Novel Chem/Bio-sensing Technologies Based On An Advanced Spectroscopic Quantitative Theory

Chembiosensing technologies have the advantages of fast detection,high specificity and sensitivity,and therefore have been widely applied to the rapid qualitative and quantitative analysis of various complex systems.Quantification of the target analytes by conventional chembiosensing technologies is generally based on the change in signal intensity.For complex systems,matrix effects,background signals,coexistent interfering substances,etc.can all cause conventional chembiosensing technologies to produce false positive/negative results.As a result,how to improve the accuracy of the qualitative and quantitative results of chembiosensing technologies for complex systems is one of the toughest‘nuts'to crack in the field of chembiosensors.Over the last decade,there have been some significant progresses in spectroscopic quantitative theories and methods in the field of Chemometrics,which can to a large extent mitigate or eliminate the detrimental effects matrix effects,background signals,coexistent interfering substances on the qualitative and quantitative results of spectroscopic techniques for complex systems.This thesis attempts to apply the advanced spectroscopi quantitative theories developed in the field of Chemometrics to the design of novel chembiosensors suitable for qualitative and quantitative analysis of complex systems.The details are as follows:1.Novel chembiosensing technologies based on mass spectrometry(Chapter 2 and Chapter 3)Given the close correlation between the expression of specific mi RNAs and the development and/or progression of some human diseases,there is a need for methods enabling highly sensitive and specific detection of mi RNAs in biological samples.In chapter 2 of this thesis,a highly specific and sensitive mi RNA detection platform was developed by combining nicking enzyme-assisted rolling circle amplification strategy,mass spectrometry and spectral shape deformation theory(SSD).The combination of nicking enzyme-assisted rolling circle amplification and mass spectrometry endows the proposed platform with high selectivity and sensitivity.The adoption of SSD facilitates the extraction of qualitative and quantitative inforamiton of the target mi RNA from the complex mass spectral signals measured by the proposed platform.Moreover,due to the usage of two padlock structures and two DNA probes,the proposed platform can not only unambigouely distinguish the target mi RNA from mismatched mi RNA based on the mass spectral response patterns defined by the relative mass spectral intensities of the two DNA probes and their fragments,but also provide the location information of the mismatched bases in non-target mi RNAs.In chapter 3,the hydrolysis patterns of DNase I against a DNA probe in both single-strand form and DNA/mi RNA heteroduplex were experimentally investigated in detail by mass spectrometry.Based on the knowledge obtained in the experiments,DNase I-assisted signal amplification strategy was proposed and used to design a mass spectrometric platform for mi RNA detection.Under the guidance of the spectral shape deformation quantitative theory,an advanced calibration model was derived to model the relationship between the complex mass spectral signals measured by the proposed mass spectrometric platform and the concentration of the target mi RNA(e.g.,mi RNA-122).With the aid of the advanced calibration model,the proposed mass spectrometric platform achieved quite accurate quantification results for mi RNA-122 in cell lysate samples of Huh-7 and Hep G2 cell lines with recovery rates ranging from 98%to 104%.It is noteworthy that the proposed mass spectrometric platform can differentiate the target mi RNA from other non-target one based on their mass spectral response patterns instead of mass spectral intensities,and hence possesses high specificity for the target mi RNA.2.Novel chembiosensing technologies based on fluorescence spectroscopy(Chapter 4 and Chapter 5)In chapter 4,a fluorescence platform for mi RNA detection was established by seamlessly integrating the duplex-specific nuclease(DSN)-assisted target recycling amplification and the strand displacement amplification in tandem.The designing of the proposed method was based on the full understanding of the hydrolysis patterns of DSN against the probe DNA in DNA/mi RNA heteroduplexe of the target mi RNA by mass spectrometry which endows it with high specificity.The proposed platform was successfully applied to the quantification of mi RNA-141 in cell lysate samples of three types of cancer cells.Its quantitative results were in consistent with those obtained by reverse transcription polymerase chain reaction method.The application of the proposed platform to the detection of other mi RNAs is rather simple and straightforward,requiring only changing the sequence of two DNA probes.Coventional fluorescence chembiosensing technologies distinguish the target DNAs from non-target ones with mismatched bases according to the intensities of fluorescence measurements.They are prone to the influences of matrix effects and coexistent fluorescence interferences and therefore liable to produce false positive results.In chapter 5,an attempt was made to realize accurate quantification of DNA in complex systems by integrating Exonuclease?assisted target recycling amplification with a pair of DNA probes labeled with two different fluorescent groups(concentration ratio equals to 1:1)and the advanced SSD model.The introduction of dual DNA probes labeled with two different fluorescent groups makes it possible to distinguish the target DNA from non-target ones according to the relative changes in the fluorescence intensities of the two fluorescent groups,and hence to accurately identify DNAs with single base mutations.3.Novel chembiosensing technologies based on surface-enhanced Raman spectroscopy(Chapter 6)In chapter 6,a SERS enhancing substrate with a high selectivity for Co²⁺were prepared by attaching(bis(pyridin-2-ylmethyl)amino)ethane-1-thiol onto the surfaces of Ag nanoparticles.A facile SERS platform for the quantification of Co²⁺in Chinese tea was developed based on this new SERS enhancing substrate.A calibration model based on the spectral shape deformation quantitative theory was adopted to address the poor reproducibility problem in quantitative SERS assay due to the practical impossibility in controlling the number and distribution of"hot spots"on or close to the surfaces of enhancing substrates.The proposed SERS platform was applied to the quantitative analysis of Co²⁺in five types of Chinese tea samples,and achieved quite satisfactory quantitative results with accuracy and precision comparable to that of the reference method?inductively coupled plasma mass spectrometry(ICP-MS).