Aptasensor-encapsulating semi-permeable proteinosomes for direct target detection in non-treated biofluids

Detecting biomarkers in biofluids directly without sample treatments makes molecular diagnostics faster and more efficient. Aptasensors, the nucleic acid-based molecular biosensors, can detect a wide range of target molecules, but their susceptibility to degradation and aggregation by nucleases and charged proteins, respectively, limits their direct use in clinical samples. In this work, we demonstrate that when aptasensors are encapsulated in proteinosomes, the protein-based liposome mimics, clinically important small molecules can be sensitively and selectively detected in non-treated specimens, such as 100 % unpurified serum. As serum albumin is used to form the membrane, the nanomeshed proteinosomes become semi-permeable and antifouling, which enables exclusive admission of small molecules while blocking unwanted large proteins. Consequently, the enclosed aptasensors can maintain close-to-optimal performance for target binding, and nucleolytic degradation and electrostatic aggregation are effectively suppressed. Three different structure-switching aptamers specific for estradiol, dopamine, and cocaine, respectively, are demonstrated to fully conserve their high affinities and specificities inside the microcapsules. The shielding effect of proteinosomes is indeed exceptional; the enclosed DNA aptasensors remain completely intact over 18 h in serum and even in an extremely concentrated DNase solution (1 mg/ml, ∼300,000× the serum level). Moreover, the proteinosome-mediated compartmentalization enables independent operation of multiple aptasensors in the same mixture. Hence, simultaneous real-time sensing of two different targets is demonstrated with different operation modes, 'recording' target appearance and 'reporting' target concentration changes. This work is the first demonstration of small-molecule-specific aptasensors operating with optimal performance in serum environments and will find promising applications in molecular diagnostics.