Signal Amplification-based Fluorescent Biosensor For Highly Sensitive Detection Of Small Biological Molecules

Biomolecules include all kinds of unique organic molecules in organisms,including nucleic acids,proteins,enzymes,cells,tissues,and small biological molecules.The simple,fast and efficient detection of biomolecules is of great significance in disease diagnosis,food and drug analysis,environmental monitoring and scientific research.As we all know,there have been various serious food safety incidents in recent years,and food contamination is a problem that deserves much attention throughout the country and the world.In addition,early diagnosis and treatment of diseases are also issues that cannot be ignored for human health and safety.The detection of various biochemical indicators such as glucose and antibiotics in human body provides an important basis for clinical analysis.Therefore,it is of great significance to sensitively detect small molecular substances related to food monitoring and disease diagnosis.Fluorescent biosensors have the advantages of being faster,more sensitive and lower cost than traditional methods,and they have been widely used in various fields today.Based on the above reasons,this work focuses on the construction of different signal amplification methods forfast and sensitive detection of small molecules as different targets.The specific research content is as follows:（1）Highly sensitive detection of melamine illegally added to food.Melamine is often added to milk to increase its protein content because of its high nitrogen content.However,excessive melamine can cause damage to human body tissues or even death,such as the kidneys,and severe kidney failure is at risk of death.So,it is very important to detect the melamine content in food.Melamine can coordinate with two thymines to form unique and stable T-melamine-T structures,therefore,we have developed a T-melamine-T base mismatch-mediated rolling circle amplification（RCA）signal amplification means for amplified sensitive and label-free fluorescent detection of melamine in milk.In this method,the presence of melamine facilitates the hybridization between two T:T base mismatch containingin DNA strands,which triggers RCA generation of many long sequences with repeated G-quadruplex segments.The fluorescent dye,thioflavin T,further binds the resulting G-quadruplexes to exhibit substantially enhanced fluorescence for sensitive detection of melamine at the low nanomolar level in a label-free manner.Besides,the developed approach is highly selective toward the discrimination and detection of melamine in milk samples.With reasonable design,this method can also be applied to the detection of other biomolecules.（2）High sensitivity detection of kanamycin in food is closely related to food safety.In this study,we have established a fluorescence sensor based on the combination of primer exchange reaction and DNAzyme amplification technology to achieve high specific detection of kanamycin in milk.The target molecule kanamycin can specifically bind to aptamer and the toehold on the hairpin probe HP1 bound to the primer strand is exposed.Next,the extension of the auxiliary chain was triggered in the presence of polymerase and numerous Mg²⁺dependent DNAzymes was obtained through the primer exchange reaction.Subsequently,DNAzyme specifically recognize and then digest the cleavage sites of hairpins 2,which resulted in the release of G-quadruplex sequences locked in the hairpin structure.The fluorescent dye ThT is subsequently embedded in the G-quadruplex sequences.Thus significantly enhanced fluorescence signal was generated.The sensor achieves high sensitivity detection of kanamycin down to the detection limit of nM.In addition,by changing the sequence of the aptamer,this method can be used for the detection of other biomolecules and provide convenience for food safety monitoring.（3）ATP is a direct energy substance of living organisms,and it plays an important role of a“molecular unit of currency”in living cells.Therefore,detection of aberrant ATP concentrations with high sensitivity and selectivity is of critical importance for monitoring many biological processes and disease stages.The chain displacement caused by the target molecule can lead to multiple cycles,we have established a highly sensitive,label-free biosensor based on aptamer for the fluorescence detection of ATP in human serum.The ATP target molecules associate with the aptamer-containing double hairpin probes and cause conformational changes of the probes.Then,the cyclic strand extension/excision processes were initiated in the presence of polymerase,endonuclease and assistance sequences for the detection of ATP and a large number of G-quadruplex sequences were produced.The organic dye thioflavin T subsequently binds these G-quadruplex sequences to yield substantially enhanced fluorescence emission for achieving highly sensitive detection of ATP down to 2.2 nM in the range of 5 to 200 nM without using any labels.The developed aptamer sensing method also exhibits high selectivity and allows the detection of ATP at low concentrations in diluted human samples,which offers promising opportunities to establish effective signal magnification means for the detection of various biomolecules at trace levels.