Construction Of Signal Amplification-based Optical Aptasensors And Their Applications In Microcystin-LR Detection

Objective:Microcystin-LR（MC-LR）is a representative microcystin with the widest distribution range,the highest detection frequency and the strongest toxicity.The development of sensitive and easy-to-use methods for detecting trace MC-LR holds significant importance in safeguarding ecological environment and human health.In this paper,MC-LR specific aptamer was used as MC-LR specific recognition elements,while the enzyme catalysis and CRISPR-Cas systems as well as nanomaterials were used as signal amplification tools,the potential of different signal amplification strategies for the construction of MC-LR detection optical sensors was studied,and then,a series of signal amplification-based optical aptasensors were designed.This work presented scientific enlightenment for the development of novel MC-LR optical sensors,and provided novel technical support for the detection of MC-LR in the environment.Methods:1.DNA hydrogel signal amplification-based colorimetric aptasensor for the detection of MC-LRA Cu/Au/Pt TNPs mimic with peroxidase-like activity was synthesized by using"one-pot method".The peroxidase-like activity and storage stability of Cu/Au/Pt TNPs was evaluated.Subsequently,a Cu/Au/Pt TNP-DNA hydrogel which specifically responsive to MC-LR was prepared in which the MC-LR aptamer was used as the cross-linker,the complementary DNA（cDNA）was used as the hydrogel backbone,and Cu/Au/Pt TNPs were embedded.The synthesis and detection conditions of Cu/Au/Pt TNP-DNA hydrogel were optimized.Then,the linear range,limit of detection（LOD）and specificity of the DNA hydrogel signal amplification-based colorimetric aptasensor were investigated.Finally,the estabilished aptasensor was used for the determination of MC-LR in food spiked samples and environmental spiked water samples.2.CRISPR-Cas12a-based signal amplification fluorescence aptasensor for the detection of MC-LRThe cDNA to the MC-LR aptamer was designed.A competition reaction system in which MC-LR competed with the cDNA for the aptamer was established.Subsequently,using cDNA as the target sequence of CRISPR-Cas12a,the competition reaction was combined with CRISPR-Cas12a to establish a fluorescent aptasensor based on CRISPR-Cas12a signal amplification for specific detection of MC-LR.The important conditions,such as competition reaction temperature,cDNA concentration,competition reaction time and Cas12a/g RNA concentration,were optimized.The linear range,LOD,and specificity for MC-LR detection were evaluated.In addition,the spike recovery experiments using water samples were conducted to validate the accuracy and potentiality of that estabilised aptasensor.3.HRP-mediated CRISPR-Cas12a dual-signal amplification colorimetric aptasensor for the detection of MC-LRThe method for the modification of horseradish peroxidase（HRP）onto single-stranded DNA（ss DNA）was explored.A novel reporter of CRISPR-Cas system,MB-ss DNA-HRP,was prepared,followed by evaluating its catalytic performance,reproducibility,and stability.Then,the HRP-mediated CRISPR-Cas12a dual signal amplification aptasensor was constructed by using MB-ss DNA-HRP as signal reporter.The main conditions for detecting MC-LR through that aptasensor were optimized.The linear range,LOD and specificity of that aptasensor were duly evaluated.Finally,the accuracy and applicability of that aptasensor were assessed in actual samples by using the standard recovery method.4.CRISPR-Cas14a combined metal-organic framework fluorescence sensor for the detection of MC-LRTwo-dimensional ultra-thin Cu-TCPP（Fe）nanosheets were synthesized through hydrothermal method,and the characteristics of Cu-TCPP（Fe）nanosheets were characterized by various techniques.The ss DNA adsorption ability and fluorescence quenching ability of Cu-TCPP（Fe）nanosheets were investigated,and the mechanism of quenching ss DNA-FAM was explored.Subsequently,a novel CRISPR-Cas fluorescent signal reporter probe,Cu-TCPP（Fe）/ss DNA,was prepared throughπ-πstacking effect between Cu-TCPP（Fe）and ss DNA-FAM.Combining the signal amplification ability of CRISPR-Cas14a system with the fluorescence quenching ability of Cu-TCPP（Fe）nanosheets,a Cas14-p MOFs fluorescence aptasensor was constructed for highly sensitive detection of MC-LR.The linear range,LOD and specificity for the detection of MC-LR were evaluated.Additionally,that aptasensor was used for the detection of MC-LR in water samples,and the accuracy of the method was evaluated by spike recovery experiments.Results:1.DNA hydrogel signal amplification-based colorimetric aptasensor for the detection of MC-LRThe synthesized Cu/Au/Pt TNPs were spherical particles with diameters of 5～10 nm.The enzymatic kinetic results indicated that Cu/Au/Pt TNPs had higher enzymatic activity compared with nature HRP and other reported peroxidase-like nanomaterials.Cu/Au/Pt TNPs exhibited good storage stability,and their peroxidase-like activity did not change significantly within 7 days of storage.SEM and TEM results showed that the prepared DNA hydrogel had a typical three-dimensional network structure and successfully encapsulated Cu/Au/Pt TNPs.The intensity of the absorbance change produced by the aptasensor based on DNA hydrogel signal amplification showed a good linear relationship with the concentration of MC-LR in the range of 4.0 ng/L to 10.0μg/L,and the LOD was 3.0 pg/m L.MC-RR,MC-YR,and common small molecule pollutants estradiol（E2）and chloramphenicol（CH）in food and environment had little interference on the detection results.The spiked recovery test results showed that the aptasensor had a spiked recovery rate of 95.34%-107.07%for fish tissue,with RSD of 5.52%～7.86%,and for tap water samples of 93.96%～105.33%,with RSD of 7.86%～12.01%.Comparative analysis with commercial ELISA kits revealed no significant differences between the eatablished sensor and ELISA kits.2.CRISPR-Cas12a-based signal amplification fluorescence aptasensor for the detection of MC-LRAn effective competition reaction was established with 20 nt length cDNA at the 3’end of the aptamer.The optimization results showed that when the competition reaction incubation temperature was 30℃,the cDNA concentration was 90 nmol/L,the competition reaction incubation time was 20 min,and the Cas12a/g RNA concentration was 10 nmol/L,the aptasensor based on CRISPR-Cas12a signal amplification achieved the most favorable analytical performance for aptasensor.This aptasensor boasted high accuracy in quantifying MC-LR,within a linear range of 0.01ng/m L～50.0 ng/m L and a LOD of 0.39 pg/m L.Additionally,the sensing system encountered mild interference from potential contaminants such as MC-RR,MC-YR and Ochratoxin A（OTA）.The standard recovery rate of tap water samples by this method was between 99.65%～100.56%,and the RSD was between 6.49%and 11.16%,while the standard recovery rate of river water samples was between 99.77%～108.77%,and the RSD was between 6.98%～11.97%.3.HRP-mediated CRISPR-Cas12a dual-signal amplification colorimetric sensor for the detection of MC-LRThe HRP was efficiently modied on ss DNA by Na IO₄ method.MB-ss DNA-HRP had high catalytic activity and excellent reproducibility.However,due to the poor physical and chemical stability of natural HRP,the catalytic activity of the prepared MB-ss DNA-HRP decreased after being stored at 4°C for 7 days.Under optimal conditions,the constructed HRP-mediated CRISPR-Cas12a dual signal amplification aptasensor showed a linear response to MC-LR in the range of 0.01 ng/m L～50.0ng/m L with a LOD of 4.53 pg/m L.The results of specificity investigation showed that MC-RR,MC-YR and OTA interfered less with the detection results of that aptasensor.The aptasensor has achieved good results in the detection of tap water samples and river water samples.The standard recovery rate of tap water samples was between 86.22%～113.02%,the RSD was less than 14.62%,and the standard recovery rate of river water samples was between 91.01%～118.45%.The RSD was less than 17.58%.4.CRISPR-Cas14a combined metal-organic framework fluorescence sensor for the detection of MC-LRThe prepared Cu-TCPP（Fe）was an ultrathin nanosheet with the average thickness of 3.4±0.8 nm.Cu-TCPP（Fe）showed good storage stability with no leakage of Cu²⁺and Fe³⁺after being stored in aqueous solution for 10 days.The Cu-TCPP（Fe）nanosheets were synthesized through the interaction of carboxyl groups in TCPP（Fe）organic ligands with Cu metal sites.Compared with other MOFs or commonly used nanomaterials,Cu-TCPP（Fe）presented better quenching efficiency and faster quenching kinetics,which quenched 99%of the fluorescence of ss DNA-FAM within 10 min.The fluorescence quenching of ss DNA-FAM by Cu-TCPP（Fe）nanosheets was attributed to fluorescence resonance energy transfer（FRET）.Under optimal conditions,the Cas14-p MOFs fluorescence aptasensor presented a linear response to MC-LR ranging from 50.0 pg/m L to 1.0μg/m L,with a LOD of 19.0 pg/m L.The sensor exhibited high specificity which MC-YR,MC-RR,and MC-LW showed no significant influence on detection.The spiked recovery results illustrated high accuracy of that aptasensor in detecting MC-LR.The spiked recoveries of tap water samples and drinking water samples were between 89.63～93.02%and 71.46～96.16%,and the RSDs were 3.16～14.61%and 7.19～18.55%,respectively.Conclusions:1.The Cu/Au/Pt TNPs were synthesized with strong peroxidase activity and high stability.A Cu/Au/Pt TNPs-DNA hydrogel was prepared by using MC-LR aptamer as the cross-linker of the hydrogel which embed the Cu/Au/Pt TNPs.This Cu/Au/Pt TNPs-DNA hydrogel exhibited selective responsiveness towards MC-LR,enabling to detect MC-LR in food and water samples.2.Through a design and selection of the cDNA,a competition reaction system,which MC-LR competed with the cDNA for the aptamer was established.This competition reaction system was then combined with the signal amplification ability endowed by the trans-cleavage activity of CRISPR-Cas12a,and a fluorescence aptasensor based on CRISPR-Cas12a signal amplification was constructed to detect MC-LR in water samples.3.The MB-ss DNA-HRP was effectively synthesized and deployed as the signal reporter probe of the CRISPR-Cas system.By integrating the trans-cleavage activity of CRISPR-Cas12a and the catalytic activity of HRP,an HRP-mediated CRISPR-Cas12a dual signal amplification aptasensor that allowed MC-LR detection was developed.4.The ultrathin Cu-TCPP（Fe）nanosheets with ss DNA adsorption ability and fluorescence quenching ability was prepared.The Cu-TCPP（Fe）nanosheets were integrated with the signal amplification capabilities of the CRISPR-Cas14a system to establish a novel Cas14-p MOFs fluorescent aptasensor to detect MC-LR.5.The four optical aptasensors based on different signal amplification strategies were ease-to-use,had high-throughput analysis potential,high specificity and sensitivity for the detection of MC-LR,which could meet the demand for rapid and efficient detection of low concentration MC-LR.