Chitosan-based fluorescent inverse opal particles for Cr(VI) sensing

Chromium and its compounds are widely used in alloy production, dyestuffs, leather tanning, electroplating, and catalysis, leading to widespread chromium pollution in soil and water. The WHO recommends 0.96 µM total chromium in drinking water, but hexavalent chromium, Cr(VI), is about 100 times more toxic than Cr(III) and is a Class I carcinogen, making selective Cr(VI) detection crucial. Existing analytical strategies (spectrophotometry, ICP-AES, ICP-MS, AAS, electrochemistry, fluorescence) offer sensitivity and selectivity but require complex pretreatment, expensive equipment, and time-consuming protocols. Chitosan (CS), a natural polymer with intrinsic fluorescence that persists after glutaraldehyde (GA) crosslinking and can be quenched by Cr(VI), presents a low-cost, simple alternative. However, bulk CS-GA structures can be nonuniform and require large amounts of material. Inverse opal materials, prepared by templating colloidal crystals, offer ordered macroporous structures, high surface area, and photonic properties that can enhance fluorescence. Few inverse opal particle (IOP) systems have targeted Cr(VI), and CS-based IOPs leveraging intrinsic fluorescence quenching by Cr(VI) had not been reported. This study develops uniform CS inverse opal particles (IOPs) templated from microfluidic silica colloidal crystal beads (SCCBs), leveraging their photonic structure to enhance CS fluorescence and enabling rapid, sensitive, and specific Cr(VI) detection. The CS IOPs show stable fluorescence across broad pH and temperature ranges and improved emission over non-structured CS particles, with rapid quenching upon exposure to Cr(VI) while being stable to other ions. The platform aims to provide a simple, low-cost, fast method for selective Cr(VI) detection in water.

The paper situates Cr(VI) detection within established methods: spectrophotometry, ICP-AES, ICP-MS, AAS, electrochemistry, and fluorescence, which, despite high performance, suffer from complex sample pretreatment, expensive instrumentation, and time-consuming workflows. Prior CS-based fluorescence has been observed and maintained after GA crosslinking, with quenching by Cr(VI) attributed to binding of Cr(VI) to CS and quenching of nonconjugated fluorophores. Inverse opal materials, formed by templating colloidal crystals, possess ordered macropores and photonic bandgaps (PBG) that can enhance emission when matched to fluorophore spectra. While diverse IOPs have been developed for biosensing, few target Cr(VI) in water and none have used simple fluorescence quenching of intrinsic CS fluorescence in CS IOPs. The study addresses these gaps by combining CS’s intrinsic fluorescence and Cr(VI)-induced quenching with IOP photonic enhancement for an inexpensive, facile sensor.

Materials: Chitosan (degree of deacetylation 80–95%), PEGDA (Mw 700), HMPP photoinitiator, GA, HF (40%), EDTA-2Na, Na2CrO4, various nitrate salts (Cr, Ce, Fe, Hg, Al, Mg, Co, Mn, Ca, Cu, Cd, Zn), Na3PO4, NaNO2, paraffin, acetic acid; ultrapure water (≥18 MΩ·cm).

SCCB template preparation: SiO2 nanoparticles in water (20% w/v) served as the inner phase (1 mL/h) and were emulsified by silicone oil (500 cSt; 6 mL/h) using a single-emulsion microfluidic platform to form droplets. Droplets were dried at 75 °C overnight to self-assemble into silica colloidal crystal beads (SCCBs). Beads were washed with n-hexane to remove silicone oil, then calcined at 800 °C for 4 h to enhance mechanical strength.

CS IOP fabrication: SCCBs were immersed for 6 h in a pregel solution of CS, PEGDA, and HMPP (1%) to infiltrate nanovoids, followed by UV polymerization to form SCCB/CS hybrid particles. Hybrids were manually stripped and CS was further crosslinked with GA (5% v/v, 4 h). Silica was removed using HF to yield CS inverse opal particles (CS IOPs). To improve visibility and mechanical robustness (pure CS IOPs were highly transparent and collapsed upon drying), PEGDA was doped; optimal pregel concentrations selected were 4% w/v CS and 10% v/v PEGDA based on fluorescence intensity and mechanical/processing considerations.

Control particles: Pure CS hydrogel particles (non-IOP) were prepared by adding a fresh mix of CS (800 µL, 4% w/v) and GA (200 µL, 5% v/v) dropwise into liquid paraffin containing Span 80 (10% w/v) under stirring (800 rpm) at 50 °C; these exhibited poor size dispersion and lower fluorescence intensity than CS IOPs of similar size.

Characterization: SEM imaged SCCBs, SCCB/CS hybrids, and CS IOPs, showing close-packed silica arrays and complementary inverse opal structures after silica removal. Optical reflectance and structural colors were assessed; SCCBs’ reflective peak A depended on particle diameter and average refractive index (A ≈ 1.633 d n_average). Hybrid particles showed slight red shifts relative to SCCBs, while CS IOPs blue-shifted due to replacement of high-index silica with water.

Optimization: Systematically varied CS (1–4% w/v) and PEGDA (0–20% v/v). Fluorescence intensity increased with CS concentration and decreased with PEGDA; 4% CS and 10% PEGDA were chosen as a compromise between emission, solubility, opacity, and mechanical strength.

Cr(VI) detection protocol: Individual CS IOPs were imaged under a fluorescence microscope to record baseline intensity. Particles were then immersed for 10 min in Na2CrO4 solutions (0, 1, 3, 5, 10, 20, 30, 50, 100, 300, 1000, 3000, 10,000 µM) containing EDTA (2 mM) as a masking agent, gently washed, and re-imaged. Average fluorescence intensities pre- and post-incubation were quantified using ImageJ to compute quenching ratios.

Specificity and anti-interference: For specificity, CS IOPs were incubated in 1 mM solutions of various ions (with 2 mM EDTA) for 10 min, washed, and fluorescence changes were measured. For anti-interference, IOPs were incubated in 20 µM Cr(VI) containing 1 mM of interferent ions (with 2 mM EDTA) for 10 min to assess deviations from Cr(VI) response. The necessity of EDTA was evaluated; without EDTA, metal cations affected fluorescence due to binding to CS.

Stability and repeatability: Fluorescence stability was tested across pH 3–9, temperatures up to >40 °C, and storage time; repeatability was assessed and mitigated by high-throughput particle generation (~3×10^4 droplets per hour).

Real sample analysis: Tap water and pond water were tested via standard addition. CS IOPs were immersed in matrices spiked with 0, 5, 10, 15, 20 µM Na2CrO4 (with 2 mM EDTA) for 10 min, washed, and fluorescence was measured to calculate Cr(VI) concentrations and recoveries. Replicates per group were n=3; error bars represent standard deviations.

• Uniform CS inverse opal particles (IOPs) were fabricated by templating microfluidically produced silica colloidal crystal beads, yielding ordered macroporous structures with photonic bandgaps that enhanced CS fluorescence emission relative to non-structured CS particles.
• Detection performance: Rapid fluorescence quenching upon Cr(VI) exposure with a usable response range up to 10,000 µM and a linear range of 1–30 µM (R^2 = 0.99617; measurement uncertainty 0.07676). Limit of detection (LOD) = 0.055 µM within the 0–30 µM linear range.
• Specificity: At 1 mM, interferent ions caused <4% fluorescence quenching, whereas Cr(VI) quenched >80%. In 20 µM Cr(VI) with 1 mM interferents (50-fold excess), deviations in quenching were <5% when EDTA (2 mM) was present as a masking agent.
• Stability: Fluorescence remained relatively stable across pH 3–9; slight decrease observed above 40 °C; minimal effect from storage time. Repeatability was relatively poor but offset by high-yield particle production (~3×10^4 droplets/hour).
• Structural benefits: CS IOPs exhibited significantly stronger fluorescence than same-size CS hydrogel particles despite containing less CS (inverse opal duty ratio ~26%), indicating photonic enhancement; estimated fluorescence enhancement ≈580% due to PBG alignment near CS emission band.
• Real samples: Determined Cr(VI) at 0.066 µM in tap water and 0.186 µM in pond water; recoveries were 86–106% (tap) and 99.7–107.3% (pond), demonstrating practical applicability.
• Controls: PEGDA-only IOPs showed no fluorescence; non-IOP CS particles showed lower intensity and poorer uniformity.
• Comparative context: Among fluorescence-based methods summarized, the CS IOPs achieved the lowest reported LOD with straightforward, low-cost operation and short assay times.

The study demonstrates that leveraging chitosan’s intrinsic fluorescence and Cr(VI)-induced quenching within an inverse opal photonic architecture enables rapid, sensitive, and selective detection of Cr(VI) in water. The microfluidics-templated inverse opal structure provides uniform particle size and an ordered macroporous network, enhancing light–matter interactions and aligning the photonic bandgap edge with CS emission to boost fluorescence intensity and quenching sensitivity. This directly addresses the need for simple, low-cost, and fast assays that avoid complex instrumentation and pretreatment. Robust performance across pH 3–9, modest temperature susceptibility, and minimal storage effects support field applicability. Specificity is ensured via EDTA masking to preclude binding of other metal cations to CS, with negligible interference even at high interferent concentrations. Real-sample analyses with high recoveries validate the method’s practical relevance. Compared to other Cr(VI) detection strategies, CS IOPs offer competitive linear range and superior LOD among fluorescence-based approaches, while simplifying operation and reducing costs. The results indicate the essential role of the inverse opal microstructure in achieving enhancement (~580%) and improved sensitivity relative to bulk CS particles, highlighting photonic engineering as a powerful approach for amplifying intrinsic biopolymer fluorescence for environmental sensing.

The work introduces chitosan-based inverse opal particles as a simple, low-cost, and rapid fluorescence-turn-off platform for Cr(VI) detection. By templating microfluidic silica colloidal crystal beads and harnessing photonic bandgap effects, the CS IOPs exhibit enhanced fluorescence, high sensitivity, and strong specificity to Cr(VI). The sensor provides a linear response from 1–30 µM with an LOD of 0.055 µM and functions across a broad application range up to 10,000 µM. Stability across pH 3–9, manageable temperature effects, and successful quantification in tap and pond waters (recoveries ~86–107%) demonstrate practical utility. Future research should broaden the linear dynamic range, reduce or eliminate sample pretreatment steps, improve repeatability, and extend the platform to detect additional heavy metal ions, further advancing environmental monitoring applications.

• Limited linear dynamic range (1–30 µM); broader ranges would expand applicability.
• Sample solutions require pretreatment (e.g., EDTA masking), adding operational steps.
• High sensitivity and specificity demonstrated primarily for Cr(VI); extension to other heavy metals is needed.
• Repeatability is relatively poor, though mitigated by high-throughput particle production.