Fluorescence Sensing Technologies Based On Nucleic Acid Probes And Advanced Data Analysis Methods

Fluorescence sensing technologies with the merits of simple operation,high detection speed,exquisite sensitivity and good selectivity,have been widely applied to the fields of biochemical analysis,medical diagnostics,environmental monitoring,food and drug safety,etc.Along with the development of other academic disciplines,fluorescence sensing technologies have been continuously evolving to become more accurate and efficient.Since many analytes are generally nonfluorescent or weakly fluorescent,their detections by fluorescence sensing technologies need the aid of fluorescent probes including organic small molecule probes,quantum dots,metal nano-probes,and nucleic acid probes.Among the probes used in fluorescence sensing technologies,nucleic acid probes are much more popular due to their low toxicity,good biocompatibility,high design flexibility,and easy availability.Quantification of analytes by fluorescence sensing technologies is generally based on the intensities of fluorescence responses.For complex real-world samples,the intensity of fluorescence response is not only related to the concentration of the target analyte but is also affected by other non-concentration factors such as background interferences and matrix effects which hiders the extraction of accurate quantitative information of the target analyte from its fluorescence response by s imple data analysis approaches.Fortunately,recent years have seen some important progress in chemometric s methods for alleviating the influence of non-concentration factors on the results of fluorescence quantitative analysis.Therefore,this thesis atte mpts to develop fluorescence chemo/biosensing technologies capable of accurate quantification of target analytes in complex samples through the combination of nucleic acid probes and advanced chemometric methods.The details are as follows:1.Quantitative polymerase chain reaction method based on internal standard technique and spectral deformation quantitative theory（Chapter 2）In real-time quantitative polymerase chain reaction（qRT-PCR）,the standard curve between the threshold cycle the and logarithm of template concentration is currently the gold standard for template quantification.The efficacy of this approach is limited by the necessary assumption that all samples are amplified with the same efficiency.To overcome this limitation,a new method has been proposed in Chapter 2 for quantitative PCR with internal standard under the direction of spectral deformation quantitative theory.Unlike existing methods based upon analysis of amplification profile position,the new method tries to determine the ini tial quantity of the target template in a sample from the fluorescence spectrum measured at a certain point during its PCR reaction.There is no unrealistic prerequisite（e.g.,constant amplification efficiency）for the successful application of the new me thod.It is reasonable to expect that the new method would have a place in real-time quantitative PCR,thanks to its features of no unrealistic prerequisite,sound theoretical basis,good performance,and implementation simplicity.2.Fluorescence chemo/biose nsing technologies based on nucleic acid probe and standard addition multivariate calibration strategy（Chapters 3 to 6）Traditional standard addition method can be used to correct the matrix effects in the fluorescence quantitative analysis of complex samples.However,it cannot address the problem of background fluorescence interferences that may exist in the samples at the same time.In Chapter 3,a fluorescence sensing method capable of the quantification of adenosine triphosphate（ATP）in biological samples such as serum and cell lysates with both matrix effects and obvious background fluorescence interferences through the combination of a fluorophore-labeled aptamer probe with the probe technique-based generalized standard addition multivariate calibration strategy(GSAM_probe).The experimental results showed that the fluorescence sensing method based on the aptamer probe and GSAM_probe could achieve accurate quantification of ATP in both serum and cell lysate samples.It is worth pointing out that the proposed method needs only one standard addition sample to mitigate both matrix effects and background fluorescence interferences,which effectively reduces the consumption of test samples.This property is very beneficial for quantitative analysis of test samples with high costs or sample volume that cannot meet the requirements of traditional standard addition methods.In Chapter 4,the probe technique-based generalized standard addition multivariate calibration strategy(GSAM_probe)was specifically modified,and combined with terminal deoxynucleotidyl transferase（TdT）to design a simple label-free fluorescence sensing method for alkaline phosphatase（ALP）.In the proposed ALP fluorescence sensing method,a DNA primer with a phosphoryl group at its 3’terminal is designed as the substrate of ALP-catalyzed hydrolysis.In the presence of ALP,the 3’-phosphoryl group of the DNA primer can be hydrolyzed to a hydroxyl group,which will initiate the elongation reaction of the DNA primer with the addition of TdT and d GTP.The resultant elongated DNA can readily fold into G-quadruplexes under the inducement of K⁺.ThT can specifically bind with G-quadruplexes to form G-quadruplex-ThT complexes,resulting in a strong increase in fluorescence emission centered at 495 nm.The modified GSAM_probe effectively mitigated the influence of matrix effects and background interference effects on the fluorescence response and realized accurate quantification of ALP in lysate samples of cancer cell lines with precision and accuracy incomparable to the commercial ALP detection kit.The limit of detection and limit of quantification of the proposed method were 0.02 and 0.07 U/L,respectively,about twenty times lower than those of the commercial ALP detection kit.Due to its merits of simplicity,sensitivity,specificity,as well as robustness to matrix effects and background interferences,the proposed method is expected to be used for ALP assays in clinical diagnosis and the screening of ALP inhibitors.In Chapter 5,a locked nucleic acid（LNA）assisted repeated fishing amplification strategy was proposed,and combined with GSAM_probe to develop a label-free and sensitive miRNA detection method with enhanced practical applicability.In the proposed method,the signal probe is captured and collected by a DNA-modified gold foil through a temperature-controlled cyclic process to achieve the accumulation and amplification of fluorescence re sponse.While GSAM_probe is adopted to mitigate the influences of both matrix effects in complex biolo gical samples and background interferences caused by the non-specific adsorption of the gold foils.Experimental results showed that the concentrations of miRNA-122 in different liver cancer cells determined by the proposed method were in good consistent with those obtained by the quantitative reverse transcription PCR method.Its limit of detection and limit of quantification values for miRNA-122 were 2.9 fM and 8.7 fM,respectively,fully satisfying the requirements for the quantification of miRNA-122 in real-world biological samples.In contrast to other methods based on enzyme-assisted signal amplification strategies,the performance of the proposed method is essentially unaffected by variation in enzyme activity.The design of the probe sequences used in the proposed method is rather simple and requires no labeling process.It is therefore applicable to the detection of any other miRNA sequences.In Chapter 6,a label-free fluorescence sensing method for kanamycin is developed by combining the strand di splacement amplification reaction triggered by kanamycin with the LNA-assisted repeated fishing amplification strategy proposed in Chapter 5.Kanamycin triggers the strand displacement amplification reaction to generate a new DNA sequence while achieving t he target cycling.The generated new DNA sequence can be used to capture and collect signal probes in the subsequent LNA-assisted repeated fishing amplification reaction,thus enabling sensitive detection of kanamycin.The proposed method realized accurate quantification of kanamycin in milk samples of different brands with average recovery rates varying from 93.2%to 110.4%.Its limit of detection and limit of quantification values for kanamycin were 18 nM and 54 nM,respectively,satisfying the requirements for kanamycin quantification in real-world samples.Its relative standard deviation values for repeated assays were less than 7.0%,significantly lower than those of the commercial ELISA kit.In the proposed method,the detection of kanamycin was translated into relatively simple hybridization reactions among DNA sequences.After some minor modifications to the target recognition sequence,it can be used to detect other small organic molecules,thus extending the application of the LNA-assisted repeated fishing amplification strategy.