Toward a Plasmon-Based Biosensor throughout a Thermoresponsive Hydrogel

Abstract

This study investigates the potential of thermoresponsive hydrogels as innovative substrates for future in vitro diagnostic (IVD) applications using AVAC technology, developed and patented by the Mecwins biomedical company. In order to convert the hydrogel in a substrate compatible with AVAC technology, the following prerequisites were established: (1) the hydrogel layer needs to be permeable to gold nanoparticles (AuNPs), and (2) the optical properties of the hydrogel should not interfere with the detection of AuNPs with AVAC technology. These two key aspects are evaluated in this work. A silicon substrate (Sil) was coated with a layer of a thermosensitive hydrogel (TSH) based on poly(N-isopropylacrylamide-co-N,N'-methylene bis(acrylamide) (PNIPAAm-co-MBA). The TSH offers the advantage of easy modulation of its porosity through cross-linker adjustments, crucial for the plasmonic nanoparticle (NP) permeation. The platforms, denominated as (Sil)-g-(PNIPAAm-co-MBA), were fabricated by changing the cross-linker concentrations and exploring three deposition methods: drop casting (DC), spin coating (SC), and 3D printing (3D); the DC approach resulted in a very homogeneous and thin hydrogel layer, very suitable for the final application. Furthermore, after physical-chemical characterization, the TSH demonstrated its functionality in regulating nanoparticle absorption, and AVAC technology's capability to precisely identify such NPs through the hydrogel matrix was validated. The proposed hydrogel platform fulfills the initial requirements, opening the possibility for employing these hydrogels as dynamic substrates in sandwich immunoassay devices. The next step in the development of the hydrogel substrate would be its functionalization with biorecognition groups to allow for biomarker detection. By leveraging their enhanced capture efficiency and the ability to manipulate particle flow thermally, we anticipate a significant advancement in diagnostic methodologies, combining the spatial benefits of three-dimensional hydrogel structures with the precision of AVAC's digital detection.

Scheme 1. Schematic Illustration of the Main Steps of Functionalization and Grafting of the Silicon Wafers

Scheme 2. Schematic Illustration of (A) the Pathways Followed for the (Sil)-g-(PNIPAAm-co-MBAx)/y Platform Preparation; (B) the Thermo-Assisted AuNP Permeation Experiments Carried out with Different TSHs; and (C) the Detection and Characterization of AuNPs inside the TSH, Using Scattering with Dark-Field Microspectrophotometry

Figure 1

Raman spectra and, in the inset, water contact angle (WCA) measurements carried out on untreated (Sil_UT) and plasma-treated (Sil_PT) silicon wafers.

Figure 2

(A) FTIR spectra showing the comparison between bands associated with the cross-linker (MBA) and the PNIPAAm copolymers obtained in the presence of different concentrations of MBA, PNIPAAm-co-MBA10 with NIPAAm:MBA = 250:10, and PNIPAAm-co-MBA100 with NIPAAm:MBA = 250:100; SEM micrographs and pore size distribution of (B, D) PNIPAAm-co-MBA10 and (C, E) PNIPAAm-co-MBA100.

Figure 3

(A) Raman spectra and corresponding optical images of (B) (Sil)-g-PNIPAAm-co-MBA10 and (C) (Sil)-g-PNIPAAm-co-MBA100.

Figure 4

(A) Illustration of the experimental setup for the thermo-assisted AuNP permeation assays; (B, C) UV–vis spectra of (Sil)-g-(PNIPAAm-co-MBA10) and (Sil)-g-(PNIPAAm-co-MBA100) platforms, respectively. Insets in parts (B) and (C) correspond to optical images of the AuNP solution distribution at T > LCST above PNIPAAm-co-MBA10 and PNIPAAm-co-MBA100 hydrogels, respectively.

Figure 5

(A) Raman spectra showing the comparison between (Sil)-g-(PNIPAAm-co-MBA10)/DC, (Sil)-g-(PNIPAAm-co-MBA10)/SC, and (Sil)-g-(PNIPAAm-co-MBA10)/3D platforms (inset: thickness of the three plasmon-based devices). Dark-field microcopy images of (B) (Sil)-g-(PNIPAAm-co-MBA10)/SC, (C) (Sil)-g-(PNIPAAm-co-MBA10)/3D, and (D) (Sil)-g-(PNIPAAm-co-MBA10)/DC, showing the AuNPs distribution.

Figure 6

AuNP identification by means of (A) SEM micrograph and EDX spectrum and (B) dark-field micrograph using AVAC technology and color 2D-histogram. Cryogenic SEM micrographs of (Sil)-g(PNIPAAm-co-MBA10)/DC at (a1) 500× and (a2) 6500× after AuNP capture; (a3) EDX elemental analysis of the (Sil)-g(PNIPAAm-co-MBA10)/DC sample; (b1) dark-field image and (b2) two-dimensional histogram corresponding to AuNPs embedded in the hydrogel layer; (b3) dark-field microscopy image corresponding to the two-dimensional histogram, where 246 AuNPs have been detected. The bottom inset is a schematic drawing of the experimental setup.