4.2.1 Structural features of dye nanoparticle-coated test strip

Recently, we reported a simple and versatile method for preparation of dye nanoparticle-coated test strips (DNTSs) for detection of inorganic ions [11]. A thin dye nanoparticle/nanofiber layer (400 nm–2 μm) located on the top surface of the support membrane provides a remarkably concentrated signaling surface composed of a 100% reagent layer. Therefore, almost all signals are available efficiently (Figure 4.1A), but in most conventional test papers, indicator dyes are distributed over the entire support (Figure 4.1B). For that reason, the signal obtained from the top surface must be less intense. It is noteworthy that the present membranes were prepared without any additives such as coating polymers or modifiers. Yet the reagent is not removed from the support by rubbing with a finger or by immersion into water. The cross-sectional SEM image indicates that the thickness of the dye layer is <1 μm (Figure 4.1C). Thus it provides a remarkably concentrated signaling surface composed of 100% pure indicator dye. In contrast, the surface concentration of dye is rather low in conventional test strips prepared by soaking of dye solution.

4.2.2 Simple yet versatile fabrication methods of DNTSs

We designed two fabrication methods of DNTSs for the purpose of producing a large number of test strips with various organic indicator dyes. These methods, including two different preparation processes of nano-dispersion solution and simple filtration process of the dispersion with a fine membrane filter having microscopic pores, are shown in Figure 4.2.

In order to fabricate DNTSs with hydrophobic indicator dyes, a simple process referred to as the “reprecipitation method” [12,13] was proposed for the preparation of aqueous dye nano-dispersion (Figure 4.2, top). The typical preparative process involves injection of a water miscible organic solution of a dye compound into water under vigorous stirring. Dispersion of nano-sized dye compounds is formed immediately upon combining the aqueous solution. The growth time of the nano-dispersion solution was determined to be the time until the average size of dye nanoparticles become more than ca. 100 nm. After the growth period ended, the nano-dispersion is simply passed through with a fine membrane filter by suction. Then, by surface filtration mechanism, dye nanoparticles are uniformly and firmly attached to a membrane filter. The thicknesses of dye nanoparticle layers were experimentally observed to be between 400 and 700 nm, which depend largely on the amount of reagents loaded as well as on the molecular size (weight). The morphology of the nanoparticles varies from particle to fibrous depending on the molecular structure (Figure 4.3).

On the other hand, the “reprecipitation method” is inapplicable to hydrophilic charged dyes such as reagents having sulfone groups and pyridinium groups. For these hydrophilic indicator dyes, nanoscale ion exchangers are used as dye absorbents to produce dispersions of hydrophilic dye/nano absorbent nanocomposites. In principle, a charged dye adsorbs onto the surface of nano-adsorbent having the opposite charges (1–100 nm in diameter), followed by decrease in the surface charge of the adsorbent, and then aggregation occurs (Figure 4.2, bottom). A preparative procedure of hydrophilic dye/nano absorbent loaded DNTS is technically easy, only by mixing individual solutions under the appropriate pH.

For example, 10 mL of TMPyP/silica nanocomposite aqueous solution was prepared by mixing 100 μL of 2 mM TMPyP and 4×10⁻⁵ wt% of silica (ca. 10–14 nm in diameter) at pH 7.8 with 0.01 M Tris buffer solution [14]. Similarly, the growth time of nanocomposite in the aqueous solution was decided as the time until the average size of nanocomposites become more than ca. 100 nm. Then, by filtration in the similar way to that of hydrophobic dyes, roughly 100% of the nanocomposite was coated as a thin layer onto the surface of the membrane filter through surface filtration. Some metal oxides and modified latex nanoparticles, which are smaller than 100 nm in diameter and having sufficient positive or negative surface charges in aqueous solution, are promising candidates as nano-adsorbents for fabricating nanocomposite-coated DNTSs. The thicknesses of dye nanoparticle layers were experimentally observed to be ca. 1–2 μm, which depends on the amount and the size of the nano-adsorbent used.

A wide variety of analytical dye compounds can be converted as nanoparticles or nanocomposites in aqueous solution by these two methods and then DNTSs can be simply fabricated from them. The versatility of the preparative procedures of DNTSs enables a screening test of reagents against one target ion. Another outcome of the versatility is that DNTS is a novel platform technology to produce highly sensitive test strips for any target with common dye indicators.

4.2.3 Detection characteristics with DNTS

The DNTS is applicable not only to immersion test but also to filtration enrichment in which target ions are concentrated by passing a sample solution through it (Figure 4.4). Immersion test is just the same detection way as using any ordinary test papers, such as urine, pH, and ions, but it takes longer to complete sufficient color development with DNTS than those with other test papers. The major reason for this is that DNTSs detect a very low concentration of a target ion at ppb level. In addition, DNTSs are water permeable and insoluble in aqueous samples, and hence trace ions are enriched on them simply by filtration of the sample solution. Filtration enrichment is a unique approach provided by DNTS and based on the fact that the dye nanoparticle layer acts as a solid phase feasible to extract a target ion mainly by complex formation between a target ion and indicator dye. Generally, filtration enrichment is more sensitive than immersion test, and one of the examples performed by PAN (1-(2-pyridylazo)-2-naphthol) nanofiber DNTS is introduced in Section 4.4.1. Moreover, filtration enrichment enables highly selective detection by optimizing the reaction conditions, such as solution pH, masking reagent, and flow rate, and the example carried out by dithizone nanofiber DNTS, described in Section 4.4.2.

4.4.1 PAN nanofiber DNTS for Zn(II) detection

Membranes coated with PAN nanofibers were applied as test strips for the detection of Zn²⁺ in a test solution (pH 8.4) [11]. Figure 4.6A shows the color changes of PAN-coated strips in immersion test. Notably 65 ppb of Zn²⁺ was detected by a naked eye color test. Sub-ppb concentrations of Zn²⁺ were successfully detected by filtration enrichment of 100 mL of the sample solution (Figure 4.6B). This filtration enrichment procedure amplifies the signal intensity capable of eye detection of ppb-level metal ions. Practically, leaking of reagents out of the sample solution was observed to be negligible during dip test and sample filtration procedures. We also monitored the color change and the relative color intensity with reflectance absorption spectrometry. The peak at λ_max=550 nm indicates that neutral [Zn(PAN)₂] is formed on the membrane filter as the major species. The increasing peak intensity with increasing Zn^(II) concentration enables quantitative determination of test samples by comparison with the calibration curve.

4.4.2 Dithizone nanofiber DNTS for Hg(II) detection

We demonstrated dithizone nanofiber thin film for highly sensitive and selective detection of Hg(II) [15,16]. Dithizone nanofiber was prepared by the reprecipitation method. Simply by filtration of the dispersion through the membrane filter dithizone nanofiber thin film of ca. 440 nm in thickness was deposited firmly and uniformly on one side of the membrane filter. Determination of Hg(II) at ppb level was achieved by filtration enrichment of a sample solution and successive colorimetric analysis. Consequently, Hg(II) was concentrated in the film as reddish brown complexes at pH 2.7. More than 90% of 10 ppb Hg(II) was retained in the film at the filtration rate of 1.3–9.3 mL/min. The presence of Na⁺ (10,000 ppm), K⁺ (5000 ppm), Ca(II) (5000 ppm), Al(III) (500 ppm), Cr(III) (200 ppm), Mn(II) (100 ppm), Fe(III) (100 ppm), Ni(II) (100 ppm), Cd(II) (10 ppm), and Pb(II) (100 ppm) in the acidic solution at pH 2.7 did not interfere with the detection of 10 ppb of Hg(II) at all (Table 4.1). The addition of the masking reagent, EDTA was very effective for Cu(II) and Zn(II) by forming water-soluble complex and passing through dithizone DNTS. Prefiltering, in combination with the addition of iodine ion, also effectively masked Ag(I) interference. The interference from Cr(VI) and Fe(II) was also eliminated based on oxidation–reduction reaction. The present method succeeded in determining simulated wastewater, river water, and seawater spiked with 10 ppb Hg(II). Calibration curves for Hg(II) were obtained when the sample volume was 100 mL, and the 3σ detection limit was calculated to be 0.057 ppb (n=10) by a thin layer chromatography (TLC) scanner at 500 nm.

Some other DNTSs were also effective in detecting targets at ppb level, for instance, TMPyP/silica nanocomposite DNTS for the determination of Cd(II) and Pb(II) ions by filtration enrichment [14], 5-Br-PADAP nanoparticle DNTS for Cd(II) by improved immersion test [17].