Controlling the Nucleation and Growth of Salt from Bodily Fluid for Enhanced Biosensing Applications

Abstract

Surface-enhanced Raman spectroscopy (SERS) represents a transformative tool in medical diagnostics, particularly for the early detection of key biomarkers such as small extracellular vesicles (sEVs). Its unparalleled sensitivity and compatibility with intricate biological samples make it an ideal candidate for revolutionizing noninvasive diagnostic methods. However, a significant challenge that mars its efficacy is the throughput limitation, primarily anchored in the prerequisite of hotspot and sEV colocalization within a minuscule range. This paper delves deep into this issue, introducing a never-attempted-before approach which harnesses the principles of crystallization-nucleation and growth. By synergistically coupling lasers with plasmonic resonances, we navigate the challenges associated with the analyte droplet drying method and the notorious coffee ring effect. Our method, rooted in a profound understanding of crystallization's materials science, exhibits the potential to significantly increase the areal density of accessible plasmonic hotspots and efficiently guide exosomes to defined regions. In doing so, we not only overcome the throughput challenge but also promise a paradigm shift in the arena of minimally invasive biosensing, ushering in advanced diagnostic capabilities for life-threatening diseases.

Keywords: SERS; biosensing; crystallization; extracellular vesicles; nucleation; plasmonics.

Figure 1

Crystallization dynamics on SERS substrate: (a) sequential progression of crystallization observed as the analyte droplet (PS in PBS) undergoes drying on a SERS substrate, illustrating the key stages of the process; (b) area covered by precipitates as the drying process progresses; (c,d) contact angle and droplet diameter.

Figure 2

Confocal FM image of Alexa 488-conjugated polystyrene beads forming a coffee ring (a,c) at the droplet center and (b,d) at the droplet edge. (e,f) At the corresponding locations: SERS mapping of the 1000 cm⁻¹ peak of polystyrene which is linked to the breathing mode of the aromatic carbon ring of styrene; (g) optical microscopy images with the locations presented in (a,b) marked in red.

Figure 3

(a–d) Progress of nucleation and growth observed on the SERS substrate at the laser spot, (e) analyte droplet containing fluorescent PS beads, (f) no localized precipitation observed on the flat-gold substrate which is non-plasmonic, (g) dried droplet on SERS substrate without the laser, (h) dried droplet on SERS substrate with the laser.

Figure 4

Cluster of Alexa 647-conjugated PS beads being pushed away by crystal nucleation and growth at the laser spot on the SERS substrate. The dashed line indicates the perimeter of a crystallite, which moves with the growth process. A True color image of the precipitate is inset on the right. A reduced focal length at higher magnification leads to variable focusing as the droplet height reduces as it dries.

Figure 5

Analysis of an analyte droplet containing sEVs: (a) composite image of a simply dried droplet, (b) areal mapping showing the low density of good SNR spectra; (c) 5× rise in the number of usable SERS signals after laser-assisted drying; (d) laser-assisted drying; (e) higher areal density of high SNR peaks observed, and there is an inverse relation to salt area coverage, which is reduced due to plasmonic precipitation; (f) overlap of 10 high-quality SERS signals observed from sample (d).