Digital colloid-enhanced Raman spectroscopy for the pharmacokinetic detection of bioorthogonal drugs

Bioorthogonal drug molecules are currently gaining prominence for their excellent efficacy, safety and metabolic stability. Pharmacokinetic study is critical for understanding their mechanisms and guiding pharmacotherapy, which is primarily performed with liquid chromatography-mass spectrometry as the gold standard. For broader and more efficient applications in clinics and fundamental researches, further advancements are especially desired in cheap and portable instrumentations as well as rapid and tractable pretreatment procedures. Surface-enhanced Raman spectroscopy (SERS) is capable of label-free detection of various molecules based on the spectral signatures with high sensitivity even down to a single-molecule level. But limited by irreproducibility at low concentrations and spectral interference in complex biofluids, SERS hasn’t been widely applied for pharmacokinetics, especially in live animals. In this work, we proposed a new method to quantify the bioorthogonal drug molecules with the signatures at spectral silent region (SR) by digital colloid-enhanced Raman spectroscopy (dCERS) technique. This method was first validated by 4-mercaptobenzonitrile in the mixture of analogue molecules, exhibiting reliable and specific identification capability based on the unique SR signature and Poisson-determined quantification accuracy. We further developed a single-step serum pretreatment method and successfully profiled the pharmacokinetic behavior of an anticancer drug, erlotinib, from animal studies. In a word, this method, superior in sensitivity, controllable accuracy, minimal background interference and facile pretreatment and measurement, promises diverse applications in fundamental studies and clinical tests of bioorthogonal drug molecules.