Multi-Factors Cooperatively Actuated Photonic Hydrogel Aptasensors for Facile, Label-Free and Colorimetric Detection of Lysozyme

Abstract

Responsive two-dimensional photonic crystal (2DPC) hydrogels have been widely used as smart sensing materials for constructing various optical sensors to accurately detect different target analytes. Herein, we report photonic hydrogel aptasensors based on aptamer-functionalized 2DPC poly(acrylamide-acrylic acid-N-tert-butyl acrylamide) hydrogels for facile, label-free and colorimetric detection of lysozyme in human serum. The constructed photonic hydrogel aptasensors undergo shrinkage upon exposure to lysozyme solution through multi-factors cooperative actuation. Here, the specific binding between the aptamer and lysozyme, and the simultaneous interactions between carboxyl anions and N-tert-butyl groups with lysozyme, increase the cross-linking density of the hydrogel, leading to its shrinkage. The aptasensors' shrinkage decreases the particle spacing of the 2DPC embedded in the hydrogel network. It can be simply monitored by measuring the Debye diffraction ring of the photonic hydrogel aptasensors using a laser pointer and a ruler without needing sophisticated apparatus. The significant shrinkage of the aptasensors can be observed by the naked eye via the hydrogel size and color change. The aptasensors show good sensitivity with a limit of detection of 1.8 nM, high selectivity and anti-interference for the detection of lysozyme. The photonic hydrogel aptasensors have been successfully used to accurately determine the concentration of lysozyme in human serum. Therefore, novel photonic hydrogel aptasensors can be constructed by designing functional monomers and aptamers that can specifically bind target analytes.

Keywords: aptamer conformational change; colorimetric sensing; multi-factors actuation; photonic hydrogel aptasensors; volume phase transition.

Figure 1

Fabrication of photonic hydrogels. PS particles were injected onto the water surface and orderly self-assembled to form a 2DPC array on water, followed by transfer to a glass slide. Then, polymerizable precursor solution was added to the 2DPC surface on glass substrate, and a coverslip was subsequently placed on the solution. The photopolymerization was performed and the 2DPC was embedded in the hydrogel network. The resultant 2DPC hydrogel was peeled off the glass slide and washed by water and PBS solution, and finally stored in PBS solution.

Scheme 1

Photopolymerization reaction of monomers and the functionalization of aptamers to fabricate the photonic hydrogel aptasensors.

Figure 2

FTIR spectra of the P(AAm−AAc−TBAm) hydrogel before and after modification of lysozyme-binding aptamer. The appearance of new absorption peaks at 1564 cm⁻¹, 1225 cm⁻¹, 1034 cm⁻¹ and 776 cm⁻¹ demonstrated the successful modification of the aptamers.

Figure 3

(a,b) SEM images of 2DPC array prepared from ~960 nm of PS microspheres on glass slide; Inset in (b) a photograph of the 2DPC array taken under illumination with a flashlight below at an angle of ~75° to the normal. The PS microspheres arrange orderly and closely on the glass slide and form a 2D monolayer array with a vivid rainbow color. (c) SEM image of the 2DPC hydrogel prepared initially. (d) SEM image of the 2DPC hydrogel equilibrated in PBS solution. The PS microspheres are still close-packed for the freshly prepared 2DPC hydrogel, while they become non-close-packed after equilibration in PBS solution due to the swelling of the hydrogel.

Scheme 2

(a) The response mechanism of the photonic hydrogel aptasensor toward lysozyme. The specific binding between the aptamer and lysozyme induces its conformational change from a single-strand stretch to a G-quadruplex structure. This shortens the distance of the polymer chains and increases the cross-linking density of the hydrogel, leading to its shrinkage. In addition, the adjacent carboxyl groups and the hydrophobic N-tert butyl groups simultaneously interact with lysozyme based on the electrostatic interaction and hydrophobic groups interaction, which is also helpful to the shrinkage of the hydrogel. The hydrogel shrinkage induces the particle spacing (d) to decrease. (b) Measurement of Debye diffraction ring. The 2DPC embedded in the hydrogel network strongly diffracts light in the forward direction upon illumination by a laser along its array normal, generating a Debye ring on the screen below. The Debye ring diameter (D) can be measured directly and used to calculate the particle spacing of 2DPC according to a formula, $d=4{\lambda }_{laser}\sqrt{{\left(D/2\right)}^2+h^2}/\left(\sqrt{3}D\right)$, in which λ_laser is the laser wavelength, and h is the distance between the 2DPC plane and the screen below. Lysozyme induces the photonic hydrogel to shrink and its particle spacing decreases. The particle spacing changes (Δd) can be obtained by measuring the Debye ring diameters of the photonic hydrogel before and after response toward lysozyme and the following calculation of d.

Figure 4

The particle spacing changes of various hydrogel films upon reaction with 500 μM lysozyme solutions. Compared with the hydrogels H1 and H2, the remarkable particle spacing decreases of the hydrogel sensors DNA−H1−960 and DNA−H2−960 with aptamers demonstrated the key actuation role of specific binding between the aptamer and lysozyme in the hydrogel shrinkage. Compared with DNA−H2−960 without N-tert-butyl groups, the larger particle spacing decrease of DNA−H1−960 showed the presence of the N-tert-butyl groups in the hydrogel network also facilitated the hydrogel shrinkage. The particle spacing change of DNA−H1−960 at pH 7.40 was larger than that at pH 5.00, showing the electrostatic interaction between carboxyl anions and lysozyme.

Figure 5

The particle spacing changes of the DNA−H1−960 after treatment with various solutions of 500 μM. The particle spacing decreases of the sensor in lysozyme and the mixture solution (1 Mixture, prepared by mixing the compounds 2–8 listed in this figure) were much higher than those in the other biomolecules’ solutions. The results showed the high detection selectivity and anti-interference of the DNA−H1−960 to lysozyme.

Figure 6

Time dependence of the particle spacing changes for DNA−H1−960 hydrogel aptasensor in 500 μM and 1 mM lysozyme solutions (Insets: the photographs of DNA−H1−960 at different response time). The scale bar “1 cm” represents the size of the photographs. The particle spacing decreased sharply within 10 min and achieved the response equilibrium at 60 min. The size of the hydrogel was obviously decreased along a longer reaction time.

Figure 7

The particle spacing changes of the hydrogel aptasensors after reaction with different concentrations of lysozyme (C_LYs): (a) DNA−H1−960; (c) DNA−H1−698; The insets are photographs of the corresponding aptasensors after achieving response equilibrium at different concentrations of lysozyme. The scale bar “1 cm” represents the size of the photographs. The linear relationship between the particle spacing changes and C_LYs: (b) DNA−H1−960; (d) DNA−H1−698. (e) The particle spacing changes of DNA-H1 aptasensors fabricated from different diameters of PS microspheres upon exposure to different concentrations of lysozyme. The particle spacing changes increased with an increase in the lysozyme concentration and the limit of detection for DNA−H1−960 and DNA−H1−698 were 1.8 nM and 56.3 nM, respectively, indicating a more sensitive detection of lysozyme for DNA−H1−960. The comparison of the particle spacing changes at the same lysozyme concentrations demonstrated that the 2DPC hydrogel aptasensor prepared from larger diameter microspheres showed a clearer particle spacing change to lysozyme, which is more beneficial to the lysozyme detection.