Towards sustainable diagnostics: replacing unstable H2O2 by photoactive TiO2 in testing systems for visible and tangible diagnostics for use by blind people

Blind and color blind people cannot use colorimetric diagnostics; the problem is especially severe in rural areas where high temperatures and the absence of electricity challenge modern diagnostics. Here we propose to replace the unstable component of a diagnostic test, H₂O₂, with stable TiO₂. Under UV irradiation, TiO₂ forms reactive oxygen species that initiate polymerization of acrylamide causing liquid-to-gel transition in an analyte-dependent manner. We demonstrate that specific DNA sequences can be detected using this approach. This development may enable the detection of biological molecules by users with limited resources, for example in developing countries or for travelers in remote areas.

Blind and color blind people cannot afford colorimetric diagnostics; the problem is especially severe in rural areas where high temperatures and the absence of electricity challenge modern diagnostics.

An ideal diagnosis, including diagnosis for infectious diseases, should meet the ASSURED criteria: (i) affordable by those at risk of infection; (ii) sensitive; (iii) specific; (iv) user-friendly; (v) rapid, and robust, for example not requiring refrigerated storage; (vi) equipment-free; (vii) delivered to those who need it.¹Analytic methods with visually detectable outputs (e.g. color change) satisfy criteria (iv) and (vi), and are therefore among the most common. Indeed, pregnancy tests, test strips for measuring acetone and glucose in urine for diabetic people, and pH strips are known to be the best to make the analysis easiest in data output. However, such methods cannot be used by the visually impaired.Recently, we described an alternative output signal that cannot only be detected by sight, but also by touch and applied it in the detection of adenosine triphosphate (ATP) and deoxyribonucleic acid (DNA).² The method is based on the analyte-dependent radical polymerization of acrylamide into polyacrylamide in the presence of hydrogen peroxide (H₂O₂). H₂O₂ serves as a source of radicals. The test uses affordable reagents and does not require any instrumentation for signal readout. Such test systems can be adopted for detection of a wide variety of biological analytes. Unfortunately, H₂O₂ is subject to light decomposition and should be refrigerated for long term storage; it is also prone to exploding at high concentrations. Therefore, substituting H₂O₂ with a more stable ingredient would increase the shelf life of the test system and make it usable in those environments with limited access to refrigeration. This work is devoted to addressing the ASSURED criterion (v): we substituted perishable H₂O₂ with stable titania (TiO₂) as an alternative source of initiators of radical polymerization.Recently we demonstrated the possibility of converting electromagnetic energy into pH gradients³ or generating reactive oxygen species (ROS) using TiO₂,^3,4 which makes this approach attractive for controlling interactions between chemical networks. Moreover, combining several functional chemical networks can result in a network with new functions. We have also shown that the pH gradient on titania can be used for regulating enzymatic reaction networks.⁵ In this work, we were interested in using photogenerated ROS (such as superoxide anion O₂˙⁻, hydroxyl radical ˙OH, and hydrogen peroxide H₂O₂) (Fig. 1) to initiate radical polymerization with the aim of making a visual and tactile portable sensor for the detection of biologically important molecules (DNA, RNA, ATP). Under ultraviolet (UV) irradiation, TiO₂ splits water, which results in the generation of a high concentration of ROS and free radicals.⁶Open in a separate windowFig. 1(i) A portable sensor based on a light-induced liquid-to-gel transition for polymer by radical polymerization on TiO₂ particles. The details of the sensor design are shown in Fig. 5; (ii) ROS formation on titania via reactions with photogenerated photohole (h⁺) and photoelectron (e⁻).The sensing system is based on deoxyribozyme sensor that produces ROS in the presence of a specific analyte, thereby triggering the radical polymerization of acrylamide into polyacrylamide. The buffer contained acetylacetone, H₂O₂, 40% acrylamide/bisacrylamide, hemin and a split deoxyribozyme sensor with peroxidase-like activity (PxR).² In the presence of the specific analyte sequence (A1 in this study), the sensor hybridized with the analyte and formed PxR, which bound hemin and decomposed H₂O₂ to ROS. The latter oxidized acetylacetone to the acetylacetone radical, which initiated the polymerization of acrylamide, resulting in liquid-to-gel conversion. Here, the aim of the research is to increase the system sustainability by changing H₂O₂ to TiO₂ particles. Noteworthy is that the system is externally controlled by UV light, since TiO₂ produces ROS only upon irradiation.Firstly, we quantified the TiO₂-derived ROS with the aim of finding the optimal conditions for the generation of the minimum amount of ROS needed for the polymerization. We used luminol chemiluminescence (CL) calibrated with hydrogen peroxide. Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) is a widely used CL reagent and has CL emission at different wavelengths depending on the conditions.⁶ Traditionally, luminol CL is observed in the presence of H₂O₂ in alkaline solutions (Fig. 2), which is catalyzed by metal ions, metal complexes or vitamins.^7–10Open in a separate windowFig. 2Mechanism of chemiluminescence of luminol and O₂⁻˙.The luminol CL signaling is conveniently used for the detection of ROS (particularly superoxide anion, hydrogen peroxide, hydroxyl radical) in biological systems.^11–16Irradiation of TiO₂ suspensions of different concentrations (0.03 M and 0.06 M) at two wavelengths (365 nm and 254 nm) revealed more ROS being formed upon irradiation at 254 nm – as was expected (Fig. 3).Open in a separate windowFig. 3Log concentration of ROS generated in TiO₂ suspensions of different concentration (0.03 M and 0.06 M) under irradiation by UV light with different wavelengths vs. irradiation time.According to the data obtained, the minimum concentration of TiO₂ needed to trigger polymerization after 5 min of irradiation at 365 nm was 1.25 mM (Fig. 4). We therefore used these conditions in the following experiments, since short irradiation time and longer wavelength of UV-A irradiation are less damaging to the DNA-based biosensor component than UV-C light with wavelength of 254 nm. It should be noted that control experiments were made without TiO₂ particles and polymerization did not occur. Next, we optimized the conditions for the analyte-dependent activation of PxR resulting in acrylamide polymerization. If polymerization occurs, the gel will stick to the bottom of the inverted tubes (Fig. 5).² To demonstrate the general applicability of the approach, we designed a sensor for an analyte of biomedical significance, nucleic acids, as an example. A sequence of 16S rRNA, which was represented in this study by synthetic A1 sequence (5′-CAT TAC TCA CCC GTC CGC CAC TCG TCA GCG AAG CAG CAA GCT GCT TCC TGT TAC CGT TCG), of pathogenic E. coli O157:H7 was chosen as the target analyte,. The binding of A1 to PxR1 and PxR2 stabilized the G-quadruplex structure, which then binds to hemin and catalyzes the polymerization (Fig. 5A).Open in a separate windowFig. 4The dependence of the log concentration of ROS generated on TiO₂ particles on TiO₂ suspension concentration. Inserts show the minimum concentration of TiO₂ particles in a suspension (therefore minimum concentration of ROS generated on TiO₂ particles), needed for the gel formation.Open in a separate windowFig. 5Polymerization-based visual and tactile detection of A1 analyte. (A) Sensor design: RNA strands PxR1 and PxR2 bind A1 sequence and form a G-quadruplex structure, which then binds to hemin (green oval) and catalyzes the radical polymerization of the acrylamide solution. The dotted lines represent triethylene glycol linkers. (B and C) Inverted test-tubes with radical polymerization of acrylamide initiated by reactive oxygen species generated on TiO₂ particles under UV light 365 nm. (B) Systems with different concentrations of hemin (