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Development of a novel fluorescently-labeled aptamer structureswitching assay for sensitive and rapid detection of gliotoxin Shunxiang Gao, Xin Zheng, Yuan Tang, Yajun Cheng, Xiaobo Hu, and Jihong Wu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05094 • Publication Date (Web): 20 Dec 2018 Downloaded from http://pubs.acs.org on December 23, 2018

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Analytical Chemistry

Development of a novel fluorescently-labeled aptamer structure-switching assay for sensitive and rapid detection of gliotoxin Shunxiang Gao,†,±,‡,§ Xin Zheng,∇,§ Yuan Tang,≠,§ Yajun Cheng,*,≡ Xiaobo Hu,*,∇ and Jihong Wu*,†,±,‡ †Eye

Institute, Eye and ENT Hospital, College of Medicine, Fudan University,

Shanghai, China ±Shanghai

Key Laboratory of Visual Impairment and Restoration, Science and

Technology Commission of Shanghai Municipality, Shanghai, China ‡NHC

Key Laboratory of Myopia (Fudan University), Key Laboratory of Myopia,

Chinese Academy of Medical Sciences, Shanghai, China ∇Department

of Clinical Laboratory, Longhua Hospital, Shanghai University of

Traditional Chinese Medicine, Shanghai, China ≠Department

of Gastrointestinal Surgery, Changzheng Hospital, Second Military

Medical University, Shanghai, China ≡Department

of Orthopedics, Changhai Hospital, Second Military Medical

University, Shanghai, China

§These

authors contributed equally to this work.

*Corresponding Authors: Jihong Wu ([email protected]), Xiaobo Hu ([email protected]) and Yajun Cheng ([email protected]).

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ABSTRACT Gliotoxin—one of the most toxic metabolites produced during the growth of Aspergillus fumigatus—can cause direct damage to the immune system, and result in infection and spread of Aspergillus, or even lead to invasive aspergillosis. Accurate, rapid and sensitive detection of the disease-specific marker gliotoxin—particularly in serum, urine, or other body fluids—is therefore an important approach to achieving early and rapid diagnosis of Invasive Aspergillus fumigatus infection (IAFI). In this study, aptamers that specifically bind to gliotoxin were successfully obtained using immobilization-free GO-SELEX technology. Furthermore, the performance of the aptamer—including

binding

affinity,

targeting

specificity,

and

structural

stability—was further improved by optimizing through truncation and mutation. Finally, the optimized aptamer APT8T1M was used to develop a novel fluorescently-labeled aptamer structure-switching assay (FLASSA) for the detection of gliotoxin. The method exhibited a good linear range from 0.1 nM to 100 nM of gliotoxin, with a lower detection limit of 0.05 nM. Moreover, FLASSA was applied to the detection of gliotoxin in spiked serum and urine samples. A good mean recovery of 98.76–110.85% and a low coefficient of variation (5.45–14.59%) were obtained indicating a high degree of selectivity for gliotoxin, good reproducibility, and stability. These results show that the developed FLASSA has significant potential and offers an alternative to the traditional analytical methods for the rapid, sensitive, and efficient detection of gliotoxin, thus providing an effective tool for the early and rapid diagnosis of IAFI. 2 / 25


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Aspergillus fumigatus is one of the most serious conditional pathogens, which can cause severe deep Aspergillus infection in patients with low immunity, and even lead to invasive aspergillosis.1,2 In recent years, with the rise of organ transplantation; the increase in patients with malignant tumors; and the widespread use of broad-spectrum antibiotics and glucocorticoids; the infection rate of invasive Aspergillus fumigatus has significantly increased—accounting for approximately 15% of the population with low immunity—and the mortality rate can be 90% or higher.3,4 However, owing to lack of characteristic clinical symptoms, and the incidence of occult being difficult to distinguish, the survival of infected patients depends on early and rapid diagnosis and timely treatment.5 Currently, the commonly used methods for detecting Aspergillus fumigatus include traditional culture methods, histopathological examinations, imaging examinations, and serological assays.6-8 Traditional microbial culture and tissue biopsy are the gold standard for the diagnosis of invasive Aspergillus fumigatus infection (IAFI), however because of the time required, low sensitivity, and the invasivity of the tissue biopsy, this approach has been unable to meet the needs of rapid clinical diagnosis.9 Imaging methods such as CT or X-ray, which have certain value for the early diagnosis of IAFI, but poor specificity, are limited to joint application.10 Serological assays—particularly the galactomannan assay—that are mostly carried out by ELISA, have the advantages of rapidity and specificity, and have been used in Europe and the United States.11 However, owing to large fluctuations in sensitivity, numerous interference factors, and the sensitivity to Aspergillus fumigatus being lower than for other Aspergillus; further application of 3 / 25



the method is limited.12 Therefore, the development of sensitive and specific rapid diagnostic methods has been a particular focus and challenge in the field of IAFI. Gliotoxin is one of the most toxic metabolites produced during the growth of Aspergillus fumigatus and can cause direct damage to the body, infection and spread of Aspergillus, or even invasive aspergillosis by reducing immune function.13 In vitro culture experiments showed that gliotoxin could be detected at 24 h and the concentration reached a peak at 48–72 h.14 Gliotoxin was also measured in the lungs and serum of mice with experimentally induced invasive aspergillosis, and levels were found to decrease with antifungal therapy.15 Furthermore, gliotoxin was present in the serum of patients with invasive aspergillosis, which suggests that the mycotoxin can act as a novel diagnostic indicator for invasive aspergillosis.15-17 Therefore, accurate, rapid and sensitive detection of gliotoxin, particularly in serum, urine, or other body fluids, is an important method for achieving early diagnosis of IAFI. Among the existing methods for the detection of gliotoxin, a semi-quantitative bioassay based on the morphological changes in cultured cells was developed.18 This method had a detection limit of 18–20 ng and allowed for the rapid screening of samples for gliotoxin in extracts using an automated microplate-reader. Gliotoxin was also measured in human serum from patients suspected of having aspergillosis by HPLC-MS/MS.19 However, most of these techniques are time consuming, limited in sensitivity, and require expensive instrumentation and complex sample preparation procedures, which limits their application in early clinical rapid detection. Therefore, there is an urgent need to develop new analytical methods that can perform rapid, 4 / 25


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simple, cost-effective, and sensitive monitoring of gliotoxin. Aptamers are ssDNA or RNA with high affinity and specificity to targets, which are generated by an in vitro selection technique termed; systematic evolution of ligands by exponential enrichment (SELEX).20,21 Aptamers are promising in the development of various new assay methods owing to their facile synthesis and labeling, high stability, excellent reusability, and being suitable for a wide range of targets.22-26 However, so far there are no reports on the screening and identification of gliotoxin aptamers, possibly because the development of small molecule aptamers like gliotoxin presents significant challenges for traditional SELEX technology. Gliotoxin has a small molecular size, which leads to inefficient size-based separation, and the lack of chemical groups for immobilization makes carrier-based washing challenging. In this study, aptamers that specifically bind to gliotoxin were successfully obtained by using immobilization-free, graphene oxide (GO)-based SELEX (GO-SELEX). To further improve and regulate aptamer function, a truncation optimization strategy was introduced and a core aptamer sequence was obtained consisting of only 24 nucleotides. Furthermore, by introducing stabilized mini-hairpin structures at the 5′ stem-loop regions and 3′ ends of the aptamer APT8T1M, structural stability, binding affinity, and targeting specificity were significantly improved. Most importantly, the APT8T1M maintained its structure-switching characteristics under the induction of a target, which was further used to develop a fluorescently-labeled aptamer structure-switching assay for sensitive, rapid and specific detection of gliotoxin. This could provide an effective tool for early and rapid diagnosis of IAFI. 5 / 25



EXPERIMENTAL SECTION Materials, Reagents, and Methods All materials, reagents, and related experimental methods are described in details in the supporting information.

In vitro selection of the DNA aptamer The GO-SELEX was performed as described previously with some modifications (Figure S1),27 following the protocol detailed in Table S1. The specified amounts of ssDNA pool in binding buffer (5 mM MgCl2, 5% DMSO in Dulbecco’s PBS) were heated to 95 °C for 10 min, cooled in an ice bath for 5 min and kept at room temperature for 30 min to form the stable three-dimensional conformations, then incubated with gliotoxin (200 pmol). Subsequently, 500 μL of GO solution (1 mg/mL) was added to the mixture and the final total volume was made up to 1 mL with binding buffer. The mixture was incubated at room temperature with end-over-end rotation, and was subsequently centrifuged at 15,000 rpm for 15 min, three times. The collected supernatant was detected by Qubit® 2.0 Fluorometry and purified by Dr. GenTLE® Precipitation Carrier, while the GO and ssDNA that was adsorbed on the GO surface were discarded. The precipitated pools were then amplified by the PCR in 40 parallel 50 μL reactions, each containing GoTaq® Hot Start DNA Polymerase, Colorless GoTaq® reaction buffer, 2 mM MgCl2, 200 µM dNTP, and 0.5 µM forward and modified reverse primers (Table S2). PCR conditions were as follows: 94 °C for 1 min, followed by 20 cycles of 95 °C for 30 s, 60 °C for 6 / 25


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30 s, 72 °C for 30 s, and a final extension step of 2 min at 72 °C. The PCR products were separated by 12% urea denaturing PAGE, and the relevant ssDNAs were recovered by boiling the gel band. Finally, the recovered ssDNAs were purified using a QIAEX® II gel extraction kit and redissolved in binding buffer and used for the next selection round. To further improve the screening efficiency and specificity, a counter selection strategy was incorporated with the GO-SELEX after the fourth round. As before, the ssDNA pool was first subjected to the heating and cooling treatment, and then incubated with counter-targets at room temperature. The GO solution was then added to the mixture and incubated for a further 1 h. After the incubation, the mixture was centrifuged and washed three times with 1 mL of binding buffer, and ssDNA that was adsorbed on the GO surface was collected, while that bound to the counter-targets in the supernatant was discarded. The target molecule gliotoxin was then added to the GO solution, which was further incubated with end-over-end rotation. When the target began to collide with and bind to the aptamer adsorbed on the GO surface, the conformation of the corresponding aptamer also changed. The aptamers could be induced and folded into unique three-dimensional conformations that bind specifically to gliotoxin. This induction of the fit process in turn breaks the π–π stacking and hydrophobic interactions between the aptamers and GO, resulting in release of the aptamers from the GO surface. The aptamers in the supernatant can therefore be recovered, amplified, and used for the next selection round. After 8 selection rounds, the ssDNA pool selected was amplified by PCR using 7 / 25



the unmodified primers. The PCR product was separated by 8% PAGE, and the gel band containing the correct PCR product was recovered and purified. The purified PCR product then was cloned and sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). The obtained ssDNA sequences were aligned and analyzed using the Clustal X software. Their secondary structures were predicted by the mfold Web Server.

RESULTS AND DISCUSSION Selection and verification of gliotoxin aptamers Aptamers that bind to gliotoxin with high affinity and specificity were successfully screened using immobilization-free GO-SELEX technology. The GO-SELEX was performed as described previously with some modifications, illustrated in Figure S1,27 following the protocol detailed in Table S1. To improve the screening efficiency, a counter selection step was introduced into the GO-SELEX after the fourth round to eliminate non-specifically bound ssDNA. Furthermore, four different counter targets, including β-1,3-glucan, okadaic acid, ATP, and D-galacto-D-mannan were gradually added to the Counter-SELEX step to further remove the weakly interacting aptamers or ssDNAs that may be present in the screening library, and bind to the related counter targets with structures similar to gliotoxin. To effectively improve the success rate of GO-SELEX, a combined strategy of melting curve analysis and DNA recovery analysis was developed to monitor the enrichment of the specific ssDNA to gliotoxin during the SELEX rounds. As shown in Figure 1A, the melting peaks increased from the initial 70.15 °C to 73.09 °C, and 8 / 25


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tended to a stable state in the eighth round. Furthermore, the ssDNA recovery rate continued to increase after the introduction of Counter-SELEX and also reached a high point in the eighth round (Figure 1B). These results indicate that the ssDNA that specifically binds to gliotoxin in the eighth pool had been significantly enriched. Therefore, the selection cycles were stopped and the enriched ssDNA from the last round was amplified for cloning and sequencing.

Figure 1. Aptamer monitoring. (A) Melting curve analysis of the enriched aptamer pools using the QuantStudio™ Real-Time PCR System. (B) Enrichment level monitoring of the specific ssDNA to gliotoxin by Qubit® 2.0 Fluorometry. Multiple sequence alignments of the sequences were obtained using GO-SELEX by Clustal X software. These sequences were grouped into 3 families (Figure S2), and the representative sequences (APT8 and APT16) with the highest homology were chosen for further study of binding affinity and specificity to gliotoxin by biolayer interferometry (BLI). As shown in Figure 2A and 2C, gliotoxin at different concentrations (5, 2.5, 1.25, 0.63, and 0.31 μM) was analyzed for association time over 150 s and dissociation time over 150 s. In addition, BSA and counter-targets, 9 / 25



which have similar structures or detected frequently markers, were used to evaluate the interaction of the aptamers (Figure 2B and 2D). Although the aptamers APT8 and APT16 showed the same high binding affinity, APT8 has a superior specificity for gliotoxin, which is important for practical diagnostic and therapeutic applications. Therefore, a high affinity aptamer APT8 (KD = 376 nM) that specifically binds to gliotoxin was selected for further improvement and optimization.

Figure 2. Aptamer verification. (A) Binding affinity analysis of the aptamer APT8 by biolayer interferometry. (B) Binding specificity analysis of the aptamer APT8. The signal intensities were detected by biolayer interferometry and the markers were 10 / 25


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calculated by normalizing the gliotoxin signal to 100%. (C) Binding affinity analysis of the aptamer APT16. (D) Binding specificity analysis of the aptamer APT16.

Optimization and identification of the aptamer APT8 To further improve and regulate aptamer functions, a truncation optimization strategy was first introduced into the aptamer APT8 sequence. Based on the prediction of the mfold web server, it was found that APT8 contains three stem-loop structures (Figure 3). When stem-loop 1 was truncated, APT8T1 not only showed a stable secondary structure, but also had higher binding affinity (KD = 196 nM, Figure 4A). Therefore, stem-loops 2 and 3 were further truncated in anticipation of obtaining a more precise binding sequence for the aptamer (Figure S3). However, binding affinity experiments showed no binding signals for APT8T2 or APT8T3 (Figure 4A). This result indicated that stem-loops 2 and 3 in aptamer APT8T1 specifically bind to the gliotoxin by folding into a unique spatial structure. Furthermore, when stem-loop 1 was removed, the binding affinity of the aptamer APT8T1 to gliotoxin increased, which may be because stem-loop 1 folded into a rigid three-dimensional structure hindering the effective binding of the aptamer to the target. Therefore, a core aptamer sequence was obtained consisting of only 24 nucleotides and the aptamer APT8T1 had a higher targeting affinity. In addition to truncation optimization, we found that ssDNA aptamers can be stabilized by introducing an exceptionally stable mini-hairpin DNA sequence into the stem-loop regions of their secondary structures.28,29 Therefore, a rational site-directed 11 / 25



mutagenesis optimization strategy was also introduced and the aptamer APT8T1M with GCGNNAGC and GGGNACC sequences (N = A, G, C, or T) formed compact hairpin-like structures containing two G–C and GTCA or GGA loops (Figure 3). Encouragingly, the binding affinity of the APT8T1M aptamer was increased by more than 18-fold (KD = 10.5 nM, Figure 4B) and it showed higher targeting specificity (Figure 4C). To better understand the mechanism of this enhancement, the structures of the aptamers were measured by circular dichroism (CD). As shown in Figure 4D, compared with the CD spectrum of the aptamer APT8T1, the characteristic absorption peak of APT8T1M showed a blue-shift based on the positive peak at 272 nm and the negative peak at 243 nm, and the ellipticity at 272 nm was also increased. These results indicate that it is possible that the folding structure of the APT8T1M aptamer was slightly adjusted, resulting in further exposure of the binding site, thereby binding more tightly to the target and causing a significant increase in binding affinity. In addition, upon gliotoxin binding, further blue-shift of the CD spectrum was observed and the absorption intensity of the negative peak at 240 nm also increased (Figure 4E). This change in the CD spectrum indicates that gliotoxin can induce the conformational transformation of the APT8T1M aptamer, and this feature is consistent with the structure-switching principle of GO-SELEX for aptamer screening. Some thermodynamic parameters of the APT8T1M aptamer with gliotoxin were also measured using the isothermal titration calorimetry. As shown in Figure S4, Gliotoxin (50 μM) was titrated into 5 μM of APT8T1M aptamer at 25 °C. The ΔH and ΔS of the aptamer APT8T1M to gliotoxin were found to be -1.602E4 cal/mol and -25.4 12 / 25


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cal/mol/deg, respectively. Thermodynamic results showed that the combination of the APT8T1M aptamer and gliotoxin was an exothermic reaction process, forming a complex system with lower energy, further indicating that gliotoxin can induce the APT8T1M aptamer to fold into a more stable spatial conformation.

Figure 3. Secondary structure of the aptamers predicted using the mfold web server. Next, we examined the structural stability of the aptamers. As shown in Figure 4F, the original aptamer APT8 exhibited high thermal stability (Tm = 78 °C). Excitingly, the truncation optimization strategy further increased the Tm of APT8T1 (Tm = 80 °C), this may be a result of the truncated aptamer folding into a relatively 13 / 25



more stable spatial structure. In addition, by introducing stabilized mini-hairpin structures at the 5′ stem-loop regions and 3′ ends of the APT8T1M aptamer, the thermal stability (Tm = 84 °C) was further increased by 4 °C. More importantly, the higher thermal stability indicated that the APT8T1M aptamer can maintain its structure more persistently in vivo, making it easier for it to exert biological functions of recognition, diagnosis, and treatment. Therefore, through the introduction of truncation and mutation optimization strategies, the performance of the APT8T1M aptamer was significantly improved—including binding affinity, targeting specificity, and structural stability—and it maintained its structural transformation characteristics under the induction of the target. This property can be used to construct related novel methods for rapid detection of gliotoxin.

Figure 4. Aptamer optimization and identification. (A) Binding affinity analysis of the truncated aptamers by biolayer interferometry. (B) Binding affinity analysis of the 14 / 25


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mutated APT8T1M aptamer. (C) Binding specificity analysis of the APT8T1M aptamer. The signal intensities were detected, and the markers were calculated by normalizing the gliotoxin signal to 100%. (D–E) Conformation analysis of the aptamers by circular dichroism. (F) Thermal stability analysis of the aptamers using an Agilent Cary 100 UV-Vis spectrometer equipped with a temperature controller. The melting profiles were normalized using the absorbance at 15 °C and 100 °C, and the first derivatives of the absorbance are shown.

Scheme

1.

Schematic

representation

of

the

fluorescently-labeled

aptamer

structure-switching assay for gliotoxin detection.

Aptamer-based structure-switching assay for gliotoxin detection To enable rapid and effective gliotoxin monitoring, a fluorescently-labeled aptamer structure-switching assay (FLASSA) was developed. As outlined in scheme 15 / 25



1, the 5(6)-carboxyfluorescein (FAM)-labeled APT8T1M aptamer was immobilized on the surface of a 96-well microplate by hybridization with complementary sequences. When the samples were transferred to the wells, the APT8T1M aptamer could be induced and folded into unique three-dimensional conformations that bind specifically to gliotoxin, which in turn broke the interaction between complementary base pairing, resulting in the aptamers being released from the surface of the microplate. Therefore, the fluorescence intensity of the aptamer structure switching can be detected. However, the length of the complementary sequences that are used for immobilization on the microplates and to generate detection signals by hybridization with the FAM-labeled aptamer, is a key factor in the FLASSA system. Theoretically, more base pairings between complementary sequences and APT8T1M limit the efficiency of aptamer structure switching, resulting in reduced sensitivity; while fewer pairings reduce the number of aptamer hybridized, resulting in a decrease in the detection range. Therefore, complementary sequences lengths from 8 to 20 nucleotides were studied, and the fluorescence signal was found to increase with increasing complementary sequence length up to 14 nucleotides (Figure 5A). Longer complementary sequences may show reduced fluorescence signal as a result of their folding into a more rigid spatial structure that inhibits the efficiency of hybridization with aptamers. More importantly, when adding gliotoxin, CS14 has the same fluorescence signal inhibition rate as CS8 and CS11, which are almost the highest (Figure 5A). However, when the length of the complementary sequence was greater 16 / 25


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than 17, the fluorescence signal of the structural transition may be lower owing to the interaction of the greater base complementary pairing. These results indicate that the efficiency of the complementary sequence CS14 used for aptamer hybridization and target-induced structural transformation was highly appropriate and it was thus selected for subsequent experiments. In addition, it is important that the structure-switching time allows gliotoxin to specifically bind FAM-labeled APT8T1M to form the equilibrium detection system without disturbing the binding complex of the aptamer and target. Generally, longer conversion times allow more of the hybridized FAM-labeled APT8T1M to be released for a greater fluorescence suppression effect. However, as FAM-labeled APT8T1M aptamer is depleted from the microplate surface over time, the structure complex may dissociate allowing the aptamer to hybridize again, disrupting the equilibrium and altering the signal magnitude. To evaluate this effect, structure-switching times from 7.5 to 90 min were studied, and the fluorescence inhibition rate was found to increase with increasing gliotoxin incubation times up to 30 min (Figure 5B). Therefore, an incubation time of 30 min was chosen to strike a balance between the two effects of system stability and signal strength in the FLASSA. To further evaluate the detection of gliotoxin using the FLASSA, dose-dependent changes in signal to gliotoxin at different concentrations (0.01 nM-10 μM) were investigated under the optimal experimental conditions. As shown in Figure 5C, an increase in the FLASSA signal was observed with increasing gliotoxin concentration, and a calibration curve of the signal versus the logarithm of gliotoxin 17 / 25



concentration was obtained. The data points in the calibration curve represent three independent measurements, and could be fitted to a sigmoidal logistic four-parameter equation: y = (Rmax –Rmin) / [1 + (x/ EC50) ^ b] + Rmin. Where Rmax, Rmin are the maximum and minimum signal values, respectively, b is the slope of the curve. EC50 is the logarithm of gliotoxin concentration leading to 50% of the maximum signal value. After generation of the experimental data, the following equation was obtained: y = (69.5 + 1.476) / [1 + (x/ 0.0056) ^ 0.5212] – 1.476, R2 = 0.986. A good linear detection range from 0.1 nM to 100 nM was also obtained, which can be represented by the equation: y = 17.02 x + 72.44, with a good correlation coefficient, R2 = 0.995. The limit of detection (LOD) of the FLASSA for gliotoxin was calculated to be 0.05 nM (S/N=3), where the noise level is the standard deviation of multiple measurements on blank samples (n=10). A straightforward comparison between this work and the previous reports is shown in Table 1. Obviously, the developed FLASSA has a broader detection window for gliotoxin, and the LOD is significantly lower than some of the previously reported typical assays such as HPLC, HPTLC, HPLC-MS/MS and bioassays (Cell-based morphological changes).15-19 In addition, the whole gliotoxin quantitative detection by FLASSA, including the pretreatment of the sample, can be completed in less than one hour. More importantly, compared with our previous biosensors used for small molecule toxin detection, this study used simpler experimental designs, more efficient detection protocols, and easier-to-operate experimental setups.30-32

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The selectivity of the FLASSA system, which is a critical property for practical applications, was further evaluated. Several markers (such as okadaic acid and ATP) and structural analogues (β-1,3-glucan and D-galacto-D-mannan), which were used as interferents, were analyzed. As shown in Figure 5D, cross-reactivity experiments showed that the fluorescence inhibition rate of the interferents (each 100 nM) was similar to the binding buffer. Furthermore, gliotoxin (100 nM) yielded an inhibition signal of 0.5613 in the mixture containing each interferent, which was comparable to the case where only gliotoxin was measured. The results show that the developed FLASSA system exhibits high selectivity for gliotoxin detection.

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Figure 5. FLASSA detection system. (A) Optimization of the length of the complementary sequences. The hybridization rate and inhibition rate of fluorescence intensity for the complementary sequences were calculated by normalizing the CS14 signal to 100%. (B) Optimization of structure switching time for the FAM-labeled aptamer. Complementary sequence CS14 was immobilized on the microplates and the FAM-labeled aptamer was hybridized. After incubation with 200 nM of gliotoxin for different time periods, the fluorescence inhibition rate was measured. Duplicate samples were analyzed and the average data are shown. (C) The calibration curve for gliotoxin, plot of fluorescence inhibition rate vs logarithm of gliotoxin concentration. (D) Selectivity of the FLASSA system for different interferents. The error bars represent the standard deviation of the measurements.

Table 1. Comparison of different detection methods for gliotoxin. Methods

Detection range

LOD

References

HPLC

25-1000 ng/ml

25 ng/ml

17

LC-MS-MS

0.25-25 ng/ml

Not reported

15

HPTLC

100-1000 ng/spot

25 ng/ml

16

Bioassay

0.2-1 μg/ml

20 ng/well

18

HPLC-MS/MS

10-120 ng/ml

3 ng/ml

19

FLASSA

0.03-32.6 ng/ml

0.016 ng/ml

This work

Analysis of gliotoxin in human serum and urine samples 20 / 25


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To further assess the feasibility of gliotoxin detection using FLASSA with real samples, clinical serum and urine samples (which were diluted 10 times using binding buffer) were spiked with free gliotoxin. Spiked samples with final gliotoxin concentrations of 1, 10, and 100 nM were analyzed using a preset calibration curve. As shown in Table S3, good recovery percentages of 98.76–110.85% were achieved, which indicated that the clinical samples did not significantly interfere with the FLASSA system. The relatively low coefficient of variation (5.45–14.59%) indicates that the detection system has good repeatability in clinical serum and urine samples. In addition, spiked samples were analyzed simultaneously using HPLC-MS and FLASSA. The results are shown in Table S4, which are highly similar with the reference results from the HPLC-MS/MS. These results demonstrate that the fluorescently-labeled aptamer structure-switching assay could achieve highly sensitive and rapid detection of gliotoxin in real clinical samples. This could provide an effective tool for early and rapid diagnosis of IAFI.

CONCLUSIONS In conclusion, this work is the first report of successful selection, optimization and identification of DNA aptamers that bind with high affinity and specificity to disease-specific marker gliotoxin. Most importantly, the optimized aptamer maintained its structure-switching characteristics under the induction of the target, and this was used to construct a novel fluorescently-labeled aptamer structureswitching assay for the detection of gliotoxin. The method exhibited a good linear 21 / 25



range from 0.1 nM to 100 nM of gliotoxin, with a lower detection limit of 0.05 nM. Furthermore, FLASSA was applied to the detection of gliotoxin in spiked serum and urine samples and showed a good reproducibility and stability. These results show that the developed FLASSA has significant potential and offers an alternative to the traditional analytical methods for the rapid, sensitive, and efficient detection of gliotoxin, thus providing an effective tool for the early and rapid diagnosis of IAFI.

ASSOCIATED CONTENT Supporting Information Materials, reagents, and related experimental methods Supplementary Figures S1 – S4 Supplementary Tables S1 – S4

Conflict of Interest Disclosure The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (81702094, 81700883, 81770925, 81470624 and 81790641) and the Western Medicine Leader Program of Shanghai Municipal Science and Technology Commission (17411971700).

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REFERENCES (1) Brown, G. D.; Denning, D. W.; Gow, N. A.; Levitz, S. M.; Netea, M. G.; White, T. C. Hidden killers: human fungal infections. Sci. Transl. Med. 2012, 4, 165rv113. (2) Krijgsheld, P.; Bleichrodt, R.; van Veluw, G. J.; Wang, F.; Muller, W. H.; Dijksterhuis, J.; Wosten, H. A. Development in Aspergillus. Stud. Mycol. 2013, 74, 1-29. (3) Sugui, J. A.; Kwon-Chung, K. J.; Juvvadi, P. R.; Latge, J. P.; Steinbach, W. J. Aspergillus fumigatus and related species. Cold Spring Harbor Perspect. Med. 2014, 5, a019786. (4) Verweij, P. E.; Ananda-Rajah, M.; Andes, D.; Arendrup, M. C.; Bruggemann, R. J.; Chowdhary, A.; Cornely, O. A.; Denning, D. W.; Groll, A. H.; Izumikawa, K.; Kullberg, B. J.; Lagrou, K.; Maertens, J.; Meis, J. F.; Newton, P.; Page, I.; Seyedmousavi, S.; Sheppard, D. C.; Viscoli, C.; Warris, A.; Donnelly, J. P. International expert opinion on the management of infection caused by azole-resistant Aspergillus fumigatus. Drug Resist. Updates. 2015, 21-22, 30-40. (5) Camargo, J. F.; Husain, S. Immune correlates of protection in human invasive aspergillosis. Clin. Infect. Dis. 2014, 59, 569-577. (6) Powers-Fletcher, M. V.; Hanson, K. E. Molecular Diagnostic Testing for Aspergillus. J. Clin. Microbiol. 2016, 54, 2655-2660. (7) Barton, R. C. Laboratory diagnosis of invasive aspergillosis: from diagnosis to prediction of outcome. Scientifica 2013, 2013, 459405. (8) Lamoth, F.; Calandra, T. Early diagnosis of invasive mould infections and disease. J. Antimicrob. Chemother. 2017, 72, i19-i28. (9) Gullo, A. Invasive fungal infections: the challenge continues. Drugs 2009, 69 Suppl 1, 65-73. (10) Nurzynska-Flak, J.; Zawitkowska-Klaczynska, J.; Kowalczyk, J. R. The radiologic diagnosis of invasive fungal infections in neutropenic children. Ann. Univ. Mariae Curie-Sklodowska, Sect. D 2004, 59, 184-188. (11) Mennink-Kersten, M. A.; Donnelly, J. P.; Verweij, P. E. Detection of circulating galactomannan for the diagnosis and management of invasive aspergillosis. Lancet Infect. Dis. 2004, 4, 349-357. (12) Vehreschild, J. J. As Galactomannan Disappoints, Our Quest for a Feasible Diagnostic Standard for Invasive Aspergillosis Continues. Am. J. Respir. Crit. Care Med. 2014, 190, 248-249. (13) Dagenais, T. R.; Keller, N. P. Pathogenesis of Aspergillus fumigatus in Invasive Aspergillosis. Clin. Microbiol. Rev. 2009, 22, 447-465. (14) Gardiner, D. M.; Howlett, B. J. Bioinformatic and expression analysis of the putative gliotoxin biosynthetic gene cluster of Aspergillus fumigatus. FEMS Microbiol. Lett. 2005, 248, 241-248. (15) Lewis, R. E.; Wiederhold, N. P.; Chi, J.; Han, X. Y.; Komanduri, K. V.; Kontoyiannis, D. P.; Prince, R. A. Detection of gliotoxin in experimental and human aspergillosis. Infect. Immun. 2005, 73, 635-637. (16) Puri, A.; Ahmad, A.; Panda, B. P. Development of an HPTLC-based diagnostic method for invasive aspergillosis. Biomed. Chromatogr. 2010, 24, 887-892. (17) Lewis, R. E.; Wiederhold, N. P.; Lionakis, M. S.; Prince, R. A.; Kontoyiannis, D. P. Frequency 23 / 25



and species distribution of gliotoxin-producing Aspergillus isolates recovered from patients at a tertiary-care cancer center. J. Clin. Microbiol. 2005, 43, 6120-6122. (18) Grovel, O.; Kerzaon, I.; Petit, K.; Robiou Du Pont, T.; Pouchus, Y. F. A new and rapid bioassay for the detection of gliotoxin and related epipolythiodioxopiperazines produced by fungi. J. Microbiol. Methods 2006, 66, 286-293. (19) Cerqueira, L. B.; de Francisco, T. M.; Gasparetto, J. C.; Campos, F. R.; Pontarolo, R. Development and validation of an HPLC-MS/MS method for the early diagnosis of aspergillosis. PloS one 2014, 9, e92851. (20) Ellington, A. D.; Szostak, J. W. In vitro selection of RNA molecules that bind specific ligands. Nature 1990, 346, 818-822. (21) Tuerk, C.; Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 1990, 249, 505-510. (22) Tang, X.; Deng, R.; Sun, Y.; Ren, X.; Zhou, M.; Li, J. Amplified Tandem Spinach-Based Aptamer Transcription Enables Low Background miRNA Detection. Anal. Chem. 2018, 90, 10001-10008. (23) Wu, H.; Liu, R. J.; Kang, X. J.; Liang, C. Y.; Lv, L.; Guo, Z. J. Fluorometric aptamer assay for ochratoxin A based on the use of single walled carbon nanohorns and exonuclease III-aided amplification. Microchim. Acta 2018, 185. (24) Tan, W.; Donovan, M. J.; Jiang, J. Aptamers from cell-based selection for bioanalytical applications. Chem. Rev. 2013, 113, 2842-2862. (25) Eissa, S.; Ng, A.; Siaj, M.; Tavares, A. C.; Zourob, M. Selection and Identification of DNA Aptamers against Okadaic Acid for Biosensing Application. Anal. Chem. 2013, 85, 11794-11801. (26) Lv, L.; Cui, C. B.; Liang, C. Y.; Quan, W. R.; Wang, S. H.; Guo, Z. J. Aptamer-based single-walled carbon nanohorn sensors for ochratoxin A detection. Food Control 2016, 60, 296-301. (27) Gao, S.; Hu, B.; Zheng, X.; Cao, Y.; Liu, D.; Sun, M.; Jiao, B.; Wang, L. Gonyautoxin 1/4 aptamers with high-affinity and high-specificity: From efficient selection to aptasensor application. Biosens. Bioelectron. 2016, 79, 938-944. (28) Kimoto, M.; Nakamura, M.; Hirao, I. Post-ExSELEX stabilization of an unnatural-base DNA aptamer targeting VEGF165 toward pharmaceutical applications. Nucleic Acids Res. 2016, 44, 7487-7494. (29) Yoshizawa, S.; Ueda, T.; Ishido, Y.; Miura, K.; Watanabe, K.; Hirao, I. Nuclease resistance of an extraordinarily thermostable mini-hairpin DNA fragment, d(GCGAAGC) and its application to in vitro protein synthesis. Nucleic Acids Res. 1994, 22, 2217-2221. (30) Gao, S.; Zheng, X.; Hu, B.; Sun, M.; Wu, J.; Jiao, B.; Wang, L. Enzyme-linked, aptamer-based, competitive biolayer interferometry biosensor for palytoxin. Biosens. Bioelectron. 2017, 89, 952-958. (31) Gao, S. X.; Zheng, X.; Wu, J. H. A biolayer interferometry-based competitive biosensor for rapid and sensitive detection of saxitoxin. Sens. Actuators, B 2017, 246, 169-174. (32) Gao, S.; Zheng, X.; Wu, J. A biolayer interferometry-based enzyme-linked aptamer sorbent assay for real-time and highly sensitive detection of PDGF-BB. Biosens. Bioelectron. 2018, 102, 57-62.

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Development of a Fluorescently Labeled Aptamer ... - ACS Publications

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