Study On Monitoring Toxins Induced Apoptosis And Apoptosic Markers Based On Gold Nanooptical Probes

Biotoxins are a large class of toxic chemicals that are derived from organisms and can't be self-replicated.Both Abrin and aflatoxin B₁?AFB₁?are highly toxic.Once they enter the human body,they will cause serious threats and harm to human life and health.Most of the current studies on Abrin and AFB₁ focus on the establishment of detection methods for toxin content,but the applicability for detecting damage to organisms is weak.Therefore,the establishment of a new method for to study the toxicity of toxins at the molecular biological level is of great significance for exploring the apoptosis induced by toxins and monitoring the process of apoptosis.Gold nanomaterials have excellent optical properties and have been widely used in food safety,analytical chemistry and biomedical fields.Gold nanomaterials can be used as substrates of surface enhanced Raman scattering?SERS?with ultra-high sensitivity.Label-free biosensing at the single-cell level has been achieved.In particular,the multi-branch gold nanomaterials can enhance the Raman signal to a greater extent due to more"hot spots".At the same time,gold nanomaterials are good fluorescent quenchers.Combining Raman with fluorescence allows for multimodal visual monitoring of cell biology processes.Based on the optical properties of gold nanomaterials,label-free Raman probes and labeled Raman-fluorescence dual-mode nanosensors were prepared for qualitative characterization of cell apoptosis and quantitative detection of markers,which provided ideas for in-depth study of toxin-induced cell apoptosis.The main contents of this paper are as follows:1.Real-time and in situ monitoring of cell apoptosis induced by Abrin based on gold nanostars?Gold nanostars,AuNSs?.To achieve real-time in situ monitoring of apoptosis,the thiol polyethylene glycol?mPEG-SH?and cell penetrating peptide?TAT?was modified on75±5 nm AuNSs to construct as a label-free functionalized SERS probe to enhance the Raman signal of the cell itself.Cell damage was induced with Abrin.The changes of intracellular substances at different time?0 h,2 h,4 h,8 h,12 h and 24 h?were characterized by SERS spectroscopy.Differential spectrum analysis was used to visually compare the specific changes of intracellular substances in adjacent injury periods.Principal component analysis?PCA?was used to identify and distinguish different cell states,and to reveal the molecular dynamics of apoptosis.The results showed that within 0 h to 24 h of Abrin-induced cell injury,the changes of intracellular substances were mainly protein and DNA damage.2.Real-time and in situ quantitative detection of cytochrome c?Cyt c?in living cells based on SERS-fluorescence dual-mode nanosensors.On the basis of in situ monitoring of apoptosis,it is important to detect the activation of specific apoptotic markers.Cyt c,as an important marker of early apoptosis,is considered as a non-regression point in apoptosis.Based on this,quantitative detection of Cyt c in living cells was achieved by SERS and Fluorescence resonance energy transfer?FRET?.The aptamer of Cyt c was first modified on the surface of Gold nanoplates?AuNTs?,and the complementary strand modified with Raman reporter and the fluorophore Sulfonic acid-Cy5 carboxylic acid?Cy5?was ligated by complementary pairing criteria to construct a SERS-fluorescent dual-mode nanosensor.AuNTs simultaneously provided both enhanced Raman signal of Cy5 and quenched its fluorescence.Apoptosis was induced by aflatoxin B₁?AFB₁?.Under the optimal conditions,the SERS and fluorescence intensity of Cy5 showed good linear relationship with the concentration of Cyt c in living cells from 0.044?M⁹.95?M.The detection limit?LOD?could reach 0.021?M.The method was specific and accurate.The error of quantitative detection of Cyt c in living cells by this method was less than 10%.3.Real-time and in situ detection of caspase 3 in living cells based on SERS-fluorescence dual-mode nanosensors.On the basis of detecting the activation of specific apoptotic markers,tracking the transmission of apoptotic signals in real time can provide more detailed information for the study of apoptosis.Caspase 3,as a downstream apoptotic factor of Cyt c,is considered to be the most important terminal shear enzyme in cell apoptosis.Based on this,a SERS-fluorescence dual-mode nanosensor was constructed by modifying the N-terminal of caspase-3-specific dodecapeptide with Raman reporter and fluorophore Rhodamine B?Rb?,and connecting AuNTs with the thiol?-SH?on the C-terminal cysteine.AuNTs provided the effect of enhancing the Raman signal of Rb and quenching its fluorescence.Active caspase 3 specifically cleaved the recognition site in the dodecapeptide,resulting in attenuation of the Raman signal of Rb and recovery of the fluorescent signal.Under the optimal conditions,the SERS and fluorescence intensity of Rb showed a good linear relationship with active caspase 3 in the concentration range of 0.05 nM⁵0 nM,and the LOD could reach 0.001 nM.AFB1 was still selected to induce cell apoptosis in caspase 3expression.When the Cyt c in the cytoplasm reached a certain level,caspase 3 was activated.The application of this sensors was achieved within particular living cells.