Determining ultratrace amounts of protein biomarkers in patient samples in a straightforward and quantitative manner is extremely important for early disease diagnosis and treatment. Here, we successfully demonstrate the novel use of zinc oxide nanorods (ZnO NRs) in the ultrasensitive and quantitative detection of two acute kidney injury (AKI)-related protein biomarkers, tumor necrosis factor (TNF)-α and interleukin (IL)-8, directly from patient samples. We first validate the ZnO NRs-based IL-8 results via comparison with those obtained from using a conventional enzyme-linked immunosorbent method in samples from 38 individuals. We further assess the full detection capability of the ZnO NRs-based technique by quantifying TNF-α, whose levels in human urine are often below the detection limits of conventional methods. Using the ZnO NR platforms, we determine the TNF-α concentrations of all 46 patient samples tested, down to the fg/mL level. Subsequently, we screen for TNF-α levels in approximately 50 additional samples collected from different patient groups in order to demonstrate a potential use of the ZnO NRs-based assay in assessing cytokine levels useful for further clinical monitoring. Our research efforts demonstrate that ZnO NRs can be straightforwardly employed in the rapid, ultrasensitive, quantitative, and simultaneous detection of multiple AKI-related biomarkers directly in patient urine samples, providing an unparalleled detection capability beyond those of conventional analysis methods. Additional key advantages of the ZnO NRs-based approach include a fast detection speed, low-volume assay condition, multiplexing ability, and easy automation/integration capability to existing fluorescence instrumentation. Therefore, we anticipate that our ZnO NRs-based detection method will be highly beneficial for overcoming the frequent challenges in early biomarker development and treatment assessment, pertaining to the facile and ultrasensitive quantification of hard-to-trace biomolecules.

Crystallographic surface-resolved examination of protein-ZnO interactions can greatly enhance the fundamental understanding of protein adsorption on these technologically important solid surfaces which, in turn, will be tremendously valuable for the emerging applications of ZnO-based biomaterials and biosensors. We examine experimentally and via computer simulations the intriguing differences in the adsorption preferences and binding behavior of whole immunoglobulin G (IgG) proteins to various, low-index ZnO crystal surfaces at the individual biomolecule level. By performing direct atomic force microscopy imaging, we determine that IgG predominantly binds to the ZnO plane of (101̅0) relative to the other three low-index planes of (0001), (0001̅), and (112̅0). This phenomenon is highly unusual, particularly considering the fact that the average binding energy of amino acids (AAs) on the ZnO (0001) facet is higher than that on the (101̅0) plane. In conjunction with combined Monte Carlo-molecular dynamics simulations, we further explain the possible origins of our unusual experimental findings with critical factors such as the specific spatial locations of strongly binding AAs in the protein and their spatial distributions on the exterior surface of the protein.

The precise effect of crystallographically discriminating biomolecular adsorption on the fluorescence intensification profiles of individual zinc oxide nanorod (ZnO NR) platforms was elucidated in this study by employing peptide binding epitopes biased towards particular ZnO crystal surfaces and isolating the peptides on given crystalline facets of ZnO NRs. Subsequently, the fluorescence emission profiles of the preferentially bound peptide cases on the basal versus prismic planes of ZnO NRs were carefully evaluated both experimentally and via computer simulations. The phenomenon of fluorescence intensification on NR ends (FINE) was persistently observed on the individual ZnO NR platforms, regardless of the location of the bound peptides. In contrast to the consistent occurrence of FINE, the degree and magnitude of FINE were largely influenced by the discriminatory peptide adsorption to different ZnO NR facets. The temporal stability of the fluorescence signal was also greatly affected by the selectively located peptides on the ZnO NR crystal when spatially resolved on different NR facets. Similarities and differences in the spatial and temporal fluorescence signal of the crystalline NR facet-specific versus-nonspecific biomolecular adsorption events were then compared. To further illuminate the basis of our experimental findings, we also performed finite-difference-time-domain (FDTD) calculations and examined the different degrees of FINE by modelling the biased peptide adsorption cases. Our multifaceted efforts, providing combined insight into the spatial and temporal characteristics of the biomolecular fluorescence signal characteristically governed by the biomolecular location on the specific NR facets, will be valuable for novel applications and accurate signal interpretation of ZnO NR-based biosensors in many rapidly growing, highly miniaturized biodetection configurations.