Fluorescence anisotropy detection methods based on nucleic acid probes
Nucleic acid probe(NAP)-based fluorescence anisotropy(FA)is an excellent biosensing method due to its high throughput,homogeneous detection ability,and accuracy.FA is a ratio determination method in which the FA value(r)is inversely proportional to the rotational speed of the fluorophore modified NAP when the temperature and viscosity of the detection solution are constant.Based on the above,the FA method has been widely utilized for detecting various biomolecules.However,the sensitivity of traditional FA approaches is limited,mostly due to the following two factors:(1)The small volume(or mass)and fast rotation speed of most target molecules make it difficult to produce significant changes in FA,resulting in poor detection sensitivity;and(2)the large volume(or mass)of the NAPs causes the background signal to be high,resulting in the change in r after addition of the target to be insignificant,thereby leading to poor sensitivity.In this review,we describe in detail the strategies for different signal amplification methods to improve the sensitivity of NAP-based FA methods.To enhance the sensitivity of FA,one strategy is to use enhancement materials of an appropriate volume(or mass)to improve the FA signal.Another is to reduce the background signal by cutting NAPs into fragments using different enzymes.As a biomacromolecule,protein not only has a large volume(or mass),but it also exhibits good biocompatibility,making it a common FA amplification material.Unfortunately,the suitability of this protein is limited,as it becomes unstable when heated and complex to operate during use.At the same time,inorganic nanomaterials,such as metal nanoparticles,carbon nanomaterials,and transition metal nanomaterials,offer advantages such as good thermal stability and easy synthesis and surface modification;as a result,they are often utilized to enhance FA signals.However,most inorganic nanomaterials have uncontrollable sizes and strong fluorescence quenching capabilities,which can reduce the accuracy of FA.DNA nanostructures are another excellent FA amplification material due to their simple synthesis,precise and controllable structure,strong addressability,good biocompatibility,and ease of adding multiple modifications.Although the sensitivity of these methods has improved,the large volume(or mass)of NAPs still results in a high background signal and a low signal-to-background ratio,so the sensitivity still needs to be improved.An ideal FA method should have both a high FA signal and low background noise.With the development of DNase signal amplification technology,researchers have reduced the background signal by cutting NAPs into fragments through various enzyme digestion reactions,thereby obtaining a highly sensitive FA method.Additionally,the emergence of CRISPR-Cas technology provides a new option for signal amplification.CRISPR-Cas proteins can bind to crRNA to form complexes that activate Cas activity when the target gene is present.Moreover,CRISPR/Casl2 and CRISPR/Cas13 proteins have excellent trans-cleavage activity,allowing them to cut not only specific targets,but also any undifferentiated non-target ssDNA.Consequently,researchers have utilized the CRISPR system's trans-cleaving capability to cut NAPs,thereby reducing background values and increasing sensitivity.In a word,we summarized the signal amplification strategy of the NAP-based FA method from two aspects of increasing signal value and decreasing background value.At the same time,we also compared the sensitivity of the FA method for detecting different biomolecules,providing a valuable reference for establishing a highly sensitive FA detection method.
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