Chem-bio Sensing Technologies Based On Mass Spectrometric Measurements

The accurate quantitative analysis of some important analytes(such as protein,miRNA,drugs,etc.)in complex biological systems is of great significance for the diagnosis,monitoring,treatment and prognosis of diseases.Chem-bio biosensors based on spectroscopic(e.g.,fluorescence spectroscopy,ultraviolet-visible spectroscopy,surface-enhanced Raman spectroscopy)and electrochemical techniques have played an important role in the detection of important analytes in complex biological systems.However,conventional chem-bio biosensors suffer from one or more of the following problems:severe matrix interferences,time-consuming operation,expensive dye labeling,miscellaneous primer design,and difficulties in simultaneous detection of multiple target analytes,and therefore have not yet been widely accepted and applied in practice.The development of novel sensing technologies with properties of high detection speed,simple operation,low cost,high sensitivity and specificity,and multi-target detection capability is still one of the research hot spots in the field of biomedical research and clinical diagnosis.Recently,mass spectrometry has attracted much attention in the field of chem-bio sensing due to its advantages of high detection speed,excellent specificity,multi-target detection capability,and the ability to detect a wide range of analytes.However,in comparison with fluorescence spectroscopy and electrochemical techniques,mass spectrometry has the problems of relatively lower sensitivity and poorer signal stability,which limit its wide application prospects in the field of chem-bio sensing.This thesis attempts to address the above mention problems of mass spectrometry through seam Lessly integrating various signal amplification strategies developed in chem-bio sensing filed and advanced chemometric data analysis methods,and then develop novel chem-bio sensing technologies based on mass spectrometric measurements to realize fast,sensitive and accurate qualitative and quantitative analysis of target analytes in complex biological systems.The details are as follows:1.Mass spectrometric platform for the quantification of chiral compounds based on chemical derivatization and spectral shape deformation quantitative theoryIn Chapter 2,a facile mass spectrometric platform for quantitative analysis of chiral compounds was developed by integrating mass spectrometry based on chemical derivatization and the spectral shape deformation quantitative theory.Chemical derivatization was employed to introduce diastereomeric environments to the chrial compounds of interest,resulting in different abundance distribution patterns of fragment ions of the derivatization products of enantiomers in mass spectrometry.The quantitative information of the chrial compounds of interest was extracted from complex mass spectral data by an advanced calibration model derived based on the spectral shape deformation quantitative theory.The performance of the proposed platformd was tested on the quantitative analysis of R-propranolol in propranolol tablets.Experimental results demonstrated that it could achieve accurate and precise concentration ratio predictions for R-propranolol with an average relative predictive error(ARPE)of about 4%,considerably better than the corresponding results of the mass spectrometric method based on conventional univariate ratiometric model(ARPE:about 12%).The limit of detection(LOD)and limit of quantification(LOQ)of the proposed platform for the concentration ratio of R-propranolol were estimated to be1.5 and 6.0%,respectively.It is reasonable to expect that the proposed method can be a competitive alternative for the quantification of enantiomers.2.Ultrasensitive Detection of Protein Biomarkers by MALDI-TOF Mass Spectrometry Based on ZnFe₂O₄Nanoparticles and Mass Tagging Signal AmplificationIn Chapter 3,a facile MALDI-TOF mass spectrometric platform for quantitative analysis of protein biomarkers was developed based on magnetic ZnFe₂O₄nanoparticles and mass tagging signal amplification.In this platform,magnetic ZnFe₂O₄nanoparticles functionalized with an aptamer of the biomarker of interest was used to magnetically separate silica nanoparticles modified with another aptamer of the target biomarker and a barcoding peptide from solution phase in the presence of the biomarker of interest.After silica nanoparticles were dissolved by KHF₂,the released barcoding peptide was detected by MALDI-TOF mass spectrometry and magnetic ZnFe₂O₄nanoparticles could also be used as assisting matrix of laser desorption ionization.Since the mass spectral intensity of the barcoding peptide is directly related to the concentration of the target biomarker,the proposed platform can be applied to the quantification of the target biomarker in complex biological samples.3.A highly sensitive and specific mass spectrometric platform for miRNA detection based on multiple metal nanoparticle tagging strategyIn Chapter 4,a highly specific and sensitive mass spectroscopic platform for miRNA detection was developed based on ligation reaction,hybridization chain reaction amplification and multiple metal nanoparticle tagging.Its high specificity is resulted from the adoption of ligation reaction and multiple metal nanoparticle tagging.Hybridization chain reaction amplification and metal nanoparticle tagging endows it with feature of high sensitivity.The proposed mass spectrometric platform achieved quite satisfactory quantitative results for Let-7a in real-world cell line samples with accuracy comparable to that of the real-time quantitative reverse-transcriptase polymerase chain reaction method.Its limit of detection and limit of quantification for Let-7a were determined to be about 0.5 and 10 f M,respectively.Furthermore,the proposed platform can unambiguously discriminate between the target miRNA and non-target ones with single nucleotide polymorphisms based on their response patterns defined by the relative mass spectral intensities among the multiple tagged metal elements,and can also provide location information of the mismatched bases.Its unique advantages over conventional miRNA detection methods make it a promising and alternative tool in fields of clinical diagnosis and biomedical research.4.Ultrasensitive detection of multiple cancer biomarkers by single particle inductively coupled plasma mass spectrometry based on a triple cascade amplification strategyIn Chapter 5,a versatile triple cascade amplification strategy was developed for ultrasensitive simultaneous detection of multiple cancer biomarkers using single particle inductively coupled plasma mass spectrometriy(sp ICP-MS).The triple cascade amplification strategy consisted of an enhanced Rec J_fexonuclease-assisted target recycling amplification module,a hybridization chain reaction amplification module and a signal amplification module based on DNA-templated multiple metal nanoclusters.In the enhanced Rec J_fexonuclease-assisted target recycling amplification module,the DNA bases at the 5'ends of aptamers for specific recognition of biomarkers were deliberately replaced by the corresponding RNA bases to enhance amplification efficiency.The signal amplification module based on DNA-templated multiple metal nanoclusters was innovatively used to amplify the signal measured by sp ICP-MS and at the same time effectively suppress possible background.The proposed sp ICP-MS platform achieved quite satisfactory quantitative results for both carcinoembryonic antigen(CEA)and a-fetoprotein(AFP)in human serum samples with accuracy comparable to that of the commercial ELISA Kits.Moreover,it has wide dynamic ranges for both CEA(0.01-100 ng/m L)and AFP(0.01-200 ng/m L).Its limit of detection values for CEA and AFP were 0.6 and 0.5 pg/m L,respectively.Compared with conventional biomarkers detection methods,the proposed sp ICP-MS platform has advantages of operational simplicity,ultrasensitivity,wide dynamic range,and low background.Therefore,it is reasonable to expect that the proposed sp ICP-MS platform can be further developed to be a promising alternative tool for biomarker detection in fields of clinical diagnosis and biomedical research.