Large-scale, unbiased proteomics studies are constrained by the complexity of the plasma proteome. Here we report a highly parallel protein quantitation platform integrating nanoparticle (NP) protein coronas with liquid chromatography-mass spectrometry for efficient proteomic profiling. A protein corona is a protein layer adsorbed onto NPs upon contact with biofluids. Varying the physicochemical properties of engineered NPs translates to distinct protein corona patterns enabling differential and reproducible interrogation of biological samples, including deep sampling of the plasma proteome. Spike experiments confirm a linear signal response. The median coefficient of variation was 22%. We screened 43 NPs and selected a panel of 5, which detect more than 2,000 proteins from 141 plasma samples using a 96-well automated workflow in a pilot non-small cell lung cancer classification study. Our streamlined workflow combines depth of coverage and throughput with precise quantification based on unique interactions between proteins and NPs engineered for deep and scalable quantitative proteomic studies.

Direct, multiphoton photolysis of aqueous metal complexes is found to play an important role in the formation of nanoparticles in solution by ultrafast laser irradiation. In situ absorption spectroscopy of aqueous [AuCl4](-) reveals two mechanisms of Au(0) nucleation: (1) direct multiphoton photolysis of [AuCl4](-) and (2) radical-mediated reduction of [AuCl4](-) upon multiphoton photolysis of water. Measurement of the reaction kinetics as a function of solution pH reveals zeroth-, first-, and second-order components. The radical-mediated process is found to be zeroth-order in [AuCl4](-) under acidic conditions, where the reaction rate is limited by the production of reactive radical species from water during each laser shot. Multiphoton photolysis is found to be first order in [AuCl4](-) at all pHs, whereas the autocatalytic reaction with H2O2, the photolytic reaction product of water, is second order.

Simultaneous spatiotemporal focusing (SSTF) of femtosecond laser radiation is used to produce gold nanoparticles from aqueous [AuCl 4 ] − solutions. Multiphoton ionization and dissociation of water produces electrons and hydrogen atoms for the reduction of [AuCl 4 ] − to Au(0) during irradiation with the temporally chirped (36 ps) pulse and produces hydrogen peroxide (H 2 O 2 ) as a long-lived reducing agent which persists after irradiation is terminated. Aqueous H 2 O 2 is found to reduce [AuCl 4 ] − , remaining in solution after the laser irradiation is terminated, leading to growth and transformation of the existing Au(0) species. The highly efficient postirradiation reduction of [AuCl 4 ] − to Au(0) by H 2 O 2 is ascribed to reactions occurring on gold nanoparticle surfaces. In the absence of added surfactant, the negatively charged gold particles formed during irradiation are a complex mixture of irregularly shaped and spherical morphologies that are only metastable as aqueous dispersions. These particles become transformed into more perfectly shaped gold crystals, as the remaining [AuCl 4 ] − is reduced in the postirradiation period. The addition of polyethylene glycol (PEG 45 ) accelerates the rate of the [AuCl 4 ] − reduction during laser irradiation and directs the exclusive formation of spherical nanoparticles. Varying the concentration of PEG 45 tunes the diameter and size distribution of the Au nanoparticles formed by laser processing from 3.9 ± 0.7 to 11 ± 2.4 nm.