Study On Gold Nanoparticle Probe Mediated Dark-Field Visual Enumeration Strategy For Chicken Bcl11b Positive Associated Circ₂420

Circular RNA（circRNA）is a type of closed circular non-coding RNA that is enriched with microRNA（miRNA）binding sites.It is capable of alleviating the inhibition of miRNA on its target gene and plays a crucial regulatory role in disease.Therefore,circRNA is regarded as having significant value in the early diagnosis of diseases.Circular RNA₂420（circ＿2420）is a newly discovered circRNA that indirectly targets the B-cell lymphoma/leukemia 11b gene（Bcl11b）in chickens,which can positively regulate the expression of Bcl11b mRNA in chickens.B-cell lymphoma/leukemia 11b gene（Bcl11b）in chickens is a recently discovered tumor-suppressor gene that can promote cell apoptosis and exhibit abnormal expression during the occurrence of chicken Marek’s disease and chicken J-subtype avian leukemia,among other tumor-related diseases.Therefore,this study suggests that circ＿2420 may have the potential to serve as an early indicator for predicting the expression levels of chicken Bcl11b mRNA,as well as have potential application value in diagnosing chicken Bcl11b-associated cell apoptosis and tumor occurrence.Reverse transcription quantitative real-time polymerase chain reaction（RT-qPCR）is currently the gold standard for quantitative detection of circRNA.However,RT-qPCR detection relies on sophisticated equipment and a strictly controlled operating environment,which limits its use for on-site rapid detection.Gold nanoparticle（GNP）,regarded as one of the most stable metallic nanomaterials,is widely utilized in biomedical diagnostics and detection due to its unique localized surface plasmon resonance（LSPR）effect and excellent surface modifiability.Dark-field microscopy（DFM）,a technique that utilizes the LSPR effect for imaging nanoscale samples,owing to its simple structure,ease of operation,and other advantages,DFM is commonly employed in biomedical research for the detection of microorganisms,antigens and antibodies,etc.,which has a good application prospect.However,there is currently lack of report on the use of GNP for dark-field detection of circRNAs.Therefore,this study aims to develop a DNA-modified GNP probe that can specifically capture circ＿2420 and achieve visual quantitative detection under DFM,which providing a new strategy for circRNA quantitative detection.After being infected with the J-subtype of Avian Leukosis Virus,chickens may develop the J-subtype of Avian Leukosis and subsequently caused tumor diseases associated with the Bcl11b gene,resulting in significant economic losses.This study demonstrated the feasibility of using circ＿2420 as a predictive indicator of chicken Bcl11b gene expression levels in inducing cell apoptosis and in a clinical animal model through sequential cell experiments.In addition,a DNA-modified GNP probe mediated Dark-field Visual Enumeration Strategy was established for rapid detection of circ＿2420,and an artificial intelligence-based Dark-field counting software was independently developed based on Python language.1.Circ＿2420 promotes Bcl11b gene mediated cell apoptosisStable overexpression cell line of circ＿2420（abbreviated as OE cell line）and Bcl11b gene knockout cell line（abbreviated as KO cell line）were obtained by lentivirus infection of DF-1 cell line,and DF-1cell line was infected with avian leukosis virus subgroup J（ALV-J）as a positive control group;the apoptosis and proliferation levels of cells in each group were detected by flow cytometry and the CCK-8 assay,respectively,and the relative expression levels of Bcl11b mRNA and circ＿2420 were quantitatively detected by RT-qPCR..The results indicate that:1)Both ALV-J infection and overexpression of circ＿2420 can induce upregulation of Bcl11b mRNA,resulting in cell apoptosis;2)In the KO cell line,the infection of ALV-J and overexpression of circ＿2420 do not cause the cell apoptosis.In summary,in DF1 cells,circ＿2420 induces cell apoptosis by positively regulating the expression of Bcl11b mRNA.As Bcl11b mRNA is a key pathway in circ＿2420-mediated cell apoptosis,it is believed that circ＿2420 has the potential to be further studied as a detection indicator for chicken Bcl 11b gene-mediated cell apoptosis.2.Clinical study on the correlation between circ 2420 and chicken Bcl11b expression levelsALV-J positive chickens were obtained after inoculation of SPF chicken embryos by yolk sac injection,the body weight,food and water intake were tracked and recorded;blood samples were collected at 1,7,15,and 30 days of age to monitor changes in the expression levels of ALV-J,circ＿2420,and Bcl11b mRNA in plasma samples.Samples of blood,liver,kidney,spleen,and bursa sac were collected at 30 days of age,and the organ indices were calculated.The expression levels of ALV-J,circ＿2420,and Bcl11b mRNA were measured 1,7,15,and 30 days of age in blood,as well as liver,kidney,spleen,and bursa sac at 30 days of age.Pathological sections were also prepared to analyze potential lesions in the liver,kidney,spleen,and bursa sac during early infection with ALV-J.The results showed that there were no significant differences in clinical indicators such as body weight,food and water intake between the ALV-J positive chickens and the blank control group.The results of RT-qPCR showed that the expression level of the ALV-J in the blood of the ALV-J-positive chickens gradually increased,and by 30 days of age,ALV-J can be detected in the liver,kidney,spleen,and bursa sac,and the highest viral load was found in the spleen,followed by the kidney.The expression levels of Bcl11 mRNA and circ＿2420 in the blood of ALV-J positive chickens gradually increased from 15 to 30 days of age.At 30 days of age,the expression level of Bcl11b mRNA and circ＿2420 in the liver,kidney,spleen,and bursa sac of ALV-J positive chickens were higher than those in the blank control group.The expression level of Bcl11b mRNA in the spleen was extremely significantly different from that in the blank control group,and the expression level of circ＿2420 also showed significant differences.In conclusion,the expression levels of Bcl11b mRNA and circ＿2420 in chicken blood,liver,kidney,spleen,and bursa sac are positively correlated with the proliferation levels of ALV-J in these tissues.Therefore,it is believed that the circ＿2420 upregulation can be further studied as a predictive indicator of the upregulation of Bcl 11b expression level in clinic.3.Establishment of GNP probe-mediated DFM Visual Enumeration Strategy for chicken Bcl11b-associated circ＿2420 detectionThe 60 nm GNP was chosen as the carrier for the capture probe and block DNA of circ＿2420 to construct the GNP probe;the detection background is composed of a 4x4 mm2 silicon chip（referred to as "DNA-modified chip"）modified with the circ＿2420 immobilized probe（binding probe）,subsequently,the modification of GNP probe and DNA-modified chip was verified by methods including full wavelength analysis,size and potential analysis,and microscopic characterization.A comparative study was conducted using total RNA from DF1 cell as a template to establish RT-qPCR and GNP probe Mediated Dark-Field Imaging Strategy for circ＿2420 detection,and both two methods were then applied to clinical samples.Furthermore,an artificial intelligence counting software for GNP probe under dark-field was developed using Python language to simplify the counting process,finally,the stability of the GNP probe was validated under different pH and temperature conditions.The results showed that the GNP probe had good monodispersity,and compared to GNP,the GNP probe had a red-shifted absorption peak,increased hydrated particle size,and lower electric potential.Atomic force microscopy（AFM）and Scanning electron microscope（SEM）characterization results indicated the successful chemical modification of the surface of the single-polished silicon wafer,and the binding of the DNA-modified chip and GNP probe was specific.The entire hybridization process could be completed in only 30 min at 50℃.It has been verified that the detection limit of RT-qPCR method for circ＿2420 is 6.44 copies/μL,while the detection limit of the gold nanoparticle probe dark field visualization method can reach 1.61 copies/μL.Compared to GNP,GNP probe showed higher alkali resistance and remained suitable for the quantitative detection of circ＿2420 even after being treated at temperatures ranging from-20 ℃,60℃ and 95 ℃.Compared with RT-qPCR,our method omits the steps of RNase R digestion of homologous mRNA and reverse transcription,which saves time and reduces costs.Moreover,our method is not affected by non-specific factors such as primer dimerization and operating environment.Our method has the potential to be used for rapid detection of cirRNA biomarkers in clinical settings.In conclusion,this study has provided preliminary evidence supporting the feasibility of circ＿2420 as a predictive indicator for the expression levels of the chicken Bcl11b gene,both at the cellular level and in a clinical context.Additionally,a novel method for the visualization and quantitative detection of circ＿2420 using a GNP probe-assisted dark-field technique has been established,offering a new strategy for the quantitative detection of circRNAs.