Quantitative Assessment of Copper(II) in Wilson’s Disease Based on Photoacoustic Imaging and Ratiometric Surface-Enhanced Raman Scattering

Abstract

Cu²⁺ is closely related to the occurrence and development of Wilson's disease (WD), and quantitative detection of various copper indicators (especially liver Cu² and urinary Cu²⁺) is the key step for the early diagnosis of WD in the clinic. However, the clinic Cu²⁺ detection approach was mainly based on testing the liver tissue through combined invasive liver biopsy and the ICP-MS method, which is painful for the patient and limited in determining WD status in real-time. Herein, we rationally designed a type of Cu²⁺-activated nanoprobe based on nanogapped gold nanoparticles (AuNNP) and poly(<i>N</i>-isopropylacrylamide) (PNIPAM) to simultaneously quantify the liver Cu²⁺ content and urinary Cu²⁺ in WD by photoacoustic (PA) imaging and ratiometric surface-enhanced Raman scattering (SERS), respectively. In the nanoprobe, one Raman molecule of 2-naphthylthiol (NAT) was placed in the nanogap of AuNNP. PNIPAM and the other Raman molecule mercaptobenzonitrile (MBN) were coated on the AuNNP surface, named AuNNP-NAT@MBN/PNIPAM. Cu²⁺ can efficiently coordinate with the chelator PNIPAM and lead to aggregation of the nanoprobe, resulting in the absorption red-shift and increased PA performance of the nanoprobe in the NIR-II window. Meanwhile, the SERS signal at 2223 cm^-1 of MBN is amplified, while the SERS signal at 1378 cm^-1 of NAT remains stable, generating a ratiometric SERS <i>I</i>₂₂₂₃/<i>I</i>₁₃₇₈ signal. Both NIR-II PA_{1250 nm} and SERS <i>I</i>₂₂₂₃/<i>I</i>₁₃₇₈ signals of the nanoprobe show a linear relationship with the concentration of Cu²⁺. The nanoprobe was successfully applied for <i>in vivo</i> quantitative detection of liver Cu²⁺ of WD mice through NIR-II PA imaging and accurate quantification of urinary Cu²⁺ of WD patients by ratiometric SERS. We anticipate that the activatable nanoprobe might be applied for assisting an early, precise diagnosis of WD in the clinic in the future.