Non-immobilized GO-SELEX of aptamers for label-free detection of thiamethoxam in vegetables

Kong, Qianqian; Yue, Fengling; Liu, Mengyue; Huang, Jingcheng; Yang, Fengzhen; Jun-jie, Liu; Li, Jiansen; Li, Falan; Sun, Xia; Zhu, Yelong

doi:10.1016/j.aca.2022.339677

Cited by 48 publications

(13 citation statements)

References 45 publications

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“…Further, the dissociation constant (K d ) of each of the aptamers was calculated by fitting the IsdA binding data using the built-in Hill-1 function in OriginLab (Figure S2). 55,56 The determined K d values are 4 ± 2, 2.2 ± 0.5, 1.0 ± 0.3, and 0.7 ± 0.4 nM for A14, T1-280, T2-139040, and T3-117558 aptamers, respectively. However, because a short portion of the aptamers (9 nts at the 5′ end) is blocked in our design (as depicted in Figure 1), the determined K d values can be an overestimation of true K d .…”

Section: ■ Results and Discussionmentioning

confidence: 95%

“…The dissociation constants ( K d ) for all four aptamers were determined using the binding curves plotted as % high FRET versus IsdA concentration. OriginLab software was used to determine K d by fitting the binding data with the built-in Hill-1 equation shown below (assuming 1:1 binding). , where HF is % high FRET, HF max is the maximum % high FRET, HF 0 is the % high FRET in the absence of IsdA, and K d is the dissociation constant.…”

Section: Methodsmentioning

confidence: 99%

“…OriginLab software was used to determine K d by fitting the binding data with the built-in Hill-1 equation shown below (assuming 1:1 binding). 55,56 where HF is % high FRET, HF max is the maximum % high FRET, HF 0 is the % high FRET in the absence of IsdA, and K d is the dissociation constant.…”

Section: ■ Introductionmentioning

confidence: 99%

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FRET-Based Single-Molecule Detection of Pathogen Protein IsdA Using Computationally Selected Aptamers

Wijesinghe,

Sabbih,

Algama

et al. 2023

Anal. Chem.

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Iron-regulated surface determinant protein A (IsdA) is a key surface protein found in the foodborne bacteriaStaphylococcus aureus (S. aureus)which is known to be critical for bacterial survival and colonization. S. aureus is pathogenic and has been linked to foodborne diseases; thus, early detection is critical to prevent diseases caused by this bacterium. Despite IsdA being a specific marker for S. aureus and several detection methods have been developed for sensitive detection of this bacteria such as cell culture, nucleic acids amplification, and other colorimetric and electrochemical methods, the detection of S. aureus through IsdA is underdeveloped. Here, by combining computational generation of target-guided aptamers and fluorescence resonance energy transfer (FRET)-based single-molecule analysis, we presented a widely applicable and robust detection method for IsdA. Three different RNA aptamers specific to the IsdA protein were identified and their ability to switch a FRET construct to a high-FRET state in the presence of protein was verified. The presented approach demonstrated the detection of IsdA down to picomolar levels (×10–12 M, equivalent to ∼1.1 femtomoles IsdA) with a dynamic range extending to ∼40 nM. The FRET-based single-molecule technique that we reported here is capable of detecting the foodborne pathogen protein IsdA with high sensitivity and specificity and has a broader application in the food industry and aptamer-based sensing field by enabling quantitative detection of a wide range of pathogen proteins.

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Section: ■ Results and Discussionmentioning

confidence: 95%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

FRET-Based Single-Molecule Detection of Pathogen Protein IsdA Using Computationally Selected Aptamers

Wijesinghe,

Sabbih,

Algama

et al. 2023

Anal. Chem.

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“…The potential molecular interaction mechanism could be clarified by analyzing the ITC data. According to the ITC results (Figure 5), the Kd value was 4.935 × 10 −5 M for Thi-5R-18 combined with the target to release heat, and the Kd values of two published thiamethoxam aptamers FAM-Thi13 [27] and seq.20 [28] were 3.661 × 10 −4 M and 2.115 × 10 −4 M, respectively, indicating that the aptamer Thi-5R-18 in this paper had the lowest Kd value and the highest affinity among the three aptamers. Therefore, Thi-5R-18 was used as the aptamer for thiamethoxam detection in the subsequent sensor performance test.…”

Section: Measurement Of Sequence Affinity By Itcmentioning

confidence: 99%

Screening and Evaluation of Thiamethoxam Aptamer Based on Pressurized GO-SELEX and Its Sensor Application

Yue

Tian

et al. 2023

Biosensors

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Thiamethoxam, a nicotinic pesticide used worldwide, can cause great harm to the environment and even to human health, and aptamers, known as chemical antibodies, have high affinity and specificity for the target, as well as great potential in detecting small molecules such as pesticides. In this paper, we report a highly sensitive biosensor system for thiamethoxam residue detection based on aptamer technology. After 15 rounds of screening with the pressurized GO-SELEX technology, we found that the aptamer libraries of the 5th and 9th rounds showed high affinity by the capture method. Four candidate aptamers were obtained by high-throughput sequencing and secondary structure prediction. Among them, the aptamer named Thi-5R-18 from the 5th round was demonstrated to possess the highest affinity by isothermal titration calorimetry, with a dissociation constant (Kd) of 4.935 × 10−5 M. The results of molecular docking showed that thiamethoxam and Thi-5R-18 were combined with bases G-15, A-19, and T-71 through hydrogen bonding and π–π interaction.Thi-5R-18 was used as a recognition element to construct a AuNPs colorimetric aptasensor, achieving an ultralow detection limit of 0.37 nM. More importantly, this colorimetric aptasensor can be used for quantitative detection of thiamethoxam on tea leaves, with a recovery of 96.94%~105.86%. This study provides a highly sensitive biosensor for detection of thiamethoxam residue.

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“…From that point of view, the GO-SELEX can be an appropriate approach for quality control during the SELEX [ 10 , 11 ]. However, the GO has a wide 2D plane with high adsorption properties that can react directly to the DNA and RNA without chemical linkers [ 12 , 13 ]. However, the qualification methods for that process are still challenging, even if the GO-SELEX is a great approach, because there is a potential to absorb even a well-fabricated aptamer.…”

Section: Introductionmentioning

confidence: 99%

Comparison of Optical and Electrical Sensor Characteristics for Efficient Analysis of Attachment and Detachment of Aptamer

Park

Dang

Jeong³

et al. 2022

Biosensors

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Nucleic acid aptamer-based research has focused on achieving the highest performance for bioassays. However, there are limitations in evaluating the affinity for the target analytes in these nucleic acid aptamer-based bioassays. In this study, we mainly propose graphene oxide (GO)-based electrical and optical analyses to efficiently evaluate the affinity between an aptamer and its target. We found that an aptamer-coupled GO-based chip with an electrical resistance induced by a field-effect transistor, with aptamers as low as 100 pM, can detect the target, thrombin, at yields as low as 250 pM within five minutes. In the optical approach, the fluorescent dye-linked aptamer, as low as 100 nM, was efficiently used with GO, enabling the sensitive detection of thrombin at yields as low as 5 nM. The cantilever type of mechanical analysis also demonstrated the intuitive aptamer–thrombin reaction in the signal using dBm units. Finally, a comparison of electrical and optical sensors’ characteristics was introduced in the attachment and detachment of aptamer to propose an efficient analysis that can be utilized for various aptamer-based research fields.

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Non-immobilized GO-SELEX of aptamers for label-free detection of thiamethoxam in vegetables

Cited by 48 publications

References 45 publications

FRET-Based Single-Molecule Detection of Pathogen Protein IsdA Using Computationally Selected Aptamers

FRET-Based Single-Molecule Detection of Pathogen Protein IsdA Using Computationally Selected Aptamers

Screening and Evaluation of Thiamethoxam Aptamer Based on Pressurized GO-SELEX and Its Sensor Application

Comparison of Optical and Electrical Sensor Characteristics for Efficient Analysis of Attachment and Detachment of Aptamer

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