Construction Of Highly Sensitive Electrochemiluminescence Sensing Platform Based On Liposome Assisted Signal Amplification

The accuracy and sensitivity of disease-related biomarker detection are critical to many areas of modern biochemistry and biomedical research.For example,the clinical quantification of cancer biomarkers is critical for early diagnosis and disease surveillance.In addition,the detection of biomarkers can help understand the basic biological processes involved in the development of the disease and monitor how patients respond to treatments.Bioactive molecules（such as nucleic acids,proteins,small molecules,etc.）play a vital role in complex life activities.Efficient and accurate acquisition of this molecular information is of great significance for early diagnosis and treatment of diseases.Electrochemiluminescence（ECL）analysis technology has become one of the frontiers of modern analytical science,with high controllability of electrochemical analysis and high sensitivity of luminescence analysis,especially suitable for the detection and analysis of complex samples and trace components of biological systems.It opens up a new way for efficient and sensitive detection of biomolecules.With the rapid development of science and technology and the higher requirements of researchers on the analytical performance of ECL biosensors,improving the analytical sensitivity has always been a research topic in the field of ECL biosensors,which has promoted the development of a series of signal amplification strategies and technologies.How to further improve the design of ECL biosensor and obtain a more sensitive and accurate sensing platform by using various signal amplification strategies is still a huge challenge.Based on this,in order to improve the stability and sensitivity of ECL biosensors,this paper synthesized a series of liposome bioprobes（Ab2-GOLL,Ab2-AALL,Ab2-GSHL）,combined with enzyme catalysis,redox cycle,energy resonance transfer and other signal amplification strategies to build a series of NEW ECL immunoassay platform.Sensitive and accurate detection of disease markers such as prostate specific antigen（PSA）and myoglobin（Myo）was achieved.The main innovations and research contents are as follows:1.Prostate cancer is one of the deadliest diseases in the world,killing thousands of people each year.PSA is an internationally recognized biomarker for clinical diagnosis of prostate cancer.Studies have shown that when PSA concentration rises to 2 ng m L-1,the invasion of prostate cancer is more likely to occur in the human immune system.Therefore,a method with high sensitivity,good selectivity and rapid dynamic response to human serum PSA concentration is urgently needed.A sensitive and efficient ECL immunoassay platform was constructed based on enzyme catalysis and liposome signal amplification technology to detect PSA.During the construction of the sensor,GOLL was synthesized and connected with a secondary antibody（Ab2）via glutaraldehyde as a signal probe.The sandwich immune response was constructed in a 96-well plate,and the concentration of GOx released by liposome cleavage increased with the increase of target PSA concentration.GOx synergies with reduced graphene oxide gold（r GO-Au）modified on the electrode to achieve signal amplification and promote ECL emission of the system.The immunosensor detected PSA sensitively and accurately with a linear range of 1.0 × 10-13 – 1.0 × 10-8 g m L-1 and detection limit of 1.7 × 10-14 g m L-1.In addition,the method was successfully applied to the determination of PSA in human serum samples with the recoveries of 89.0%-113.0%,and the relative standard deviation（RSDs）was not higher than 6.6%.This immunoassay method makes liposome signal amplification technology have great application potential in ECL bioanalysis.2.The liposome cooperative signal amplification technique was combined with the chemical-chemical（CC）redox cycle signal amplification mechanism,and the ultra-sensitive ECL immunoassay was constructed based on the redox cycle and splitting strategy.The principle is as follows: A sandwich immune reaction was constructed in a 96-well plate and a liposomal probe（AALL）coated with ascorbic acid（AA）was attached.A large number of AA signaling molecules were released by liposome cleavage.Au NCs/Ti O2 NTs electrodes were implanted with Au NCs as seed,reducting the Au3+ to produce gold nanoparticles（Au NPs）.With the introduction of Tris[2-carboxyethyl] phosphine hydrochloride（TCEP）into the system,TCEP reduced the oxidation product dehydroascorbic acid（DHA）of AA to AA again,and the regenerated AA continued to promote the generation of Au NPs on the Ti O2 NTs electrode,thus forming an efficient CC redox cycle.Signal amplification is realized.Using PSA as the detection target,liposome and CC redox cycle signal amplification were used to achieve ultra-sensitive split ECL bioassay,with a linear range of 1.0 × 10-14 – 1.0 × 10-8 g m L-1 and a detection limit of 6.7 × 10-15 g m L-1.This method was used to determine the content of PSA in human serum.The recoveries were 90.0 – 109.1%,and the RSDs was less than 6.1%.This work provides a new vision for the further research and development of ECL bioanalysis based on liposome signal amplification technology.3.Signal amplification is very important for the construction of sensitive ECL biosensors.Therefore,it is the common goal of researchers to explore efficient and simple signal amplification strategies.A sensitive and accurate ECL-RET biosensor for Myo detection was constructed based on liposome and RET.Black phosphorus nanosheets（BP NSs）have better ECL performance and can be used as energy donor.Manganese dioxide nanosheets（Mn O2 NSs）can be formed by in-situ reduction of potassium manganate（KMn O4）on BP NSs,and BP/Mn O2 NSs can be formed.Because BP NSs and Mn O2 NSs match in spectrum,efficient RET can occur,and thus quench the ECL signal of BP NSs.At the same time,the glutathione loaded liposome（GSHLL）was connected in a 96-well plate by sandwich immune reaction.The GSH released from liposome lysate could redox with Mn O2,which reduced Mn O2 to Mn2+ and destroyed RET,thus restoring the ECL signal of BP NSs.Taking Myo as the analysis object,a sensitive split immunoassay platform was constructed by combining liposome with RET signal amplification strategy with a linear range of 1.0 × 10-13 – 1.0 × 10-7 g m L-1 and detection limit of 2.5 × 10-14 g m L-1.The method was used to determine Myo content in human serum samples successfully.The recoveries were in the range of 105.4% – 116.0%,RSDs was less than 6.0%.This work has universality and will effectively expand the application of ECL immunoassay based on liposome signal amplification technology.