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Non-enzymolytic adenosine barcode-mediated dual signal amplification strategy for ultrasensitive protein detection using LC-MS/MS

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Abstract

A method is described for the determination of proteins with LC-MS/MS enabled by a small molecule (adenosine) barcode and based on a double-recognition sandwich structure. The coagulation protein thrombin was chosen as the model analyte. Magnetic nanoparticles were functionalized with aptamer29 (MNP/apt29) and used to capture thrombin from the samples. MNP/apt29 forms a sandwich with functionalized gold nanoparticles modified with (a) aptamer15 acting as thrombin-recognizing element and (b) a large number of adenosine as mass barcodes. The sandwich formed (MNP/apt29-thrombin-apt15/AuNP/adenosine) can ben magnetically separated from the sample. Mass barcodes are subsequently released from the sandwiched structure for further analysis by adding 11-mercaptoundecanoic acid. Adenosine is then detected by LC-MS/MS as it reflects the level of thrombin with impressively amplified signal. Numerous adenosines introduced into the sandwich proportional to the target concentration further amplify the signal. Under optimized conditions, the response is linearly proportional to the thrombin concentration in the range of 0.02 nM to 10 nM, with a detection limit of 9 fM. The application of this method to the determination of thrombin in spiked plasma samples gave recoveries that ranged from 92.3% to 104.7%.

Schematic representation of a method for the determination of thrombin with LC-MS/MS. The method is based on a double-recognition sandwiched structure. With LC-MS/MS, mass barcodes (adenosine) are detected to quantify thrombin, which amplifies the detection signal impressively.

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Acknowledgments

We are grateful for the financial support of the National Natural Science Foundation of China (No.81573387, No.81703472, No.81603072 and No.81560695). Also thanks for the support from the Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. KYCX17_0683).

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Authors and Affiliations

  1. Department of Pharmaceutical Analysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, China

    Wen Yang, Tengfei Li, Chang Shu, Shunli Ji, Lei Wang, Yan Wang, Duo Li, Michael Mtalimanja & Li Ding

  2. Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, China

    Wen Yang, Tengfei Li, Chang Shu, Shunli Ji, Lei Wang, Yan Wang, Duo Li, Michael Mtalimanja & Li Ding

  3. College of Pharmacy, Anhui University of Chinese Medicine, Hefei, 230012, China

    Lei Wang

  4. College of Pharmacy and Chemistry, Dali University, Wanhua Road, Dali, 671000, China

    Yan Wang

  5. Research Division of Clinical Pharmacology, First Affiliated Hospital with Nanjing Medical University, 300 Guangzhou Road, Nanjing, 210009, China

    Luning Sun

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  1. Wen Yang
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  10. Li Ding
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Correspondence to Li Ding.

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Yang, W., Li, T., Shu, C. et al. Non-enzymolytic adenosine barcode-mediated dual signal amplification strategy for ultrasensitive protein detection using LC-MS/MS. Microchim Acta 185, 293 (2018). https://doi.org/10.1007/s00604-018-2832-4

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