**1. Introduction**

Polyglutamine (PolyQ) diseases, including Huntington's disease, spinobulbar muscular atrophy and spinocerebellar ataxias, are a group of inheritable neurodegenerative disorders characterized by the expansion of a CAG trinucleotide repeat of the affected genes and the expression of polyQ proteins with a glutamine-enriched tract, in which the number of CAG repeats in patients or carriers can reach from 40 to over 100 as compared to that of 35–50 in healthy individuals [1–5]. The polyQ proteins are neurotoxic and can cause a wide range of cellular dysfunctions in neurons. Taking Huntington's disease as an example, the mutant *HTT* gene encodes the toxic and aggregation-prone mutant huntingtin (mHTT) protein with an expanded polyQ tract translated from the expanded CAG repeats, and the accumulation of the mHTT protein will lead to neuronal cell death due to altered neural circuitry, mitochondrial dysfunction, transcriptional dysregulation, disrupted protein homeostasis, impaired protein degradation, and aberrant activation of stress responses [6–10]. In addition to the toxicity due to the misfolding, accumulation, and aggregation of the polyQ proteins, recent studies are showing that the expanded CAG RNA repeats are also neurotoxic [11–14]. A recent work has demonstrated that cellular expression of short CAG (sCAG) RNAs can hybridize with and silence the CUG-containing mRNA in the *Nudix hydrolase 16* (*NUDT16*) gene and lead to DNA damages and cellular apoptosis [15]. Small molecule inhibitors that selectively bind to the CAG RNA repeats

**Citation:** Lau, M.H.Y.; Wong, C.-H.; Chan, H.Y.E.; Au-Yeung, H.Y. Development of Fluorescent Turn-On Probes for CAG-RNA Repeats. *Biosensors* **2022**, *12*, 1080. https:// doi.org/10.3390/bios12121080

Received: 8 November 2022 Accepted: 21 November 2022 Published: 25 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

are therefore implicated in not only the development of therapeutic strategies for treating polyQ diseases, but also their rapid detection and diagnosis.

Designing small molecule binders and related fluorescent probes for specific RNA sequences is, however, very challenging because of the highly diverse, dynamic and flexible structures of RNA [16–18]. Recently, we have identified the bis(amidinium) compound, **DB213**, as a small molecule drug candidate for treating polyQ RNA toxicity. Detail binding and NMR studies showed that **DB213** binds with a *K*<sup>d</sup> of 3.8 μM to the major groove of an sCAG hairpin (*rGG-(CAG)6-CC*) and interacts specifically with two consecutive A·A mismatches via a combination of hydrogen bonds, electrostatic, and other interactions (Figure 1) [15]. The binding is selective, and a much weaker binding was observed for CUG RNA [19–21] and the rCAG/CUG heteroduplex formed from *rGG-(CAG)4-(CUG)2-CC* (*K*<sup>d</sup> = 19.2 μM) [15]. Further cellular studies and biochemical analysis also showed that **DB213** did not affect the transcription level of the mRNA of a CAG repeat disease gene, and that it could suppress toxicity due to CAG RNA but not polyQ proteins, showing that **DB213** is specific to the CAG RNA over the corresponding DNA and proteins [15]. With these promising binding properties, we propose that **DB213** could be turned into a fluorescent probe for CAG RNA repeats by appending an environment sensitive fluorophore on the bis(amidinium) core, such that the probe would elicit a fluorescent response upon binding to the target CAG RNA as a result of a change in the local environment. In this work, we present our results on this proof-of-concept study including the design, synthesis and solution characterizations of three fluorophore-appended **DB213** derivatives as potential fluorescent probes for CAG RNA. In particular, **DB-TPE** which features a tetraphenylethylene fluorophore was found to exhibit a strong fluorescence enhancement upon binding to CAG RNA via aggregation-induced emission (AIE).

**Figure 1.** Chemical structure of **DB213** that binds selectively to CAG RNA, and the design of a fluorescent probe for CAG RNA.

## **2. Materials and Methods**

All reagents were purchased from commercial suppliers (Aldrich, Dkmchem, Energy, J & K) and used without further purification unless otherwise specified. All solvents were of analytical grade (ACL Labscan, DUKSAN Pure Chemicals). Dried solvents were distilled over CaH2 (CHCl3, EtOH, MeOH). Thin layer chromatography was performed on silica gel 60 F254 (Merck) and column chromatography was carried out using silica gel 60F (Silicycle), neutral or basic aluminium oxide Brockmann I (Acros Organics). Detailed synthetic procedures and characterization of all other starting materials are described in the Supplementary File S1. 1H and 13C{1H} NMR spectra were obtained from a Brucker DPX 400 or Brucker DPX 500 spectrometer and signals were referenced to solvent residues. LC-MS analyses were performed on a UPLC-MS system with a Waters UPLC coupled to a 2489 UV/Vis detector and an ACQUITY QDa MS detector. High resolution ESI-MS data was obtained from a Waters Micromass Premier Q-ToF tandem mass spectrometer.

Synthesis of **DB-Res** was carried out as follows. Under an argon atmosphere, a mixture of **DB-N3** (10 mg, 0.03 mmol), **Res-alkyne** (8 mg, 0.03 mmol), and [Cu(CH3CN)4](PF6) (1 mg, 0.003 mmol) in DMSO (3 mL) was stirred at room temperature for overnight. The resulting mixture was purified by a neutral alumina column with CH2Cl2/MeOH (*v*/*v* = 8:2) as the eluent. Fractions containing the product was combined and concentrated using a rotary evaporator, to which a saturated HCl solution in dry EtOH (1 mL) was added. The mixture was stirred at room temperature for 3 h and centrifuged (4000 rpm, 3 min). The solution was decanted and the remaining solid was washed with dry EtOH (10 mL × 1) and dry diethyl ether (10 mL × 3). The orange-red solid obtained was dried under vacuum. Yield = 15 mg, 73%. 1H NMR (500 MHz, CD3OD, 298 K): δ 8.27 (s, 1H), 7.99–7.95 (m, 2H), 7.95–7.91 (m, 2H), 7.78 (d, *J* = 8.9 Hz, 1H,), 7.54 (d, *J* = 9.8 Hz, 1H), 7.17 (d, *J* = 2.6 Hz, 1H), 7.13 (dd, *J* = 8.9, 2.7 Hz, 1H), 6.85 (dd, *J* = 9.8, 2.1 Hz, 1H), 6.28 (d, *J* = 2.1 Hz, 1H), 5.35 (s, 2H), 4.62 (t, *J* = 6.0 Hz, 2H), 3.57 (t, *J* = 6.7 Hz, 2H), 3.26 (t, *J* = 6.7 Hz, 2H), 2.96 (t, *J* = 6.0 Hz, 2H), 2.84–2.78 (m, 2H), 2.56 (s, 9H), 2.38–2.34 (m, 2H), 2.06 (quint, *J* = 7.2 Hz, 2H), and 1.85 (quint, *J* = 6.8 Hz, 2H). 13C{1H} NMR (125 MHz, CD3OD, 298 K): δ 188.3, 164.9, 164.6, 164.2, 151.9, 147.1, 146.6, 143.9, 136.6, 135.0, 134.8, 134.7, 133.0, 130.1, 130.0, 130.0, 126.4, 115.7, 106.9, 102.3, 63.3, 57.2, 56.7, 55.1, 44.6, 44.5, 42.3, 42.3, 42.2, 26.2, and 25.5. HR-ESI-MS (+ve) calculated for C34H42N10O3 [M+H]+ (*m*/*z*): 639.3514, found: 639.3504.

Synthesis of **DB-Dan** was carried out as follows. Under an argon atmosphere, a mixture of **DB-N3** (43 mg, 0.11 mmol), **Dan-alkyne** (35 mg, 0.12 mmol), and [Cu(CH3CN)4](PF6) (4 mg, 0.01 mmol) in MeCN/MeOH (2 mL/0.5 mL, *v*/*v* = 8:2) was stirred at room temperature for overnight. Solvents were removed using a rotary evaporator, and the residue was purified by a basic alumina column using a gradient mixture of CH2Cl2/MeOH (*v*/*v* = 9:1 to 8:2) as the eluent. Fractions containing the product were combined and concentrated using a rotary evaporator, to which a saturated HCl solution in dry ethanol (1 mL) was added. The mixture was heated at 60 ◦C for 3 h, cooled to room temperature, and centrifuged (4000 rpm, 3 min). The solution was decanted and the remaining solid was washed with dry ethanol (10 mL × 1) and dry diethyl ether (10 mL × 3). The resulting solid was dried under vacuum to obtain the product as a pale-yellow solid. Yield = 20 mg, 21%. 1H NMR (400 MHz, CD3OD, 298 K): δ 8.52 (d, *J* = 8.4 Hz, 1H), 8.32 (d, *J* = 8.7 Hz, 1H), 8.15 (d, *J* = 7.2 Hz, 1H), 7.71 (s, 4H), 7.60–7.48 (m, 3H), 7.25 (d, *J* = 7.5 Hz, 1H), 4.31 (t, *J* = 6.1 Hz, 2H), 4.11 (s, 2H), 3.33 (t, *J* = 7.1 Hz, 2H), 3.18 (t, *J* = 6.7 Hz, 2H), 2.87 (s, 6H), 2.74 (t, *J* = 6.2 Hz, 2H), 2.54–2.38 (m, 4H), 2.27 (s, 6H), 2.25 (s, 3H), 1.87 (quint, *J* = 7.2 Hz, 2H), and 1.75 (quint, *J* = 6.9 Hz, 2H). 13C{1H} NMR (125 MHz, CD3OD, 298 K): δ 163.8, 157.3, 153.1, 146.0, 139.0, 138.6, 137.2, 131.1, 131.0, 130.9, 130.2, 129.1, 128.5, 128.4, 124.8, 124.3, 120.6, 116.3, 58.2, 57.6, 56.0, 45.8, 45.4, 42.9, 42.5, 42.2, 39.2, 27.6, and 27.3. HR-ESI-MS (+ve) calculated for C34H49N11O2S [M+H]+ (*m*/*z*): 676.3864, found: 676.3870.

Synthesis of **DB-TPE** was carried out as follows. Under an argon atmosphere, a mixture of **DB-alkyne** (18 mg, 0.05 mmol), **TPE-N3** (24 mg, 0.06 mmol), and [Cu(CH3CN)4](PF6) (2 mg, 0.005 mmol) in MeCN/MeOH/CH2Cl2 (2 mL/1 mL/1 mL, *v*/*v* = 2:1:1) was stirred at room temperature for overnight. The solvents were removed using a rotary evaporator, and the residue was washed with dry diethyl ether (10 mL × 2). A saturated HCl solution in dry ethanol (1 mL) was added to the residue, and the mixture was heated at 60 ◦C for 3 h, cooled to room temperature, and centrifuged (4000 rpm, 3 min). The solution was decanted and the remaining solid was washed with dry ethanol (10 mL × 1) and dry diethyl ether (10 mL × 3). The resulting solid was dried under vacuum to a obtain the product as a yellow solid. Yield = 42 mg, 93%. 1H NMR (400 MHz, CD3OD, 298 K): δ 8.39 (s, 1H), 8.03 (s, 4H), 7.16–7.01 (m, 12H), 7.01–6.88 (m, 7H), 5.58 (s, 2H), 4.59–4.37 (m, 2H), 3.72–3.55 (m, 2H), 3.53–3.39 (m, 4H), 2.94 (s, 6H), 2.90 (s, 3H), 2.39–2.28 (m, 2H), and 2.28–2.18 (m, 2H). 13C{1H} NMR (125 MHz, CD3OD, 298 K): δ 165.1, 144.8, 144.7, 143.1, 141.5, 134.8, 132.9, 132.3, 132.2, 130.3, 128.9, 128.8, 128.7, 127.7, 127.7, 56.5, 43.9, 41.7, and 24.2. HR-ESI-MS (+ve) calculated for C47H53N9 [M+H]<sup>+</sup> (*m*/*z*): 744.4497, found: 744.4494.

Fluorescence spectra were recorded on an Edinburgh Instruments FS5 Spectrofluorometer equipped with a 150 W CW ozone-free xenon arc lamp and a Photomultiplier R928P detection unit with spectral coverage of 200–870 nm. Fluorescence studies were performed in quartz cuvettes of 1 cm path length and 1.5 mL cell volume, and double

distilled water was used in all measurement. Stock solutions of **DB-Res**, **DB-Dan**, and **DB-TPE** in water were diluted to 10 μM with H2O and/or THF to the required THF/H2O ratios. **DB-Res**, **DB-Dan**, and **DB-TPE** were excited at 475 nm, 330 nm, and 300 nm, respectively. Synthetic RNAs, *rGG-(CAG)6-CC* (*5 -rGG CAG CAG CAG CAG CAG CAG CC-3* ), *rGG-(CAG)3-CC* (*5 -rGG CAG CAG CAG CC-3* ), and *rGG-(CUG)3-CC* (*5 -rGG CUG CUG CUG CC-3* ) were purchased from Integrated DNA Technologies, and stock solutions at 100 μM were prepared by dissolving the RNA in UltraPure™ DNase/RNase-free distilled water. For measurements in the presence of RNA, RNA sample were diluted with water, heated at 95 ◦C for 2 min, and slowly cooled to room temperature and added to a sample solution containing 10 μM of the fluorescent probe in 20 mM MOPS buffer (pH 7.0) in the presence of 300 mM NaCl.

Dissociation constant (*Kd*) of **DB-TPE** towards *rGG-(CAG)6-CC* was determined by non-linear curve fitting of the fluorescence data using the following equation:

$$F\_R = F\_0 + \frac{F\_{MAX} - F\_0}{2[P]\_0} \times \left[ \left( [P]\_0 + [R] + K\_d \right) - \sqrt{\left( [P]\_0 + [R] + K\_d \right)^2 - 4[P]\_0[R]} \right]^2$$

where *FR* is the fluorescence intensity at 463 nm for a given concentration of RNA [*R*], *F*<sup>0</sup> is the fluorescence intensity in the absence of RNA, *FMAX* is the maximum fluorescence intensity at saturation, [*P*]<sup>0</sup> is the total concentration of **DB-TPE** (10 μM), and [*R*] is the total concentration of RNA.

#### **3. Results and Discussions**

Three bis(amidinium) derivatives have been designed and synthesized as potential fluorescent probes for CAG RNA based on the structure of **DB213** (Figure 2). In particular, three different fluorophores, namely resorufin, dansyl, and tetraphenylethylene (TPE), were chosen as the fluorescent reporters that are sensitive to the local environment for obtaining a fluorescence response upon RNA binding. The probes were synthesized via a coppercatalyzed azide–alkyne cycloaddition between **DB-N3** or **DB-alkyne** and the corresponding fluorophore-containing click partners. Of note, compared to oligonucleotide-based probes that can engage in sequence-specific hybridization with the target DNA or RNA [22,23], small molecule probes are more versatile with diverse structure, and their photophysical, chemical, and biological properties can be readily tuned via chemical modifications [24,25]. However, due to the highly diverse and dynamic structures of RNA, small molecular probes that can selectively recognize a specific RNA sequence are scarce [16–18]. In addition to RNA binding, an appropriate mechanism to induce a change in the fluorescent properties upon binding is also essential for a successful RNA probe. While the resorufin and dansyl fluorophores are known to be sensitive to the local environment and could elicit a change in their emission due to a different degree of the intramolecular charge transfer (ICT) [26–29], TPE is a representative AIEgen that the restricted intramolecular motions upon binding will block the non-radiative decay pathway and result in an emission turn-on [30].

All the three probes have been characterized by 1H NMR, 13C NMR, HR-ESI-MS, and UV–Vis. Fluorescent properties of the probes in response to environment changes were evaluated to assess their applicability in fluorescent RNA sensing. Figure 3 shows the emission spectra of 10 μM solutions of **DB-Res**, **DB-Dan**, and **DB-TPE** in water/THF mixtures of various ratios and ionic strengths (from 3 mM to 3 M NaCl). For **DB-Res**, weaker fluorescence was observed when there is an increase in the percentage of THF, and a 7.5-fold decrease in the emission intensity at 574 nm was found at 90% THF when compared to that at pure water. The weaker emission can be explained by the formation of non-emissive aggregates of the probe when the solvent becomes more non-polar. Consistent with this, weaker fluorescence was also observed when solvent ionic strength increased, and there was a 3.2-fold decrease in fluorescence in the presence of 3 M NaCl. On the other hand, an increase in fluorescence, along with a blue shift of the emission maximum, was observed for **DB-Dan** upon increasing the THF percentage. At 90% THF, a 6.8-fold emission enhancement and a blue shift of the emission maximum by 41 nm (from 562 nm

to 521 nm) was resulted, and these solvatochromic and fluorogenic effects are consistent with an ICT mechanism [31,32]. No significant change in the emission maximum and intensity was found for **DB-Dan** at different ionic strengths. Consistent with the AIE effect of TPE, a 10 μM solution of the aqueous soluble, cationic **DB-TPE** in water was weakly emissive and can be ascribed to the rapid non-radiative decay due to intramolecular rotations of the phenyl units [33,34]. Upon increasing the THF content to 90%, a strong emission enhancement by 85-fold and a blue shift in the emission maximum by 42 nm were observed. The decrease in solubility of **DB-TPE** upon addition of THF likely resulted in the aggregation of the probe and restricted the intramolecular rotations that activated the TPE emission. Compared to the emission intensity in the presence of 3 mM NaCl, a 24-fold increase in fluorescence was observed in the presence of 3 M NaCl, showing that the salting-out effect at high ionic strength can also trigger the AIE.

**Figure 2.** Synthesis of **DB-Res**, **DB-Dan**, and **DB-TPE**.

Fluorescence response of the probes was further tested using a synthetic RNA with the sequence *rGG-(CAG)6-CC* RNA as a model of CAG repeats. Previous studies have shown that this particular sCAG RNA will form a hairpin structure in solution with which **DB213** can selectively bind with a strong affinity (*K*<sup>d</sup> = 3.8 μM) [15,35]. Fluorescence response to the RNA was studied with 10 μM of the probe in the presence of 300 mM NaCl in 20 mM MOPS buffer at pH 7. As shown in Figure 3, only a minimal change in the emission was observed in the presence of 1 eq. of the (CAG)6 RNA for **DB-Res** and **DB-Dan**, suggesting that the change in the local environment upon binding may not be significant enough to result in an obvious fluorescent response. On the contrary, a 19-fold emission enhancement of **DB-TPE** was observed under the same condition, suggesting that the AIE probe has a promising analytical potential for CAG RNA.

Response of **DB-TPE** towards the (CAG)6 RNA was studied in more details. Figure 4 shows the concentration dependent response of the probe, and the relative emission intensity at 463 nm was found to increase from 3.5-fold to 18.6-fold when the amount of (CAG)6 RNA increased from 0.05 eq. to 1 eq. Binding strength of **DB-TPE** to the (CAG)6 RNA was obtained from the fluorescence titration data, and a *K*<sup>d</sup> of 2 μM was found by fitting the binding isotherm to a 1:1 binding model. The *K*<sup>d</sup> of **DB-TPE** is comparable to that of the binding of **DB213** to the same (CAG)6 RNA, suggesting that both bis(amidinium) compounds share a similar binding mode that targets the major groove and overall secondary structure of the sCAG RNA with A·A mismatches in the hairpin structure. Consistent with this, a much weaker fluorescence, likely due to the non-specific electrostatic interactions between the cationic bis(amidinium) and anionic nucleic acid, was observed when the sCAG

RNA was denatured by urea (Figure 4b), or in the presence of 1 eq. of the canonically-paired duplex consisting of *rGG-(CAG)3-CC* and *rGG-(CUG)3-CC* with complementary A·U pairs (Figure 4c). Overall, these results clearly demonstrate the favorable analytical performance of **DB-TPE** as a turn-on fluorescent probe for the selective sensing of RNA with CAG repeats that contain A·A mismatches.

**Figure 3.** Fluorescence spectra of 10 μM (**a**) **DB-Res** (λex = 475 nm), (**b**) **DB-Dan** (λex = 330 nm), and (**c**) **DB-TPE** (λex = 300 nm) in different water/THF ratios (upper panel), ionic strengths with various NaCl concentrations (middle panel), and the presence of 1 eq. of a (CAG)6 RNA (lower panel).

**Figure 4.** Fluorescence spectra of 10 μM **DB-TPE** (λex = 300 nm) in the presence of (**a**) increasing amount of (CAG)6 RNA from 0.05 to 1 eq., (**b**) 1 eq. of (CAG)6 RNA denatured by 1 M urea, and (**c**) 1 eq. of complementary (CAG)3/(CUG)3 RNA duplex.

#### **4. Conclusions**

In summary, a fluorescent turn-on probe that selectively binds to sCAG RNA with a strong fluorescence enhancement is successfully developed. Despite the various challenges in specific RNA recognition due to their dynamic structures, by using tetraphenylethylene as an AIE-active fluorophore to decorate **DB213**, a known small molecule inhibitor that selectively binds to the major groove of CAG RNA with A·A mismatches, **DB-TPE** was obtained and found to display a strong (~19-fold) fluorescence enhancement upon binding to sCAG RNA with an apparent *K*<sup>d</sup> of 2 μM. Comparative fluorescence studies showed that **DB-TPE** binds and reports the presence of sCAG RNA with a specific secondary structure. With this promising analytical performance, **DB-TPE** not only could be employed in the sensitive detection of CAG RNA for diagnostic applications of CAG repeats-related neurodegenerative diseases, but may also allow for the real-time monitoring of the CAG RNA for further understanding of the disease in cell models. Further works on applying the probe in biological samples and specimens are currently underway in our laboratory.

**Supplementary Materials:** The following supporting information can be downloaded at https://www. mdpi.com/article/10.3390/bios12121080/s1, File S1: synthetic procedures of precursors, NMR, MS and UV-Vis spectra. References [36,37] are citied in the Supplementary Materials.

**Author Contributions:** Conceptualization, M.H.Y.L., C.-H.W., H.Y.E.C. and H.Y.A.-Y.; methodology, M.H.Y.L. and H.Y.A.-Y.; investigation, M.H.Y.L. and H.Y.A.-Y.; resources, C.-H.W., H.Y.E.C. and H.Y.A.-Y.; writing—original draft preparation, M.H.Y.L. and H.Y.A.-Y.; writing—review and editing, M.H.Y.L., C.-H.W., H.Y.E.C. and H.Y.A.-Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the CAS-Croucher Funding Scheme for Joint Laboratories and a Collaborative Research Grant (C7075-21G). M.H.Y.L. acknowledges the support of the Hong Kong Postgraduate Fellowship.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in supplementary material.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


**Koena L. Moabelo 1,2, Teresa M. Lerga 3, Miriam Jauset-Rubio 3, Nicole R. S. Sibuyi 2, Ciara K. O'Sullivan 3, Mervin Meyer <sup>2</sup> and Abram M. Madiehe 1,2,\***


**Abstract:** Retinol-binding protein 4 (RBP4) has been implicated in insulin resistance in rodents and humans with obesity and T2DM, making it a potential biomarker for the early diagnosis of T2DM. However, diagnostic tools for low-level detection of RBP4 are still lagging behind. Therefore, there is an urgent need for the development of T2DM diagnostics that are rapid, cost-effective and that can be used at the point-of-care (POC). Recently, nano-enabled biosensors integrating highly selective optical detection techniques and specificity of aptamers have been widely developed for the rapid detection of various targets. This study reports on the development of a rapid gold nanoparticles (AuNPs)-based aptasensor for the detection of RBP4. The retinol-binding protein aptamer (RBP-A) is adsorbed on the surface of the AuNPs through van der Waals and hydrophobic interactions, stabilizing the AuNPs against sodium chloride (NaCl)-induced aggregation. Upon the addition of RBP4, the RBP-A binds to RBP4 and detaches from the surface of the AuNPs, leaving the AuNPs unprotected. Addition of NaCl causes aggregation of AuNPs, leading to a visible colour change of the AuNPs solution from ruby red to purple/blue. The test result was available within 5 min and the assay had a limit of detection of 90.76 ± 2.81 nM. This study demonstrates the successful development of a simple yet effective, specific, and colorimetric rapid assay for RBP4 detection.

**Keywords:** retinol-binding protein 4; type 2 diabetes mellitus; aptasensor; colorimetric; gold nanoparticles; diagnosis; biosensing

#### **1. Introduction**

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease with debilitating effects on human health. It is a growing epidemic, that affects over 536.6 million adults worldwide [1]. If undiagnosed or untreated, T2DM can lead to microvascular (retinopathy, neuropathy and nephropathy) and macrovascular (stroke and acute coronary syndrome) complications [2]. Many of these complications can go un-noticed for years and are often subclinical at the onset, making the early diagnosis of T2DM difficult [3]. Therefore, an urgent need to develop novel and early diagnostic strategies to address this epidemic is warranted.

Retinol-binding protein 4 (RBP4), an adipokine responsible for obesity-induced insulin resistance, has been identified as one of the reliable biomarkers for the early diagnosis of T2DM [4]. RBP4 binds to retinol and transports it in the blood stream to the liver. Elevated levels of RBP4 have been found in insulin-resistant mice and humans with obesity and T2DM, causing dysfunctions in the production of glucose transporter 4 (GLUT4) and consequently leading to a failure of glucose uptake from the blood. This suggests that RBP4 could serve as a potential serological biomarker for the early diagnosis of T2DM [5–9].

**Citation:** Moabelo, K.L.; Lerga, T.M.; Jauset-Rubio, M.; Sibuyi, N.R.S.; O'Sullivan, C.K.; Meyer, M.; Madiehe, A.M. A Label-Free Gold Nanoparticles-Based Optical Aptasensor for the Detection of Retinol Binding Protein 4. *Biosensors* **2022**, *12*, 1061. https://doi.org/ 10.3390/bios12121061

Received: 3 October 2022 Accepted: 16 November 2022 Published: 22 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The detection of RBP4 is usually performed through different biotechnological assays, such as enzyme-linked immunosorbent assay (ELISA) and Western blot [10–12], which rely on the interaction between an antibody and an analyte. However, the use of antibodies in diagnostic assays have several inherent limitations such as high production costs, instability, and poor specificity, and their use requires longer incubation periods and different substrates for detection [13]. Recently, the use of aptamers in diagnostic assays have been shown to alleviate these limitations. Aptamers, which are single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules that bind with high affinity and specificity to their target molecules, exhibit significant advantages relative to antibodies in terms of their high specificity, selectivity, low molecular weight, and ease of production [13–15]. Furthermore, aptamers present a more favourable and desirable molecular recognition element (MRE) and have been employed in the development of various sensors for diagnostic purposes [16–20].

Lee et al. reported on the development of the first RBP-aptamer (RBP-A) and its application in a surface plasmon resonance (SPR) biosensor for the detection of RBP4 in serum. The SPR biosensor specifically detected RBP4 and had a limit of detection of 75 nM (1.58 μg/mL), which is sufficiently sensitive to probe for RBP4 in the serum of people who are at risk of developing T2DM [21]. For instance, it has been reported that the detection range of RBP4 in normal individuals is 23.0–24.3 μg/mL (1–1.06 μM) and ranges from 24.9–50.3 μg/mL (1.08–2.187 μM) in individuals who are diagnosed with T2DM [22]. Using the same aptamer, an enzyme-linked antibody-aptamer sandwich (ELAAS) method was developed for more convenient and sensitive detection of RBP4. Compared to the SPR-based biosensor, this method was between 20 and 68 times more sensitive [23]; however, the assay involved multiple incubation and washing steps, making it impossible for it to be applied in point-of-care testing (PoCT). Torabi et al. developed an ultrasensitive chemiluminescent aptasensor for the detection of RBP4 with an LOD of 951 fg/mL [24]. Similarly to the ELAAS, this assay involved multiple washing and incubation steps, rendering it unsuitable for use in PoCT [22].

Colorimetric assays have been extensively used for the rapid detection of various diseases due to their simplicity, quick response, and high sensitivity [25–31]. Nanomaterials, especially gold nanoparticles (AuNPs), exhibit strong localized surface plasmon resonance (LSPR) properties which have been widely leveraged for the fabrication of colorimetric sensors [32]. AuNPs give off a colourful signal that can be visualized with the naked eye and without using advanced instruments [33]. Generally, a solution of colloidal AuNPs have a ruby-red colour due to their LSPR phenomenon that is highly dependent on interparticle distance [32,34,35]. Upon addition of salt, the colour of the AuNP solution changes from ruby red to purple/blue due to the shift of the LSPR to a higher wavelength. In contrast, when the AuNPs are bound to molecules, such as MRE for example, they will retain their ruby-red colour in the presence of salt. If the analyte for the specific MRE is present, the MRE binds to the analyte instead of the AuNPs and the colour of the AuNPs will change from ruby red to purple/blue. Based on this principle, several AuNP-based optical aptasensors have been developed [36–40]. In this study, using this principle, a simple yet effective, specific, and rapid RBP-A-aptasensor for the detection of RBP4 was developed. The assay is user-friendly as it does not require any specialised instruments and it is capable of producing a test result within 5 min, highlighting the potential applicability of this assay in PoCT and in resource-limited areas.
