Development Of A Label-Free Fluorescence Method For Apoptosis-Associated Caspase Assay Based On Branched Rolling Circle Amplification

Apoptosis is a crucial programmed cell death process that regulates a series of important physiological processes and stress responses through the elimination of unwanted cells.The dysregulation of cysteine aspartyl-specific proteases(caspases)activity may disturb the normal apoptosis regulation system and leads to the occurrence of many human diseases including Alzheimer's disease,hepatobiliary disease,acquired immunodeficiency syndrome,and cancer.Therefore,the development of an efficient caspase assay is of great importance to caspase-related apoptosis research and disease diagnosis.Conventional methods for caspase assays include mass spectrometry,high-performance liquid chromatography,immunohistochemical analysis,and western blotting.Alternatively,a series of new strategies based on electrochemistry,surface-enhanced Raman scattering,colorimetry,bioluminescence,and fluorescence have been developed for caspase assay.The available methods often have unsatisfactory sensitivity for the detection of low-abundance targets due to the lack of efficient signal amplification approaches.The development of simple,cost-effective,and sensitive methods for caspase assays is urgently needed.Nucleic acid amplification represents the most direct and effective way for target signal amplification.However,to the best of our knowledge,the use of nucleic acid amplification for caspase detection has not been reported so far,because the signal of target caspase and its cleavage product cannot be directly amplified like nucleic acid targets.In this research,we demonstrate the label-free and amplified detection of apoptosis-associated caspase activity using rolling circle amplification(RCA).RCA can generate long and repetitive single-stranded DNA(ss DNA)products by using a single DNA polymerase to achieve more than 10⁹-fold signal amplification at a constant temperature.In the proposed assay,a bifunctional peptide-DNA detection probe is rationally designed to convert the target caspase activity signal into the amplifiable DNA signal,which is subsequently amplified by branched RCA reaction and activity is detected in a label-free manner.This should be the first application of nucleic acid amplification technology to the detection of caspase activity.Due to the high amplification efficiency of the branch rolling circle amplification reaction,this assay has ultra-high sensitivity.This assay can achieve a low detection limit of 1.6×10^-4 U/mL and a wide dynamic range from 0.0005 to 0.4 U/mL,and it can be further applied for enzyme kinetic analysis,inhibitor screening,and the measurement of endogenous caspase activity at the single-cell level.The significant increase in the sensitivity of this assay can be attributed to:(1)The activity signal of caspase-8 can be effectively converted into DNA signal by cleaving the peptide-DNA detection probe;(2)The exponential signal amplification is achieved through the branched rolling circle amplification reaction with high amplification efficiency.Moreover,this method can be easily extended to the detection of other members of the caspase family by simply changing the peptide sequence of the detection probe,holding great potential in caspase-related apoptosis research,disease diagnosis and drug discovery.