In this study, we apply catalytic hairpin assembly (CHA) as the signal amplification strategy for the quantification of carcinoembryonic antigen (CEA) and cytokeratin fragment antigen 21-1 (CYFRA21-1) with a surface-enhanced Raman scattering (SERS) microfluidic chip (LoC-SERS) as the carrier. Herein, antibody–DNA conjugates are designed to assist the application of CHA amplification in protein detection. In the presence of protein biomarkers, antibody–DNA conjugates can specifically bind to the target proteins, forming the antigen@antibody–DNA conjugates. The terminal free part of the DNA on the conjugates can trigger the CHA events to connect SERS nanotags to capture nanoprobes. Then, micro-magnet can gather the CHA products in a rectangular chamber, resulting in the aggregation of SERS nanotags, which can ultimately generate abundant “hot spots” for SERS signal enhancement. Using this strategy, CEA and CYFRA21-1 can be successfully determined with a limit of detection (LOD) as low as pg mL−1, much lower than recently reported methods. Meanwhile, a non-small cell lung cancer (NSCLC)-xenografted mouse model was established, and SERS was applied to analyze the expression level of CEA and CYFRA21-1 in tumorigenesis and development. The comparison between SERS results and those of the ELISA method demonstrated a high degree of consistency, suggesting that the proposed CHA-assisted LoC-SERS device has satisfying accuracy. Thus, introducing the CHA strategy via the design of antibody–DNA conjugates opens new gates to ultra-sensitive and specific SERS detection of protein biomarkers.
Yupin Cao,Jinsu Feng,Lifu Tang,Guichun Mo,Weiming Mo,Biyang Deng
A new method for simultaneously determining cytokeratin fragment antigen 21–1 (CYFRA21-1), carcinoembryonic antigen (CEA), and carbohydrate antigen (CA15–3) was developed using single-particle, ICP-MS-based, magnetic immunoassay . Gold nanoparticles (AuNPs), ZnSe quantum dots (QDs), and Silver nanoparticles (AgNPs) were employed to tag antibodies. A highly efficient capture of CYFRA21-1, CEA, and CA15–3 was achieved using biotinylated, antibody-coated magnetic beads. AuNPs/ZnSe QDs/AgNPs-tagged second antibodies helped realize sensitive quantification of the target antigen using single particle ICP-MS. Under optimum conditions, CYFRA21-1, CEA, and CA15–3 linear concentration ranges were 0.05–50 ng mL −1 , 0.02–100 ng mL −1 , and 0.6–250 mU mL −1 . The correlation coefficients were 0.9999, 0.9987, and 0.9970, respectively. CYFRA21-1, CEA, and CA15–3 detection limits were 0.02 ng mL −1 , 0.006 ng mL −1 , and 0.25 mU mL −1 , respectively. Relative standard deviations (RSD) for CYFRA21-1, CEA, and CA15–3 were 3.2%, 4.4%, and 4.2%, respectively. The method simultaneously detected CYFRA21-1, CEA, and CA15–3 in human lung cancer serum samples with recoveries of between 89.8% and 101%.
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