Development Of Novel Fluorescent Probes For Metal Nanoclusters Based On Biomarker Detection

Nowadays,biomarkers have become important indicators for early prevention,mid-term diagnosis and prognosis monitoring of diseases in clinical practice.They can also be used for the evaluation of the effectiveness and safety of clinical new drugs or diagnostic methods,which greatly shortens the time of clinical diagnosis and disease evaluation,and covers a wide range of biochemical entities such as proteins,biological small molecules,enzymes,nucleic acids and so on.With the emergence of various analytical methods,fluorescence analysis is in the ascendant.Its performance indicators are improving day by day,such as detection sensitivity,specificity and detection range.It has become an efficient biochemical analysis method.This method is simple and fast,which makes its application in the fields of biosensor,environmental monitoring,cell imaging and so on.Among them,metal nanoclusters（MNCs）and quantum dots（QDs）stand out from many fluorescent nano materials benefit from their superior optical properties.The application in biomarker detection is mainly to achieve the purpose of quantitative analysis by changing the fluorescence intensity of MNCs or QDs caused by the target analyte.The synthesis of stable and functional MNCs is the basis for the construction of fluorescent nano probes and their application in biological detection.In this thesis,three fluorescent nanomaterials,gold nanoclusters（Au NCs）,silver nanoclusters（Ag NCs）and QDs were selected to study and develop three fluorescent probes with different functions as optical sensing platforms.Through their fluorescence changes,they could identify different biomolecules closely related to human health,namely disease biomarkers,and realized the real-time and rapid detection of musculoskeletal disease（WMSD）,acute myocardial infarction（AMI）and osteoarthritis（OA）.The specific research contents were as follows:（1）In view of the close correlation between biological mercaptan and WMSD,the fluorescence enhanced monochromatic probe Au NCs was rapidly synthesized with glutathione（GSH）as stabilizer,and the rapid detection of biological mercaptan was realized based on aggregation induced luminescence effect（AIE）.The results showed that the optimal excitation wavelength of Au NCs was 420 nm and the emission wavelength was 600 nm.The probe had excellent sensitivity and selectivity to cysteine（Cys）and GSH in biological mercaptan.The detection ranges were 2.49μM～0.80 m M and 1.99μM～0.44 m M respectively,and the minimum detection limits were 0.42μM and 0.27μM respectively.The detection experiment of Cys in human serum samples exhibited that the probe could be applied to the actual sample detection of WMSD,which provided a new and effective detection method for the early prevention and treatment of WMSD.（2）Based on the study of monochromatic fluorescent probe,a ratio fluorescent probe for the detection of myoglobin（Myo）was constructed.Bovine serum albumin（BSA）-modified gold nanoclusters（BSA-Au NCs）were synthesized by hydrothermal method,and then gold nanoclusters-quantum dots（Au NCs-QDs）ratio fluorescent probes were constructed by coupling with cadmium selenide/zinc sulfide quantum dots（Cd Se/Zn S QDs）.The experimental results showed that the two emission peaks of Au NCs-QDs were located at 468nm and 630 nm respectively.Based on the fluorescence resonance energy transfer effect（FRET）,the fluorescence of Au NCs-QDs was sensitive to Myo,and the fluorescence intensity ratio(F₄₆₈/F₆₃₀)was well linear with the concentration of Myo.The linear detection range of the probe for Myo was 4.99 ng/m L～2.23 mg/m L,the detection limit was as low as 4.99ng/m L,and it had excellent specificity for Myo.The experimental sensing analysis of actual urine samples showed that the developed ratio fluorescence probe could be applied to the detection of AMI.The detection test paper combined with the ratio fluorescence probe and chromatography test paper was designed and prepared.Combined with the auxiliary detection of smart phone and software,the results displayed that the detection test paper realized the real-time,visual and intuitive detection of Myo.（3）A ratio fluorescence probe for the detection of reactive oxygen species hydrogen peroxide（H₂O₂）was developed,which relied on the unique fluorescence characteristics of MNCs.Low cost Ag NCs was synthesized by chemical reduction with sodium borohydride（Na BH₄）as reducing agent.Silver nanoclusters-quantum dots（Ag NCs-QDs）were formed by combining Cd Se/Zn S QDs.The effects of the volume ratio of Cd Se/Zn S QDs and Ag NCs on the fluorescence ratio of Ag NCs-QDs were investigated,and their spectral and fluorescence stability,morphology and potential were characterized.The experimental results clarified that the two emission peaks of Ag NCs-QDs were located at 473 nm and 661 nm respectively.The ratio fluorescence probe had good fluorescence stability and realized the sensitive detection of H₂O₂based on FRET effect.The fluorescence intensity ratio F₆₆₁/F₄₇₃had a good linear relationship with H₂O₂concentration.The ratio fluorescent probe could specifically recognize H₂O₂with a detection range of 3.32μM～2.7 m M,the minimum detection limit was 3.32μM.At the same time,it had excellent anti-interference performance.The above results showed the reliability of the probe in the analysis and detection of reactive oxygen free radicals and the diagnosis of OA.In this paper,three new fluorescent probes based on MNCs and QDs were constructed.On account of their excellent optical properties,fluorescence analysis was used as the detection method,and the fluorescence intensity of nano materials was used as the response signal to realize the real-time and quantitative analysis of target analytes.Biosensor platforms based on MNCs fluorescence signal were established,which realized the highly sensitive detection of biomarkers of WMSD,AMI and OA.They had the practical application value and application prospect of biosensor,provided efficient and accurate method for the early diagnosis of diseases,and provided experimental reference and research basis for the development of related new fluorescent probes.