A signal-enhanced and sensitive lateral flow aptasensor for the rapid detection of PDGF-BB

Abstract

Platelet-derived growth factor BB (PDGF-BB) is a potential biomarker of tumor angiogenesis. For the first time, we developed a highly sensitive aptasensor for PDGF-BB with an enhanced test line signal by using two different gold nanoparticles (AuNPs). Herein, we describe a highly sensitive biosensor for PDGF-BB detection that combines biotinylated aptamer on a sample pad and poly thymine-Cy3-AuNP-monoclonal antibody complexes against PDGF-BB immobilized on conjugate pad A. Streptavidin (SA) and rabbit anti-mouse polyclonal antibody were also immobilized in the nitrocellulose membrane at the test and control zones, respectively. When the target PDGF-BB protein was added, it first bound the aptamer, and later the monoclonal antibody to form a biotinylated complex that was captured by SA, resulting in a visual red line on the test zone. In addition, to enhance the sensitivity, another monoclonal antibody against Cy3 was conjugated on AuNP B and immobilized on conjugate pad B to form a AuNPs (A&B)-antibody-(PDGF-BB-Cy3)-aptamer-biotin-SA complex on the test line when a loading buffer was subsequently added. This approach showed a linear response to PDGF-BB from 3 ng mL^-1 to 300 ng mL^-1 with a limit of detection as low as 1 ng mL^-1 obtained in 10 minutes. Our biosensor displayed results through red lines readable by the naked eye. Interestingly, our approach has been successfully applied for real sample verification, proving its applicability for cancer monitoring and diagnosis.

Fig. 1. Schematic illustration of the highly sensitive lateral flow biosensor (LFA). (A) The biotinylated aptamer was immobilized on the sample pad, while COOH-poly Ts-Cy3-AuNP A anti-PDGF BB complex and AuNP B-anti-Cy3 complex were immobilized to both conjugate pads (CPA/CPB), respectively. (B) The complexes were assembled to the biosensor's nitrocellulose membrane (NM) containing streptavidin on the test line (TL) and rabbit anti-mouse on the control line (CL). (C) Upon PDGF BB recognition by the aptamer, the target-aptamer complex flow and react with CPA's Mouse anti-PDGF BB, which is subsequently captured by mouse anti-Cy3. The AuNPs' complex is captured on the TL zone by the immobilized streptavidin, and excessive complexes are captured on the CL by the rabbit anti-mouse. Other parts of the proposed biosensor: waterproof membrane (WPM), chasing buffer well (CBW), absorbent pad (AP), backing pad (BP).

Fig. 2. (A) Biosensor loaded with the corresponding intensities in the presence of different concentrations of NaCl: 100 mM, 150 mM, 200 mM in loading buffer. (B) Comparison of test line peak areas responding to different concentrations of NaCl in loading buffer (*p < 0.05, n = 3).

Fig. 3. (A) Illustration of the LFA detection response (left) and the corresponding semi-quantitation intensities (right) loaded with 0, 1, 3, 30, 100 and 300 ng mL⁻¹ of purified PDGF-BB protein. (B) Comparison of peak areas of the test lines corresponding to different concentrations of PDGF-BB (*p < 0.05, n = 3).

Fig. 4. (A) LFA (left) and its corresponding semi-quantitation (right) loaded concentrations with PDGF-BB protein, PDGF-AA protein, PDGF-AB protein, EGF protein, a nonspecific oligonucleotide, and BSA protein. (B) Comparison of test line peak areas corresponding to PDGF-BB protein, PDGF-AA protein, PDGF-AB protein, EGF protein, a nonspecific oligonucleotide, and BSA protein (*p < 0.05, n = 3).

Fig. 5. (A) Representative LFA picture (left) and corresponding semi-quantitation (right) of the assay loaded with clinical sera and negative controls. (B) Comparison of test line peak areas corresponding to clinical serum and negative control (*p < 0.05, n = 3).

Fig. 6. The traditional antibody double sandwich gold nanoparticles detection method for PDGF-BB protein. (A) The monoclonal antibody against PDGF-BB binds to the gold nanoparticles. (B) Schematic of the biosensor. (C) PDGF-BB protein and excessive sample pad complexes are respectively captured on the test and control lines. (D) Results with the traditional antibody double sandwich gold nanoparticles detection method. Sample pad (SP), conjugate pad (CP), nitrocellulose membrane (NM), test line (TL), control line (CL), absorbent pad (AP).

Fig. 7. ELISA sensitivity for purified PDGF-BB protein (*p < 0.05, n = 3).