2, Houston Methodist Research Institute, Houston, Texas, United States
Early detection of melanoma is important to improve survival, however, the technology for accurate early diagnosis is still challenging. C-reactive protein (CRP) is an acute phase reactanthas produced by hepatocytes when inflammation occurs in body, and it was found to be associated with melanoma and could serve as an independent prognostic biomarker in melanoma patients. Particularly, increased levels of CRP in plasma were correlated with disease stage in patients with melanoma. Here we report a novel polymeric biosensor that could provide superior sensitivity in the detection of potential early biomarkers (e.g. CRP) of melanoma. The biosensor is composed of a highly specific molecular recognition core and a highly responsive transducer made of conductive polypyrrole (PPy) nanofibers.
We prepared a polymeric matrix by polymerization of acrylamide (AM), methylenebisacrylamide (MBAA), N-Isopropylacrylamide (NIPAAm) and CRP/CRP-aptamer complex first. Next, taking advantage of the porous structure of this NIPAAm-AM-CRP-aptamer/CRP polymeric matrix, we synthesized a polypyrrole-based conductive nanofiber structure using copper phthalocyanine-3,4',4",4""-tetrasulfonic acid tetrasodium salt (CuPT) as a dopant counterion in situ to achieve uniformly dispersed nanofibers within the polymeric network of NIPAAm-AM-CRP-aptamer/CRP. After removing CRP recombinant protein, we can obtain a robust CuPT-PPy/NIPAAm-AM polymeric sensor for detection of CRP with high sensitivity and selectivity. A two-step signal amplification cascades are involved in this CRP-specific polymeric sensor: 1) CRP binding-induced polymeric network shrinkage; 2) shrinkage-triggered conductance change of the polymeric network. Therefore, serum CRP levels could be quantitatively analyzed through monitoring the conductance change caused by polymeric network shrinkage upon aptamer-CRP binding. The limit of detection (LOD) of the polymeric sensor for detection of human recombinant CRP reached 10−19 M. The Fourier transform infrared (FT-IR) spectra confirmed the chemical structure of the polymeric network, and the morphology was observed using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The results of FT-IR reveal that CuPT-PPy nanofiber was successfully synthesized in situ. The nanostructure of the nanofibers was clearly observed using SEM and AFM, indicating that the diameter of nanofiber is about 20 nm. This biosensor and a commercial CRP ELISA kit were used to perform side-by-side measurement of serum CRP in melanoma patients.
Our results indicated that this CRP-specific conductive polymeric senor is highly sensitive and selective in accurately discriminating melanoma patients from healthy controls. The serum CRP levels detected using sensor are correlated well with that obtained using ELISA. Collectively, such a flexible conductive polymeric biosensor may hold great promise as a point-of-care device in the diagnostics of melanoma and other cancers.