Label-free Aptasensor for the Ultrasensitive Detection of Insulin Via a Synergistic Fluorescent Turn-on Strategy Based on G-quadruplex and AIEgens

Insulin, the only hormone regulating blood glucose level, is strongly associated with diabetes and its complications. Specific recognition and ultrasensitive detection of insulin are of clinical significance for the early diagnosis and treatment of diabetes. Inspired by aggregation-induced emission, we presented a turn-on label-free fluorescence aptasensor for insulin detection. Quaternized tetraphenylethene salt was synthesized as the fluorescence probe. Guanine-rich aptamer IGA3 was selected as recognition element. Graphene oxide was chosen as the quencher. Under optimized conditions, the fluorescence aptasensor displayed a wide linear range (1.0 pM–1.0 μM) with a low limit of detection (0.42 pM). Furthermore, the aptasensor was successfully applied to detect insulin in human serum. Spiked recoveries were obtained in the range of 96.06%–104.26%. All these results demonstrated that the proposed approach has potential application in the clinical diagnostics of diabetes.


Introduction
Diabetes is a common metabolic disease that becomes a serious threat to human health and generally leads to a series of complications, such as diabetic ketoacidosis, cardiovascular disease, diabetic nephropathy, peripheral neuropathy, and retinopathy [1]. Diabetes results from insulin deficiency or resistance in the human body. Insulin is the only hormone reducing the level of blood glucose, a protein hormone produced by pancreatic β cells upon stimulation by exogenous and endogenous substances, such as glucose and glucagon. The level of insulin in the body is an important index in the diagnosis of diabetes [2]. Monitoring the level of insulin might provide valuable evidence for evaluating pancreatic beta cell function and for the early diagnosis, timely treatment, and disease mechanism study of diabetes complications. Thereby, an accurate, sensitive, and convenient method for insulin detection must be developed to provide some indication of the status of diabetes and improve the treatment technology.
To date, various methods have been developed for insulin detection, including immunoassays [3], chromatography [4], surface-enhanced Raman scattering [5], electrochemistry [6], and fluorescence [7]. Moreover, many studies have been conducted on insulin detection [8][9][10][11][12]. In recent years, a new fluorescent strategy, fluorescence aptasensor based on aptamer and fluorophore, has received attention [13]. It combines the excellent selectivity of aptamer and the high sensitivity of fluorescence. As a recognition element, aptamer can specifically recognize the target molecules through van der Waals, hydrogen bonding, or electrostatic interaction, forming a stable secondary or tertiary structure, including hairpin, stem loop, pseudoknot, and G-quadruplex [14]. In addition to the recognition unit, the critical moiety of fluorescence aptasensor is fluorophore, which can convert the binding information between aptamer and the targets into a detectable and quantifiable fluorescent signal.
In the fabrication of fluorescent aptasensor, aptamer can be linked with fluorophore via two strategies: 1 3 fluorophore-labeled or label-free [15,16]. Compared with the complicated fluorescence-labeled procedure through the covalent bond modification of aptamer with fluorophore, the label-free strategy via non-bonding interaction between aptamer and fluorophore is simpler, more rapid, and more cost-effective. Therefore, the label-free fluorescent aptasensor is highly desirable for biosensing applications. Various fluorophores with advantages of water solubility and environmental friendliness, including aggregation-induced emission (AIE) luminogens (AIEgens) [17], transition metal complex [18], and rare earth Europium complex [19], have been utilized in label-free aptasensor construction. Among them, AIEgens, which were first reported by Tang's group [20], have received more attention. These kinds of fluorophores show no or low fluorescence in dilute solution but display intense fluorescence in aggregated state owing to the restriction of intramolecular motion (RIM) [21]. The concept of AIE opens up a new approach for the construction of label-free fluorescent aptasensor and has been successfully applied in the detection of metals [22], organic molecules [23], and biomolecules [24]. Among the reported AIEgens, tetraphenylethene (TPE) derivatives have got considerable attentions owing to their high fluorescence quantum yield, low toxicity, and high photostability. In addition, TPE derivatives have the advantages of facile synthesis and easy functionalization. Importantly, their excellent AIE properties could significantly improve the sensitivity. TPE derivatives have been widely applied in biosensors and chemical sensors [25].
Inspired by the above works, we introduced an AIE-based fluorescence aptasensor for insulin detection. In which, AIE molecule named quaternized tetraphenylethene salt (TPE-Z) was synthesized as the fluorescence probe. Guanine-rich aptamer IGA3 was selected as the recognition unit [26]. Graphene oxide (GO) was chosen as the fluorescence quencher with a strong absorption capacity for aptamer and a powerful ability to quench fluorescent materials [27]. In the presence of insulin, the fluorescence of this aptasensor changed from "off" to "on." The fluorescence recovery was proportional to the insulin concentration, which served as the basis for sensitive insulin sensing. Moreover, this fluorescence aptasensor exhibited high selectivity and sensitivity toward insulin in human serum.
All fluorescence spectra were recorded on a F-7000 spectrofluorometer (Hitachi, Japan). Nuclear magnetic resonance spectra (NMR) were recorded on a 400 MHz NMR spectrometer (Varian, USA). Circular Dichroism (CD) spectra were measured on a 420SF CD spectrometer (Aviv, USA). Electrophoretic gel images were obtained by a Universal Hood II gel imager (Bio-Rad, USA).

Synthesis of TPE-Z
TPE-Z was synthesized following previous methods with some modifications [28,29]. TPE-MB (129.6 mg, 0.25 mmol) and anhydrous acetonitrile (20 mL) were added into a 100 mL round-bottom flask. After ultrasonic dispersion, the mixture was added with trimethylamine (15 mL), refluxed for 24 h, and naturally cooled to room temperature. The precipitate was filtrated and washed with anhydrous ether. A pale yellow solid was obtained in 60% yield (180 mg). 1

Optimization of the Concentrations of TPE-Z and GO
The optimum concentration of TPE-Z was evaluated. The IGA3 concentration was set at 0.2 μM. Different TPE-Z concentrations ranging 5.0-60 μM were added into the IGA3 solution. Upon mixing, the fluorescence spectra were measured. For GO concentration optimization, the IGA3 solution (0.2 μM) was mixed with a series of GO concentrations at 3.0, 6.0, 9.0, 12.0, and 15.0 μg/mL. The mixture was incubated at 37 °C and slowly cooled to room temperature. TPE-Z was added to the mixtures to measure the fluorescence spectra.

Determination of Insulin
IGA3 (0.2 μM) and GO (9.0 μg/mL) were mixed in the Tris-HCl buffer solution (10 mM, pH = 7.4), and the mixture was incubated at 37 °C for 40 min. A series of concentrations of insulin ranging from 1.0 pM to 1.0 μM was added into the mixture and incubated at 37 °C for 60 min. Finally, TPE-Z (30 μM) was added into the mixture to measure the fluorescence spectra. The total volume of solution was set at 200 μL.

Selectivity Analysis
IGA3 (0.2 μM) and GO (9.0 μg/mL) were dispersed in the Tris-HCl buffer solution (10 mM, pH = 7.4) and incubated at 37 °C for 40 min. Interferents, i.e., BSA, HSA, AFP, CEA, and PSA were added to the mixture and incubated at 37 °C for 60 min, and the solution was slowly cooled to room temperature. Finally, TPE-Z (30 μM) were added to the mixtures to measure the fluorescence spectra.

Detection of Insulin in Human Serum
The human serum samples collected from the First Hospital of Jiaxing were diluted 100 times with Tris-HCl buffer solution (10 mM, pH = 7.4) and centrifuged in an ultrafilter tube (10 k, Millipore) for 10 min. The solutions in the outer tube were collected and stored at 4 °C for later use. IGA3 (0.2 μM) and GO (9.0 μg/mL) were added into the diluted human serum samples. The mixtures were incubated at 37 °C for 40 min. Then three different concentrations of insulin standard solution (10, 10 3 , 10 5 pM) were added into the mixture and incubated at 37 °C for 60 min. Finally, TPE-Z (30 μM) was added into the mixtures to measure the fluorescence spectra. The fluorescence measurements were consistent with the procedure of insulin determination.

Agarose Gel Electrophoresis
Agarose gel electrophoresis was carried out on 2% agarose gel prepared from 0.5 g of agarose, 6.0 mL 4 × TBE, 19.0 mL of water, and 2.5 μL of GelRed. The mixture containing 5.0 μL of sample and 1.0 μL 6 × loading buffer was injected into the gel wells and run at 120 V for 35 min. The image was obtained by a gel imaging system.

Design Strategy and Feasibility of the Fluorescence Aptasensor
The strategy of this fluorescence aptasensor for insulin detection was illustrated in Scheme 1. Fluorescence quenching and recovery were involved in the detection. A certain amount of GO was introduced into the IGA3 solution. Owing to the excellent IGA3 absorption ability of GO via hydrogen bonding and π-π stacking interaction, IGA3 was assembled effectively on the GO surface. After TPE-Z addition, the cationic probe TPE-Z formed a complex with the negatively charged IGA3 through strong electrostatic interaction, causing the efficient fluorescence quenching of TPE-Z by fluorescence resonance energy transfer (FRET) from TPE-Z to GO. In the presence of insulin, IGA3 recognized and specifically bound Scheme 1 Schematic of the procedure for detecting insulin with insulin. Consequently, the complex formed by IGA3 with insulin departed from the GO surface. Meanwhile, the fluorescence probe TPE-Z was still attached on IGA3, resulting in the fluorescence recovery of TPE-Z due to the AIE effect. Accordingly, the value of fluorescence recovery varied linearly with the concentration of insulin. Hence, a quantitative approach for the selective and sensitive determination of insulin was constructed.
The fluorescence spectra of TPE-Z, TPE-Z-IGA3, TPE-Z-IGA3@GO, and TPE-Z-IGA3@GO + insulin were investigated to evaluate the feasibility of the fluorescence aptasensor for insulin detection, and the results were displayed in Fig. 1a. The fluorescence of TPE-Z in solution was relatively weak. In the presence of IGA3, the complex formed by TPE-Z with IGA3 through electrostatic interaction increased the fluorescence of TPE-Z by approximately four times in the aggregated state compared with that in the dispersed state owing to RIM and TPE-Z nonplanar conformation. With the introduction of GO, the fluorescence of TPE-Z decreased significantly, indicating that GO has a powerful ability to quench the fluorescence of TPE-Z. When the analyte insulin was added, the specific binding between IGA3 with insulin reduced the interaction between IGA3 and GO. This binding pushed the TPE-Z-IGA3 complex away from the GO surface. Therefore, the fluorescence of TPE-Z was recovered. This variation of the fluorescence of TPE-Z in the absence and presence of insulin could be applied for insulin detection.
Agarose gel electrophoresis was also carried out to further validate the feasibility of the aptasensor for insulin detection. As shown in Fig. 1b, lane 2 (IGA3 + insulin) and lane 5 (TPE-Z-IGA3@GO + insulin) moved shorter distances than lane 1 (IGA3) and lane 4 (TPE-Z-IGA3@GO). These results further confirmed that the proposed aptasensor formed a complex with insulin and could be applied in the detection of insulin.

Optical Properties of TPE-Z
The fluorescence emission spectra varying with different excitation wavelengths were investigated. As shown in Fig. 2, the fluorescence emission intensity of TPE-Z increased first and then decreased with the increase in the excitation wavelength ranging 300-360 nm. The maximum excitation and emission wavelengths were located at 325 and 475 nm, respectively.

Optimization of Experimental Conditions
Several essential parameters were systematically investigated to obtain the optimized analytical performance of the aptasensor for insulin detection. First, the concentration of IGA3 was set at 0.2 μM, and the other experimental   Fig. 3a, when TPE-Z was added to the IGA3 solution, the fluorescence intensities were enhanced gradually with the increasing concentration of TPE-Z, reached the maximum at 30.0 μM, and then remained unchanged with further increase in TPE-Z concentration. Accordingly, 30.0 μM TPE-Z concentration was adopted. The quenching efficiency of GO concentration on the fluorescence intensity of TPE-Z was then studied. As displayed in Fig. 3b, ∆F increased first and then decreased with the increasing GO concentrations. The maximum ∆F was found under 9.0 μg/mL GO. The influences of incubation temperatures such as 4 °C, 25 °C, 37 °C, and 50 °C on ∆F were studied. As shown in Fig. 3c, the maximum ∆F was obtained at 37 °C. Hence, 37 °C was selected in the following experiments. Figure 3d shown the quenching time of GO toward the fluorescence of TPE-Z. The fluorescence intensities of TPE-Z decreased with time from 0 to 40 min and then remain basically unchanged with prolonged quenching. Therefore, 40 min was chosen as the optimum quenching time. Finally, the effect of incubation time on fluorescence intensity in the presence of insulin was investigated. As shown in Fig. 3e, the best incubation time was 60 min.

Sensitivity
Under optimized conditions, the fluorescence spectra of TPE-Z varying with the concentrations of insulin were investigated to evaluate the analytical performance of the aptasensor toward insulin. As displayed in Fig. 4a, the fluorescence intensities recovered gradually with the increasing insulin concentration from 1.0 pM to1.0 μM. The value of fluorescence recovery was dependent on the concentration of insulin and was ascribed to the specific binding between the aptamer and insulin. As shown in Fig. 4b, a broad linear relationship was established between ∆F value and the concentration of insulin. The linear regression equation was obtained as ∆F = 13.1832 lgC + 7.7169 with a correlation coefficient (R 2 ) of 0.998. The limit of detection (LOD) of insulin determined by this aptasensor was evaluated by the representation of 3σ/K, and the LOD was calculated to be 0.42 pM. These results indicated that this aptasensor exhibits excellent sensitivity for insulin, even at trace levels. Table 1 shows that compared with those in previous works, the proposed fluorescence aptasensor has the advantages of lower LOD and broader linear range. Moreover, this aptasensor does not require fluorescence labels and contains no heavy metals.
As a specific molecular recognition element, the sensitivity of an aptasensor depends on the nucleotide sequence. IGA3 is a guanine-rich DNA sequence that forms G-quadruplex conformation through Hoogsteen base-pairing when binding to the target insulin. Therefore, circular dichroism (CD) spectroscopy was performed to explore the change of the secondary structure of IGA3 in the absence and presence of insulin. The CD spectra of IGA3, TPE-Z-IGA3, TPE-Z-IGA3@GO, and TPE-Z-IGA3@GO + insulin were shown in Fig. 5. The CD spectrum of IGA3 displayed two positive peaks centered at 260 and 208 nm and a negative peak at 237 nm. IGA3 folded into a parallel G-quadruplex structure, and this finding agreed with literature [32]. Comparison of the CD spectra of TPE-Z-IGA3, TPE-Z-IGA3@GO and TPE-Z-IGA3@ GO + insulin with that of IGA3 showed the same minor changes in the positive peak at 260 nm and the negative peak at 237 nm. Meanwhile, a distinct blue-shift was observed in the positive peak at 208 nm. These CD spectra indicated that IGA3 approximately kept a parallel G-quadruplex structure before and after interacting with TPE-Z, being absorbed on the GO surface, and complexing with insulin. These results revealed that this fluorescence aptasensor shows good sensitivity toward insulin.

Selectivity
Selectivity is an essential parameter in appraising the property of a fluorescence aptasensor. Several potential interference substances, i.e., BSA, HSA, CEA, AFP, PSA, and the mixture of these interferents with insulin were applied to assess the selectivity of the aptasensor toward insulin under the same experimental procedures. The concentrations of these interferants were tenfold higher than that of insulin (1.0 nM). As shown in Fig. 6, only a considerable fluorescence recovery was observed upon the addition of insulin, and for the interferents, a slight fluorescence recovery was obtained. The fluorescence recovery value of insulin increased by a factor of 12.0 in comparison with that of the potential interferents. Furthermore, the recovery value in the presence of the mixtures was comparable with that in the presence of insulin alone. The contributions of the interferents to the fluorescence recovery value were almost negligible. Therefore, the aptasensor displays excellent selectivity toward insulin and has potential application in detecting insulin from real samples.

Detection of Insulin in Human Serum
Under the optimized experimental conditions, the aptasensor was applied to detect insulin in human serum through spiked experiments to determine the feasibility of our method for practical samples. For this propose, three human serum samples collected from the First Hospital of Jiaxing were diluted 100 times with Tris-HCl buffer solution (10 mM, pH = 7.4). Insulin was not detectable in the diluted serum samples. Then, a certain amount of standard insulin solution (10, 10 3 , and 10 5 pM) were added into the diluted serum samples.

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The recovery was calculated as the measured concentration in the spiked serum sample divided by the spike concentration and then multiplied by 100%. As listed in Table 2, the detected values were approximately close to the spiked concentrations. The average spiked recoveries were in the range of 96.06%-104.26%, and relative standard derivations were between 2.03% and 5.24%. The results shown that this aptasensor exhibited good recovery and reproducibility, which indicated that the aptasensor has potential application for insulin detection in serum samples.

Conclusions
We presented a facile, ultrasensitive, and label-free fluorescence aptasensor for the detection of insulin. The aptasensor was combined with IGA3, TPE-Z, and GO, each of which played a role in recognition element, fluorescence signal, and quenching platform, respectively. The conditions were optimized as follows: TPE-Z concentration of 30 μM, GO concentration of 30 μM, incubation temperature of 37 °C, quenching time of 40 min, and incubation time of 60 min. CD spectra showed that IGA3 maintained a G-quadruplex structure during fluorescence aptasensor fabrication and detection. The values of fluorescence recovery exhibited a good linear relationship with the insulin concentration ranging from 1.0 pM to 1.0 μM with a low LOD of 0.42 pM. Moreover, the fluorescence aptasensor was successfully applied to detect insulin in human serum with good recoveries between 96.06% and 104.26% and RSD of less than 5.3%. Therefore, the proposed assay has potential application in the clinical diagnosis of diabetes.