Aptamer/Antibody Functionalized Nanomaterials For The Seperation And Analysis Of Disease Protein Biomarkers

Biomarkers are a group of biologically significant molecules that reflect the pathological state of certain diseases. The identification, quantification and structure analysis of disease protein biomarkers can not only help with the elucidation of the mechanisms of certain diseases, but also promote early diagnosis, monitor disease progression, and facilitate follow-up therapy, thus it is of significant importance. However, the concentrations of many important protein biomarkers in body fluid are thought to range from low ng/mL to pg/mL, or even lower, which are very hard to detect. Antibody based immunoassays such as ELISA, have been long accepted as effective methods for selective detection of such protein biomarkers at trace levels due to their ability to extract target molecules with high affinity and specificity from complex matrices. But the limitation in sensitivity, speed and price of such immunoassays encourages researchers to develop more easy and powerful detection platforms. Recently, nucleic acid aptamers have been emerging as effective substitutes for antibodies as capture molecules in biosensors. They have a number of advantages over antibodies that make them very promising in analytical and diagnostic applications. One of the biggest advantages is that oligonucleotides can be chemically synthesized with ease and extreme accuracy at very low cost. In addition, aptamers are thermally stable, easier to be chemically modified, more stable during long-term storage, and they show a wider range of targets that can be recognized from small ions to even whole cells compared with antibodies. On the other hand, nano-materials provided with unique optic, electrochemical, fluorescent, catalytic property and large surface area have been extensively employed in the design of bio-diagnostic assays, and nano-diagnostics have become a new frontier over the past decades. In this thesis, we developed a series of novel method based on aptamer and antibody functionalized nano-materials for the enrichment of disease biomarkers. By using MALDI-TOF MS as readout system, fast and sensitive detection was achieved. This thesis is divided into five chapters.In chapter1, the recent progress in antibody and aptamer based diagnostic assays were reviewed. The applications of affinity MALDI-TOF MS in the development of such assays were also summarized. The limitations of these methods were discussed. At last, the purpose and significance of this thesis was demonstrated.In chapter2, we describe a sensitive and specific method for thrombin detection with aptamer functionalized core-shell Fe3O4@C@Au magnetic microspheres (Au-MMPs). Firstly, Au-MMPs were synthesized through surface adsorption of gold nanoparticles onto PDDA functionalized Fe3O4@C magnetic microspheres. Then, the as-synthesized Au-MMPs were developed as new substrate for immobilization of thrombin binding aptamer (TBA) through easy formation of Au-S bond. After that, the prepared aptamer functionalized Au-MMPs (TBA@Au-MMPs) were used as effective magnetic absorbent to extract trace level of thrombin from dilute solutions. Finally, enriched thrombin was digested by trypsin and analyzed by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Taking advantage of the efficient affinity extraction ability of our TBA@Au-MMPs and the sensitive mass readout of MALDI-TOF, highly sensitive detection of thrombin was achieved. The limit of detection was as low as18fmol, corresponding to0.36nM thrombin in50μL original solution. Linear relation was observed within a concentration range from0.5nM to10nM with linear correlation R2=0.998. Other proteins including human serum albumin (HSA), Ig G, transferrin, oval albumin (OVA) and fetal calf serum did not interfere with thrombin detection. We have also achieved the successful retrieve of thrombin from diluted human serum. This simple method holds great potential for analyzing, sensing, purification and preconcentration of proteins in biological fluids.In chapter3, an aptamer microarray was directly fabricated on gold modified MALDI target plate for high-throughput and sensitive insulin detection. We used an easy yet effective method to modify the target plate with dense, stable and porous gold layer by directly dropping gold colloid solution onto the surface of target plate and calcinate at200℃for2h. Then the as-modified target plate was immobilized with thiol group modified insulin aptamer through the easy formation of covalent bond between gold atoms and thiol group. After MALDI-TOF MS analysis, high sensitivities were observed both in standard solutions (5ng/mL,0.86nM) and serum sample (20ng/mL,3.4nM), which were higher than the previously reported aptamer based insulin detection assays. By using porcine insulin as internal standard, linear curve can be constructed. This method shows great promise in the field of biomarker detection.In chapter4, we report a novel aptamer immobilized on-target MALDI-TOF MS strategy for the high-throughput detection of lysozyme with laser accelerated proteolysis. Up to our knowledge, we are the first group to fabricate such platform. First, we modify the target plate with stable and porous gold layer as we did in chapter3.Then, thiol group (SH-) modified aptamer was immobilized onto the spot surface through the easy formation of covalent bond with gold atoms. After that, the target plate was used for the enrichment of lysozyme in trace level. With the assistance of laser irradiation lysozyme can be efficiently digested in30s. At last, the sample was subjected to MALDI-TOF MS analysis. Peptide with m/z=1400.6were used for the identification and quantification of lysozyme. Liner calibration curves with good linealities were both obtained in standard solution and human urine by imploying peptide with one amino acid difference with the signal peptide of lysozyme. LODs of detecion at14ng/mL and66ng/mL were achieved seperately in standard solutions and undiluted human urine. We have also succesfully managed the detection of endogenous lysozyme in human serum and urine. The concentration of endogenous lysozyme in human urine was calculated based on the calibration curve to be125ng/mL, which is in accordance with the normal lysozyme range in human urine. Because of the simplicity, high-speed, sensitiveness and versatility of our method, we believe it has a bright future in the large-scale detection and analysis of proteins.In chapter5, we developed antibody based sandwich assay with peptide as barcode for the amplified MALDI-TOF MS detection of cancer biomarker AFP. First, antibody was immobilized onto the surface of magnetic microspheres for the capture of AFP. The experimental conditions were optimized. Random and self-oriented immobilization methods were compared, and at last we choose self-oriented immobilization of antibody through its carbohydrate chain to obtain the best recovery of AFP protein in low concentration. After careful optimization of experimental conditions, the succesful coadsorption of the second antibody and barcode peptide onto gold nanoparticles were achieved with little disturbance in the stability of the gold colloid solution. By eluting the barcode peptides for the MALDI-TOF MS, signal amplification will be achieved theoretically. However, the expriment was not finished due to limited time. We are trying to conque the problems encounterd and continue the research. If it succeded in the future as we expected, the method can greatly enhance the sensitivity of MALDI-TOF MS, even compareble with the most sensitive methods and will have a bright future in the large scale detecion and even clinical applications.In summary, this thesis focused on developing affinity based sensitive and high-throughput assays for the detection of disease biomarkers because of the challenges existed in this area. We first developed novel gold coated magnetic microspheres with large fuctionality sites for the immobilization of aptamer. We have also developed on-target aptamer microarray for the fast and sensitive detection of small proteins and peptides. By applying laser-accelerated digestion, the on-plate aptamer microarray can further achieve the real high-throughput ability for large protein detection. The methods described in this thesis provide good solutions and strategies to analysis of important proteins. What’s more, the idea of using peptide as barcode for the amplified biomarker detection by MALDI-TOF MS, if succesful, could lower the detection limit at least2orders of magnitude and become very promising technique in the field of biodiagnostics.