New Optical Sensing Methods For Detecting The Activity Of Enzymes Based On The Nanomaterials And Functional Nucleic Acids

Recently, biosensors have greatly promoted the development of clinical diagnosisand drug screening, because of their high sensitivity, good selectivity, short analysistime and low-cost. Optical detection technology has attracted a great attention in theconstruction of biosensors, due to many advantages such as easy operation, noseparation and the detection can be implemented in situ, real-time and in vivo.Moreover, the development of functional nucleic acids and nanomaterials providemore novel strategies and platforms for the design of biosensing technology. Thisdoctoral thesis concerns on the research hotspot in the enzyme activity detection anddrug screening, focusing on how to improve the sensitivity, reduce the cost and so on.Combination of the advantages of nanomaterials and functional nucleic acids, severaloptical detection methods have been developed for the detection of alkalinephosphatase, polynucleotide kinase,adenosine deaminase, base excision repair enzymeand their inhibitors. Compared with the traditional methods, the proposed detectionmethods are sensitive, convenient and cost-effective. The practicability of thesedeveloped methods was also verified. The detailed contents are described as follows.Alkaline phosphatase (ALP) plays an important role in the clinical diagnosis ofrelated diseases. In chapter2, based on the inhibition of the synthesis of dsDNA-templated fluorescent copper nanoparticles (CuNPs) by pyrophosphate (PPi), a novellabel free turn-on fluorescent strategy for detecting ALP under physiologicalconditions has been developed using PPi substrate. This method relies on the stronginteraction between PPi and Cu2+, which would hamper the effective formation offluorescent CuNPs, leading to low fluorescence intensity. The ALP-catalyzed PPihydrolysis would disable the complexation between Cu2+and PPi, facilitating theformation of fluorescent CuNPs through the reduction by ascorbate in the presence ofdsDNA templates. Thus the fluorescence intensity was recovered and the fluorescenceenhancement was related to the concentration of ALP. This method is cost-effectiveand convenient without any labels or complicated operations. The present strategyexhibits a high sensitivity with a detection limit of0.1nM. The turn-on mode alsoprovides a high selectivity for ALP assay. Additionally, the inhibition effect ofphosphate on ALP activity was also studied. It also exhibited a good assayperformance in complex samples and satisfactory recoveries in diluted serum samples were obtained.Phosphorylation of DNA by polynucleotide kinase (PNK) is important in DNAdamage repair, replication and recombination. The evaluation of PNK activity hasreceived an increasing attention due to the significance of PNK. In chapter3, wepresented a label free fluorescent method for PNK activity assay using double strandDNA (dsDNA)-templated copper nanoparticles (CuNPs) as a fluorescent indicator. AdsDNA probe was introduced to act as both enzyme’s substrate and template forCuNPs formation. Upon the PNK reaction, the dsDNA template was phosphorylatedand then digested by λ exonuclease immediately, prohibiting the formation offluorescent CuNPs due to the lack of dsDNA template. This homogeneous PNKactivity assay does not require any other additional modifications of DNA substrate orcomplex design, making the proposed strategy simple, cost-effective and highthroughput. The proposed strategy is selective and sensitive with a detection limit of0.49U/mL. It also worked well in complex biological samples.The evaluation of adenosine deaminase (ADA) activity and its inhibitors takes animportant part in clinical diagnostics and drug screening. In chapter4, combination ofthe high specificity of aptamer and different adsorption ability of graphene oxide fornucleic acids with different conformation, using SYBR green I as an indicator andadenosine deaminase (ADA) as a model analyte, a label free fluorescent biologicaldetection method was designed based on the graphene oxide–aptamer platform. Twoparts of split aptamer can specifically recognize adenosine together, SYBR green Icould then stained in the duplex region of the aptamer/adenosine complex. Theformation of aptamer/adenosine complex made the aptamer to be released form thesurface of graphene oxide, resulting in a high fluorescence signal. After thedeamination of adenosine by ADA, the aptamer could not recognize the productinosine, and the aptamer and fluorescence dye were adsorbed on the surface ofgraphene oxide, leading to the fluorescence quenching. This method did not requirechemical modification on aptamer probes, making the analysis costless. Additionally, ahigher sensitivity was also obtained due to the strong fluorescence quenching ability. Italso provided a usefull technology for the detection other biomolecules usingaptamer-based methods. In chapter5, a novel label free colorimetric assay has beendeveloped for convenient and sensitive detection of ADA activity and its inhibitorbased on the enzyme-regulated aggregation of unmodified gold nanoparticles (AuNPs).This strategy relied on the strong interaction between exocyclic amino group ofadenosine and AuNPs which could diminish the stability of citrate-capped AuNPs by the displacement of citrate ions from the AuNPs surface, resulting in the aggregation.The deamination of adenosine by ADA prohibited the adenosine-dependentaggregation. This enzyme-regulated-aggregation strategy allowed a visual andhomogeneous assay of ADA activity without any other coupling enzymes, aptamers oradditional modifications, making the proposed strategy simple, cost-effective and highthroughput. The present strategy is highly selective and sensitive for ADA assay with adetection limit of0.8227U/L. Moreover, the evaluation of inhibition for ADA activityusing this colorimetric method was also successfully demonstrated.The base excision repair (BER) pathway plays a key role in resisting DNA lesions,and the activity of BER enzyme is connected to several diseases. In chapter6, usinguracil-DNA glycosylase (UDG) as a model analyte, two novel fluorescent methodswere developed for the detection of the BER enzyme based on the autocatalyticDNAzyme amplification strategy. UDG reaction could activate the DNAzyme whichcatalyzed the cycling cleavage of a molecular beacon (MB) structured probe in thepresence of cofactor, resulting in signal amplification in the detection of UDG activity.First, based on the decrease of melting temperature of substrate upon UDG reaction, adouble-stranded substrate was used to demonstrate this target-activated autocatalyticDNAzyme amplification strategy. DNAzyme was released from the double-strandedsubstrate after the treatment by UDG. The released DNAzyme could then catalyze thecycling cleavage of MB substrate. The present method exhibits a wide dynamic rangeand a high sensitivity, with a linear detection range from0to1.0U/mL and a detectionlimit of0.023U/mL. Additionally, this method could also be applied to evaluate theinhibition of UDG. Second, a rolling circle amplification (RCA) process wasincorporated into the DNAzyme strategy. This RCA-assisted CAMB strategy relied onthe digest of abasic site generated by UDG treatment using endonuclease IV. Thiscleavage produced a new primer probe with3′hydroxyl end that could initiate a linearRCA reaction. The RCA product had a tandem repeated sequence of DNAzyme, andeach DNAzyme sequence could cyclically cleave the MB probe and generated anincreased fluorescence signal. A remarkable enhancement of sensitivity (limit ofdetection=0.002U/mL) is obtained due to the coupled signal amplification cascade.