Microchimica Acta

, 185:293 | Cite as

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

  • Wen Yang
  • Tengfei Li
  • Chang Shu
  • Shunli Ji
  • Lei Wang
  • Yan Wang
  • Duo Li
  • Michael Mtalimanja
  • Luning Sun
  • Li Ding
Original Paper


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%.

Graphical abstract

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.


Signal transduction Nanocomposites Mass barcodes Aptamer Proteins 



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).

Compliance with ethical standards

The author(s) declare that they have no competing interests.

Ethical approval

Institutional Review Board approval was not required because the study is not on animals.

Informed consent

Written informed consent was not required for this study because the study is not on human subjects.

Supplementary material

604_2018_2832_MOESM1_ESM.doc (400 kb)
ESM 1 (DOC 399 kb)


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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Pharmaceutical AnalysisChina Pharmaceutical UniversityNanjingChina
  2. 2.Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of EducationChina Pharmaceutical UniversityNanjingChina
  3. 3.College of PharmacyAnhui University of Chinese MedicineHefeiChina
  4. 4.College of Pharmacy and ChemistryDali UniversityDaliChina
  5. 5.Research Division of Clinical PharmacologyFirst Affiliated Hospital with Nanjing Medical UniversityNanjingChina

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