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A novel real sample assisted SELEX strategy was proposed to rationally select SAR aptamer. Based on this real SELEX strategy, we obtained an aptamer Apt-F2 with higher specificity and SAR binding affinity than Apt-B4 selected by the standard SELEX strategy. Moreover, Apt-F2-based aptasensor showed higher detection sensitivity and slightly better recovery rate of SAR from spiked milk samples than Apt-B4-based aptasensor. All of these results indicated the advantage of the real SELEX strategy superior to traditional SELEX strategy. [Display omitted] • Real sample assisted SELEX strategy can rationally select more fitted aptamer. • Real SELEX strategy can make aptamer pre-adapt to real sample. • Apt-F2 with higher specificity and SAR binding affinity was selected by this strategy. • An ease operated fluorescence aptasensor was fabricated based on Apt-F2. • The aptasensor had high sensitivity, specificity and low cost for detection of SAR. The aptamers screened by the classic SELEX strategy or other approaches do not undergo the real sample analysis during the selection process, and the environmental inconsistency between the selection and final detection systems might influence the aptamer characteristics and detection performance. Herein, a novel real sample assisted SELEX selection strategy has been proposed to rationally select the suitable aptamers with high detection performance, using the selection of a sarafloxacin (SAR) specific aptamer as an example. Based on the developed strategy, the aptamer Apt-F2 with higher specificity and SAR binding affinity was obtained as compared to the aptamer Apt-B4 selected by the standard SELEX strategy. Apt-F2 also exhibited a stronger conformational change after binding as compared to Apt-B4. Apt-B4 and Apt-F2 were used as a platform for developing the fluorescent aptasensor. Under optimized conditions, the limit of detection of Apt-B4- and Apt-F2-based aptasensors for SAR was 0.19 and 0.12 ng/mL respectively. Moreover, Apt-F2-based aptasensor also revealed a slightly superior SAR recovery rate from the spiked milk samples. The findings obtained in the study confirmed the advantage of the real SELEX strategy as compared to the traditional version. Further, the characteristics of the aptasensor proposed here also revealed its application potential for detecting SAR in the real milk or other animal-sourced food samples. [ABSTRACT FROM AUTHOR]

[Display omitted] • Study on the mechanism of aptamer-based fluorescent DNA biosensors. • Antibiotic detection is facilitated by the construction of aptamer-based fluorescent DNA biosensors using both label-free and labeled methods. • Signal amplification are harnessed to pursuit the heightened sensitivity of the antibiotic detection. The inappropriate employment of antibiotics across diverse industries has engendered profound apprehensions concerning their cumulative presence within human bodies and food commodities. Consequently, many nations have instituted stringent measures limiting the admissible quantities of antibiotics in food items. Nonetheless, conventional techniques employed for antibiotic detection prove protracted and laborious, prompting a dire necessity for facile, expeditious, and uncomplicated detection methodologies. In this regard, aptamer-based fluorescent DNA biosensors (AFBs) have emerged as a sanguine panacea to surmount the limitations of traditional detection modalities. These ingenious biosensors harness the binding prowess of aptamers, singular strands of DNA/RNA, to selectively adhere to specific target antibiotics. Notably, the AFBs demonstrate unparalleled selectivity, affinity, and sensitivity in detecting antibiotics. This comprehensive review meticulously expounds upon the strides achieved in AFBs for antibiotic detection, particularly emphasizing the labeling modality and the innovative free-label approach. It also elucidates the design principles behind a diverse array of AFBs. Additionally, a succinct survey of signal amplification strategies deployed within these biosensors is provided. The central objective of this review is to apprise researchers from diverse disciplines of the contemporary trends in AFBs for antibiotic detection. By doing so, it aspires to instigate a concerted endeavor toward the development of heightened sensitivity and pioneering AFBs, thereby contributing to the perpetual advancement of antibiotic detection methodologies. [ABSTRACT FROM AUTHOR]

A simply novel S1 nuclease digestion-based strategy was proposed to rationally truncate the PD-L1 aptamer Apt80. Based on this novel strategy, Apt38 with lower K d value and higher PD-L1 binding-associated conformational change superior to Apt80 was successfully truncation optimized. Moreover, the fabricated signal dual amplification aptasensor based on the truncated Apt38 has slighted higher detection sensitivity, wider linear range, lower detection cost and less time for detection of PD-L1 in human sample than ELISA assay. • S1 nuclease digestion-based strategy can rationally truncate PD-L1 aptamer Apt80. • S1 nuclease digestion-based strategy can directly identify the core aptamer sequence. • Truncated Apt38 had higher PD-L1 binding affinity and conformational change. • A signal dual amplification fluorescence aptasensor was fabricated based on Apt38. • The aptasensor had high sensitivity, specificity and low cost for detection of PD-L1. Truncation optimization of aptamers can improve both specificity and robustness. At present, aptamer truncation is mainly performed based on predictions from molecular docking simulations, but this usually requires tedious trial-and-error and may result in false predictions. Here, we propose a strategy based on digestion by S1 nuclease to rationally truncate the aptamer, using the PD-L1-binding aptamer Apt80 as an example. Due to steric hindrance, recognition and binding regions between Apt80 and PD-L1 are not digested by the enzyme. The truncated form, Apt38, shows higher affinity and larger conformational change after binding, when compared to Apt80. The truncated Apt38 was used as a platform for developing a signal dual amplification fluorescence aptasensor using targeted recycling assisted by exonuclease I and qRT-PCR analysis. This aptasensor exhibited a high sensitivity toward PD-L1 with a limit of detection of 0.076 ng/mL in buffer system and 0.3625 ng/mL in human serum. Owing to its high sensitivity, specificity, ease operation and low cost for detection of PD-L1, this aptasensor should be useful in assisting clinicians to evaluate the status of cancer patient and to decide whether inhibitor drugs are needed. [ABSTRACT FROM AUTHOR]