Plasmonic Sensing Assay for Long-Term Monitoring (PSALM) of Neurotransmitters in Urine

Abstract

A liquid-based surface-enhanced Raman spectroscopy assay termed PSALM is developed for the selective sensing of neurotransmitters (NTs) with a limit of detection below the physiological range of NT concentrations in urine. This assay is formed by quick and simple nanoparticle (NP) "mix-and-measure" protocols, in which Fe^III bridges NTs and gold NPs inside the sensing hotspots. Detection limits of NTs from PreNP PSALM are significantly lower than those of PostNP PSALM, when urine is pretreated by affinity separation. Optimized PSALM enables the long-term monitoring of NT variation in urine in conventional settings for the first time, allowing the development of NTs as predictive or correlative biomarkers for clinical diagnosis.

Figure 1

(a) Two PSALM protocols used for sensing NTs: PreNP: (1) AuNPs preincubated with Fe^III, (2) then aggregated using NaCl, (3) NT samples added and mixed thoroughly. PostNP: (1) NTs preincubated with Fe^III, creating Fe^IIINT, Fe^IIINT₂, and Fe^IIINT₃ complexes, (2) AuNPs separately aggregated using NaCl, and then (3) Fe^IIINT_n complexes added to the aggregate solution and mixed thoroughly. In both cases, the final step (4) focuses a 785 nm laser into the solution to obtain SERS. (b–d) Collected baseline-corrected SERS signals vs spiked DA concentration (color scale) (b) with Fe^III omitted, (c) PostNP PSALM assay, and (d) PreNP PSALM assay. Gray lines show negative control spectra using water as the analyte. (e) Scores of first-principal component from three repeats of spectra in panels (b)–(d) vs spiked DA concentrations. (f) Log–log plot of panel (e), with noise levels indicated as 2σ.

Figure 2

Evolution of the absorption spectra of the complexes of DA and Fe^III in (a) acidic to basic (A to B, red to blue) and (b) basic to acidic (B to A, blue to red) pH titration. (c) Extinction spectra peak wavelengths from panels (a, b) vs pH. Regions where Fe^IIIDA, Fe^IIIDA₂, and Fe^IIIDA₃ dominate are shaded red, green, and blue.

Figure 3

(a) Evolution of Raman signals from Fe^III–DA complexes vs pH at 50 mM DA with 50 mM Fe^III. (b) Raman intensity of 530, 591, 641, 1270, and 1489 cm^–1 lines (arrows in panel a) and the photoluminescence background (measured at 2000 cm^–1), as solution pH changed from acid to base. (c) Shift of Raman peaks initially at 1270, 1321, and 1489 cm^–1vs pH. Red, green, and blue regions indicate Fe^IIIDA, Fe^IIIDA₂, and Fe^IIIDA₃ dominating species. Lines are guides to the eye. (d) SERS of DA obtained from PreNP PSALM at pH 3.1 and 6.7. (e) SERS intensity of DA at 632 cm^–1 as pH changed from neutral (N) to base (red squares, then to acid, dashed) and from N to acid (black circles, then to base, dashed).

Figure 4

Proposed sequestration of NTs in optical hotspots between aggregated AuNP comparing PreNP and PostNP PSALM. In PreNP: (A) Fe^III chelates surfactant citrate on AuNPs, (B) NTs diffuse to these sites and chelate to Fe^III, (C) Fe^IIINT complex migrates to hotspots. In PostNP: Fe^IIIDA, Fe^IIIDA₂, and Fe^IIIDA₃ formed in solution diffuse to citrates and migrate to the hotspots. (D) Single NT can potentially chelate with two Fe^III only in the gap regions.

Figure 5

(a) Calibrated SERS intensity (cts/mW/s) of NTs, where NEPI, EPI, DOPA, and SERO are norepinephrine, epinephrine, l-DOPA, and serotonin, respectively (offset for better visualization). (b) Scores of first-principal components for NTs in PSALM using PreNP on linear and log plots. Lines are fits to the Hill–Langmuir equation. The shaded red region gives physiological concentrations of NTs in urine. (c) Measured limits of detection (LODs) of NTs using PreNP.

Figure 6

(a) Affinity separation procedure for extracting NTs via their retention on boric acid gel. The retained NTs are sequentially released for PSALM using successive HCl eluents. (b) The percentage of total captured+released DA vs mass of boric acid gel used in the column. (c) Normalized PSALM SERS (PreNP) from a sample of 1 mL containing 10 μM DA after affinity separation into eluent fractions. The inset graph shows the percentage of DA extracted in each eluent fraction. (d) Normalized PSALM SERS (PreNP) of 2 mL first eluent from affinity-separated 1 and 10 mL samples of fresh urine through 174 mg of boric acid gel. The lower graph shows extracted PSALM NT spectra (after subtracting water response), after baseline correction. Note that eluent is pH-buffered with NaOH to pH 6.5–7.5 for PSALM.