Strain-Modulated and Nanorod-Waveguided Fluorescence in Single Zinc Oxide Nanorod-Based Immunodetection

Abstract

Mechanical strain has been shown to be a versatile and tunable means to control various properties of nanomaterials. In this work, we investigate how strain applied to individual ZnO nanorods (NRs) can affect the fluorescence signals originated from external sources of bioanalytes, which are subsequently coupled and guided onto the NRs. Specifically, we determine how factors such as the NR length and protein concentration can influence the strain-induced changes in the waveguided fluorescence intensity along the NRs. We employ a protein of tumor necrosis factor-α (TNF-α) and a fluorophore-labeled antibody in a model immunoassay reaction, after which Alexa488-TNF-α immunocomplex is formed on ZnO NRs. We elucidate the relationships between the types as well as amounts of strain on the NRs and the fluorescence intensity originated from the Alexa488-TNF-α immunocomplexes. We show that tensile (compressive) strain applied to the NR leads to an increase (decrease) in the waveguided fluorescence signals. By assessing important optical phenomena such as fluorescence intensification on nanorod ends (FINE) and degree of FINE (DoF), we confirm their linear dependence with both the types and amounts of strain. Furthermore, the strain-induced changes in both FINE and DoF are found to be independent of protein concentration. We determine that NR length plays a critical role in obtaining high strain-dependence of the measured fluorescence signals. Particularly, we ascertain that longer NRs yield larger changes in both FINE and DoF in response to the applied strain, relative to shorter ones. In addition, longer NRs permit higher linear correlation between the protein concentration and the waveguided fluorescence intensity. These outcomes provide valuable insight into exploiting strain to enhance the detection of optical signals from bioanalytes, thus enabling their quantifications even at ultra-trace levels. Coupled with the use of individual ZnO NRs demonstrated in our measurements, this work may contribute to the development of a miniaturized, highly sensitive biosensor whose signal transduction is best optimized by the application of strain.

Keywords: ZnO nanorod; biosensor; compression; fluorescence; immunodetection; protein sensor; strain; subwavelength waveguiding; tension.

Figure 1

(A) The scheme illustrates the overall fabrication process of the TNF-α sandwich immunoassay based on ZnO NRs. The NR immunoassay platform was integrated onto a PDMS elastomer for the application of uniaxial tensile (blue) and compressive (red) strain with a microvice during fluorescence measurements. (B) Representative SEM (top) and fluorescence (bottom) images of ZnO NRs are shown. On both images, the left-end, middle, and right-end positions of the NR are denoted as LE, MD, and RE, respectively. In the fluorescence image, the emission from the Alexa488-TNF-α immunocomplex is pseudo-colored green. (C) The two plots show changes in waveguided fluorescence intensity as a function of position on a ZnO NR when uniaxial strain was applied to the NR. The left plot displays a decrease in the overall fluorescence intensity with compressive (red) strain, compared to the neutral state (black). The right plot presents the case for tensile (blue) strain where an increase in the fluorescence intensity at both NR ends was observed relative to the neutral state (black).

Figure 2

Representative plots of normalized fluorescence intensity with respect to position along individual (A) ZnO NPs, (B) short ZnO NRs, and (C) long ZnO NRs under neutral (black), compressive (red), and tensile (blue) strain. (A) ZnO NPs (Group 1) show no changes in the waveguided fluorescence intensity under compressive and tensile strain compared to the strain-free case. (B) Short ZnO NRs (Group 2) present a decrease in fluorescence intensity with compressive strain, while a slight increase in fluorescence intensity is observed with tensile strain. (C) Long ZnO NRs (Group 3) display similar trends as Group 2, but they exhibit greater FINE that is clearly observed at the two NR ends compared to the shorter NRs in Group 2. The fluorescence intensity of the NRs in Group 3 decreased with compressive strain. For the case of tension, the Group 3 NRs exhibited pronounced signal increases at the two NR ends.

Figure 3

The plots display normalized fluorescence intensity with respect to TNF-α concentration on (A) NR main body and (B) NR ends from the long NRs in Group 3 (red square). The Group 3 NRs show an increasing trend in fluorescence intensity with increasing TNF-α concentration on both (A) NR main body and (B) NR ends. The red dashed lines are linear fits for the data points, while the solid black lines above and below the data points represent the 95% confidence intervals. For comparison, the NPs in Group 1 (purple circle) and short NRs in Group 2 (green triangle) were similarly evaluated. Plots showing normalized fluorescence intensity as a function of TNF-α concentration on (C) NR main body and (D) NR ends are then presented. Group 1 NPs and Group 2 NRs do not exhibit any significant correlations between the fluorescence intensity and TNF-α concentration either from (C) NR main body or (D) NR ends.

Figure 4

(A) The plots display % FINE versus % strain in NR length for TNF-α concentrations of 100, 10, 1, and 0.1 fg/mL. Squared data points in blue correspond to the NRs undergoing tension, while circled data points in red refer to the NRs undergoing compression. The solid black lines represent the linear fits for the data points, while the black dashes show the 95% confidence ellipses. Positive values on the x axis of % strain in NR length denote tension, while negative values indicate compression. (B) The plot combinedly shows regressions from % FINE versus % strain for all TNF-α concentrations in (A). The overlaid regression lines exhibit linear relationships of similar slopes between % FINE and % strain in NR length.

Figure 5

(A) The plots display % DoF versus % strain in NR length for TNF-α concentrations of 100, 10, 1, and 0.1 fg/mL. Squared data points in blue correspond to the NRs undergoing tension, while circled data points in red refer to the NRs undergoing compression. The solid black lines represent the linear fits for the data points, while the black dashes show the 95% confidence ellipses. Positive values on the x axis of % strain in NR length denote tension, while negative values indicate compression. (B) The plot combinedly shows regressions from % DoF versus % strain for all TNF-α concentrations in (A). The overlaid regression lines exhibit linear relationships with similar slopes between % DoF and % strain in NR length.

Figure 6

The effect of the NR length was correlated to the strain-induced changes in both FINE and DoF. (A,B) The plots display % FINE as a function of % strain in NR length for the short NRs in Group 2 (green triangle) and the long NRs in Group 3 (red square). (C,D) % DoF values are plotted as a function of % strain in NR length for the Group 2 NRs (green triangle) and the Group 3 NRs (red square). The red and green circles in (A,C) belong to 95% confidence ellipses of the Group 2 and Group 3 NRs, respectively. The solid lines in (B,D) are the respective linear fits through the data points.