Fluorometric Assays For Rapid And Sensitive Determination Of Acinetobacter Baumannii Based On Aptamers And Nanomaterials

Acinetobacter baumannii（A.baumannii,Ab）is the most common non-fermenters,Gram negative robs in clinics.The past two decades have witnessed a dramatic rise in the incidence of nosocomial infections by A.baumannii with a worrisome morbidity and mortality.As a major nosocomial pathogen,recent surveillance has reported its high prevalence and causative agent in ventilation-associated pneumonia（VAP）,bloodstream infection,abdominal infection and surgical site infection.However,the majority of those techniques either require costly and cumbersome instruments or only applicable for pure cultured strains that require at least 8-10 hours’incubation.Thus,development of fast,sensitive and reliable techniques for Ab determination is highly desired.Recently,developed biosensors have fulfilled these critical requirements of bacterial detecting performance due to the dramatic advancement in biotechnology and nanotechnology.Aptamers are single-stranded DNA or RNA with unique tertiary structures and can specifically bind with various targets,such as proteins,DNA,RNA,antibiotics,bacteria and small biomolecules.With the advantages of smaller sizes,lower manufacturing costs,none or low immunogenicities and versatile chemical modifications,aptamers have been well adopted in the fabrication of biosensors,which have been named as aptasensors with the combination of nanoparticles.Despite good performance has been frequently reported,aptasensors still have several drawbacks to overcome:（1）The sensitivity of aptasensor for bacteria detection fails to meet the requirement of early diagnosis in bacterial infection due to the low signal amplification efficiency;（2）The modification efficiency of aptamers is relatively low and it is difficult to standardize batch-to-batch reproducibility of the modification;（3）the aptamers is unstable and prone to be destroyed in biological samples.To solve these problems,we launched three study proposals and aimed to fabricate two fluorometric aptasensors to detect A.baumannii by exploiting the superiorities of aptamers,nanomaterials and biomaterials.The detailed contents of these studies are as follows:1 The signal amplifying mechanism of Zirconium-based MOFMetal-organic frameworks（MOFs）are synthetic polymeric hybrid materials comprised of metal clusters and organic linkers,and have attracted increasing attention in the development of biosensors for nucleic acid,tumor markers,antibiotics and other small biomolecules.Zirconium-based MOFs（Zr-MOFs,e.g.,UIO-66-NH₂,UIO-67,509-MOF,etc.）have demonstrated superiority for biosensor design due to high affinity of Zirconium node toward the phosphate groups of biomolecules.Several MOFs have been explored as signal amplification labels based on their merits of high porosity and specific surface area,while the potential role of Zr-MOFs in signal amplification is under development,due to the previously reached-consensus on their excellent chemical stability in the integrity of crystallinity structures.To fill this gap,in this work,we fulfilled proof test of our speculation that high concentration of phosphate would destroy the crystal structure of UIO-66-NH₂,release the fluorescence signal preloaded in UIO-66-NH₂,and thus amplify the fluorescence signal.The competitive binding efficiency of phosphate with NH₂-BDC linker to Zr node is positively correlated with its concentration.After treatment of 1 M Na₂HPO₄,clear collapse and aggregation of the octahedral structure are observed and the milky color of the UIO-66-NH₂suspension even turns almost transparent.The featured sharp crystalline peaks disappear in XRD spectrum.The fluorescence intensity of NH₂-BDC linker in F@UIO-66-NH₂ at 426 nm reaches the maximum almost immediately under the excitation wavelength of 328 nm.The capped fluorescein molecules are released and thus produce enhanced fluorescence intensity at 512 nm（under the excitation wavelength of 492 nm）in less than 2 min.Collectively,those observations support the feasibility of our amplification strategy by exploring the unstable structure of UIO-66-NH₂in high concentration of anionic phosphate ions.This study presents critical and inspiring evidence for the application of Zr-MOF in the fabrication of biosensors,nano-imaging agents and nanodrugs.2 Fluorometric determination of A.baumannii based on aptamers and Zr-MOFsSince Zirconium-based MOFs have demonstrated high affinity of Zirconium node toward the phosphate groups of biomolecules,it facilitates the immobilization of terminal phosphate-labeled DNA/RNA strands on their outer surface via strong Zr-O-P bonding in a preferential“End-On”manner and with high modification efficiency.However,the surface function of Zr-MOFs for bacteria determination is unknown.Therefore,we fabricated a core-shell magnetic capture probe by utilizing Zr-MOFs as functional coat of magnetic Fe₃O₄ nanoparticles（MSNs）to attach terminal phosphate-labeled aptamers as capture probes for bacteria assay for the first time.Based on the aforementioned signal amplifying mechanisms,UIO-66-NH₂ was preloaded with fluorescein modification of LPS specific aptamers to fabricate signal probes.After successive incubation with A.baumannii in blood samples and magnetic separation,the sandwich-type composite of capture probe/A.baumannii cells/signal probe was treated with high concentration of anionic phosphate ions to destroy the nanostructure of UIO-66-NH₂ in the signal probe and fast release of fluorescein to produce amplified fluorescence signals.Due to the high aptamer modification efficiency of Zr-mMOF-p-Ab-Apt（93.30%）and its strong affinity to A.baumannii,the enrichment efficiency and specificity of this capture probe has reached to 96.79%and 94.31%,respectively.Further,due to the high fluorescein loading efficiency of UIO-66-NH₂ and our novel amplification strategy to destroy F@UIO-66-NH₂-p-LPS-Apt to release and amplify fluorescein signals at 512 nm in the presence of high concentration of anionic phosphate ions,the sensitivity of this method has reached 10 CFU/mL.This method allows enrichment and determination of A.baumannii within 2.5 h.The limit of detection of A.baumannii in blood samples is 10 CFU/mL with a linear range of 10¹-10⁵ CFU/mL.Owing to strong Zr-O-P bonding between Zr-MOFs and phosphory-lated aptamers,the core-shell structured magnetic capture probe offers rapid and specific enrichment of target bacteria.This strategy not only ensures the stability of the secondary structure of aptamers,but also increase the attach efficiency of aptamers to nanomaterials and the batch to batch reproducibility,which solve the aforementioned problems of aptasensors.Moreover,alteration of the types of aptamers in there probes may offer good enrichment efficiency and sensitivity to various pathogens,such as virus,bacteria and fungi.Due to novel amplification strategy,our low limit of detection indicates the potential of this assay for laboratory diagnosis of microbial infections.3 Fluorometric determination of A.baumannii based on aptamers and CRISPR-Cas12a systemSince the bacterial loading in infection sites is lower than 10 CFU/mL,especially in the early stage of bloodstream infections,the sensitivity of the aforementioned study fails to satisfy the requirement of clinical diagnosis.The possible reason is that the volume of bacteria is micrometer-level and our Zr-MOF nanoparticles are nanometer-level.The potential steric effect hinders the combination of them and decreases the sensitivity.Therefore,we further explored novel signal amplifying strategy to enhance the sensitivity of fluorometric assays to determine A.baumannii.In this work,the detection platform applies CRISPR-Cas12a as signal amplifier to enhance the fluorescence output upon activation by aptamer intermediates,generated only in the presence of target bacteria.Ab aptamer（Ab-Apt）was immobilized on MBs via streptavidin-biotin binding.Ab-Apt was then hybridized with a TS sequence-containing strand（c-Ab-Apt）partially complementary to the aptamer,the higher affinity of which toward Ab than the short complementary strand would lead to the release of c-Ab-Apt from the beads when Abs were presented,thus triggers subsequent CRISPR-Cas12a platform.The r/z ratio（Calculated by dividing the cleavage rate in the presence of Ab by that with buffer only）suggests that location of the complementary strand with 21 nt complementary to 5’-end of Ab aptamer is much more vulnerable to discharge than the other two position in the presence of Ab and lead to the highest r/z ratio.The theoretical limit of detection of A.baumannii in buffer is 3 CFU/mL with a linear range of 10¹-10⁵ CFU/mL.The selectivity of the assay was examined using E.coli,Pseudomonas aeruginosa（P.aeruginosa）,Enterococcus faecalis（E.faecalis）and Staphylococcus aureus（S.aureus）.While 10³ CFU/mL Ab in PBS buffer initiated clear cleavage of reporters,up to 10⁷ CFU/mL of the interfering bacteria did not really activated the CRISPR-Cas12a.Although approximately 30%loss of activity was observed in untreated serum sample,over 87%cleavage speed retained when 0.1 Unit/mL RNase inhibitor added into serum.This study successfully detects non-nucleic acid targets by transforming aptamer based bacterial detection signal to nucleic acid signal,which thus triggers CRISPR-Cas12a signal amplifying system.These results collectively demonstrate the potential of the CRISPR-Cas12a-based assay for detection of pathogens in clinical early diagnosis of microbial infection in singl-cell level.