical detection. Serum samples from pregnant women with/without PE have also been employed to confirm the performance of our method in analyzing clinical samples.Results:1. Optimization of experimental conditions:(1). The supramolecular recognition of the surface immobilized probe by the primer-cucurbituril complex needs 16 h to fully accomplish.(2). The optimal cleavage time for corin is 30 min.(3). The optimal reaction time of RCA reaction is 60 min.2. Signal response is more evident at greater activity of corin, until a plateau has been reached.3. The detected corin level in the serum of pregnant women correlates with the severity of PE.Conclusion:The attained sensitivity, specificity and reproducibility in detecting corin activity indicate a great potential of our method for the clinical analysis of corin, as well as other proteases, as disease biomarkers.