The Mycobacterium Ag85 complex is the major secretory protein of M. tuberculosis. It is a potential marker for early diagnosis of tuberculosis (TB). The authors have identified specific aptamers for Ag85A (FbpA) via protein SELEX using magnetic beads. After twelve rounds of selection, two aptamers (Apt8 and Apt22) were chosen from different groups, and their binding constants were determined by flow cytometry. Apt22 (labeled with Atto 647N) binds to FbpA with high affinity (Kd = 63 nM) and specificity. A rapid, sensitive, and low-cost fluorescent assay was designed based on the use of Apt22 and graphene oxide, with a limit of detection of 1.5 nM and an analytical range from 5 to 200 nM of FbpA.
Tuberculosis (TB), caused by Mycobacterium tuberculosis, is still the major global health infectious disease that is responsible for high morbidity, especially in developing countries. Thesurvey in 2014 showed 9 million people fell ill with TB and 1.3 million M. tuberculosis deaths were occurred . Emergence of multi-drug resistant (MDR) strains and co-infections with human immunodeficiency virus (HIV) cause high prevalence of TB . Early diagnosis of TB is important for efficiently controlling the epidemic disease.
Conventional diagnostic systems of TB rely on the mycobacterial culture and smear microscopy of acid fast bacilli (AFB) . These approaches have major drawbacks. AFB staining sensitivity is variable, ranging from 20% to 80% . Traditional culture detection, which is “Gold standard” of clinical TB diagnosis requires extended incubation times, 6–8 weeks . Also assays such as DNA hybridization, fluorescent antibody test, and polymerase chain reaction (PCR) have been developed for detection of TB [6, 7]. However, these methods need costly equipment, well-established laboratory, and highly trained personnel. Consequently, it is necessary to develop rapid and accurate new diagnostic tools for TB detection to improve TB control and allow the initiation of treatment. Among new methods for diagnosis of TB, detecting abundantly secreted proteins of M. tuberculosis is the most significant approach.
Researchers have identified potential secreted TB marker proteins to achieve an earlydiagnosis of TB, the 6-kDa early secreted antigenic target (ESAT 6) , the 10-kDa culture filtrate protein (CFP 10) , and the antigen 85 (Ag85) complex . The Mycobacterium Ag85 complex is the major secretory proteins of M. tuberculosis and composed of three variant Ag85 proteins, A, B and C, at the ratio of 2:3:1, respectively , but this ratio varies in response to changes in the environment. This complex involved in biological processes such as enzymatic mycolyltransferase for cell wall biosynthesis in the final stage of mycobacterial growth . Other studies confirmed the presence of the Ag85 complex in the serum of patients and offered a trustworthy TB diagnosis without false positives of other non-TB diseases . Median serum Ag85 level is 50–150 fold higher in patients with active tuberclusis compared to healthy people . Moreover, Ag85 proteins are found in cerebrospinal fluid of tuberculous meningitis patients . Consequently, Ag85 proteins are expected to be efficient markers for TB diagnosis . The application of Ag85 complex for development of enzyme-linked immunosorbent assay (ELISA) can significantly reduce the detection time of M. Tuberculosis compared to the standard culture . Also, Saengdee and his colleagues presented the silicon nitride Ion Sensitive Field Effect Transistor (ISFET) based immunosensor for the detection of Ag85 . The result indicates that this assay is a useful technique for use in a mycobacterial culture system for real-time growth monitoring of M. tuberculosis. As a result, the development of fast and effective methods for M. tuberculosis Ag85 can be necessary to implement appropriate health interventions for decreasing the TB infection cases.
Aptamers are short single-stranded DNA or RNA oligonucleotides, isolated from combinatorial libraries by Systematic Evolution of Ligands by EXponential enrichment (SELEX) . Due to folding into three-dimensional structures, aptamers can specifically bind to their targets, such as small molecules, proteins , toxins, virus, and pathogens  with high affinity. Aptamers possess several significant advantages over antibodies including high specificity and stability, simple synthesis with high purity, easy modification, lack of immunogenicity, and resistance to denaturation and degradation. Based on these properties, aptamers are considerable candidates in the fields of research, therapeutics, diagnostics and detection of pathogens, especially bacteria, in the environment, food or clinical samples . However, the numbers of aptamers for M. tuberculosis detection are still scarce.
So far, a few aptasensors were constructed for specific detection of some secrested proteins of M. tuberculosis, such as Au-IDE/CFP10-ESAT6 aptamer/DNA-AuNPs MSPQC  and aptamer-based voltammetric biosensor for the detection of MPT64 .
Graphene oxide (GO), as a novel carbon nanomaterial provides grand opportunities for developing novel sensors with powerful functions, due to its unique characteristics including large surface area, electrical conductivity, dispersion in water and its fluorescence quenching ability [23, 24]. Also, GO is an ideal nanomaterial for the development of biosensor-based devices with a low cost and low environmental impact .
In this study, for the first time aptamers for FbpA were introduced and used for the establishment of a highly sensitive and selective GO-based aptasensor for detection of FbpA, in which the generated aptamer (Apt22) was labeled with the fluorophore ATTO647N, as the energy donor and GO acted as the energy acceptor.
Materials and methods
Chemicals and materials
Recombinant FbpA protein of M. tuberculosis (His Tag#2023 M) was provided by Creative biomart (USA) (http://www.creativebiomart.net). Recombinant IL2Rα/CD25 protein (His Tag-#10,165-H08H), PD1 (#10377-H08H-50) and PDL1 (#10084-H08H-200) were purchased from Sino biological (China) (www.sinobiological.com). Magnetic beads (#36111) and Ni-NTA HisSorb Stripes (#35023) were ordered from the Qiagen Company (Germany) (https://www.qiagen.com). AccuPrep gel purification kit (#k-3035-1), lambda exonuclease (#EN0562), GeneJET Plasmid Miniprep Kit (#K0502) and InsTAclone PCR Cloning kit (#k1214) were ordered from Thermo Fisher Scientific (USA) (https://www.thermofisher.com). Polymerase chain reaction (PCR) reagents were obtained from Sina Clon (Iran) (http://www.sinaclon.com). All other chemicals such as NaCl, KCl, tris were supplied by Merck Company (Canada) (http://www.merck.com). The synthetic ssDNA library containing a sequence with 43 random nucleotides (5′-GCTGTGTGACTCCTGCAA-N43-GCAGCTGTATCTTGTCTCC-3′) and both forward and reverse primers with below sequences (5′-GCTGTGTGACTCCTGCAA-3′ and 5’GGAGACAAGATACAGCTGC-3′, respectively)  were synthesized by Bioneer Company (South-Korea) (http://www.bioneer.com). Oligonucleotides labeled with ATTO647N were ordered from Microsynth Company (Switzerland) (http://www.microsynth.ch).
Immobilization of recombinant FbpA-his tag protein on the magnetic beads
40 μL homogenous Ni-NTA magnetic beads were washed by PBS-T (50 mM K2HPO4 pH 7.5, 150 mM NaCl, 0.05% Tween-20). Then, 80 μL of 0.2 mg.mL−1 His-tag FbpA protein was added to the sample, resuspended in PBS-T and incubated with gentle rotation for 30 min at 4 °C. The bounded proteins to magnetic beads were washed and diluted in 100 μL PBS-T and stored at 4 °C for the next steps.
In vitro selection of aptamers (SELEX)
Aptamers were generated through the SELEX process to separate specific sequences for FbpA. Before each round of SELEX, FbpA-coated magnetic beads (FMBs) were washed three times by PBS-T. For the first round of selection, 1.5 nmol ssDNA library (initial pool approximately contained 1015 random oligonucleotide) was dissolved in 100 μL of binding buffer (PBS-T containing 1 mg.mL−1 bovine serum albumin (BSA)) and incubated for 10 min at 90 °C, then immediately cooled at 4 °C for 15 min. Next, FMBs were mixed with the oligonucleotide library with the total volume of 1 mL and incubated at 22 °C for 45 min by mild shaking. In order to remove the unbound ssDNAs, tubes were placed in a magnet field (Dexter) and the beads were washed in two steps with 200 μL of PBS-T. To recover the bound sequences, the ssDNA/FMBs complex was suspended in 30 μL of elution buffer (20 mM Tris pH 7.5, 500 mM imidazole) and eluted oligonucleotides were amplified with PCR. The PCR thermocycling parameters were 94 °C (5 min) for initial activation and denaturation, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and elongation at 72 °C for 30 s, finally extension step was at 72 °C (3 min). The PCR product was electrophoresed on 3% agarose gels in 1 × TAE (Tris-acetate-EDTA) buffer to select the optimum PCR cycle without non-specific products. The amplicon which amplified at the optimum PCR cycle, purified using Nucleo Spin® Extract II kit (Macherey-Nagel, Germany). Lambda exonuclease enzyme was used to convert dsDNA to ssDNA. Then, the resulted ssDNA molecules were purified using a NucleoSpin® Extract II kit (Macherey-Nagel, Germany (and its concentrations was measured using a NanoDrop spectrophotometer (NanoDrop UV-Vis spectrophotometer, USA). These purified ssDNAs were applied in the next round of SELEX. To remove nonspecifically adsorbed ssDNAs, the amount of protein was gradually reduced, also the incubation time reached to 30 min from 45 min, while washing time was increased. In order to remove nonspecific adsorbed sequences to the Ni-NTA magnetic beads, counter selection was performed after 3th round of SELEX. The counter selection was performed by using bare magnetic beads. In this step, 30 μL of Ni-NTA magnetic beads was incubated with 100 μL of ssDNA in PBS-T (total volume 900 μL) for 45 min with rotation, and then the supernatant was used for the next round. After 12 rounds of protein-SELEX, the aptamers of 7th, 10th, 11th and 12th rounds were amplified using ATTO647N-labeled forward primer and 5′-phosphorylated reverse primer. Then, purified ssDNA was subjected to SELEX process like the last round and ATTO647N–sequences were applied to flow cytometry to select the best round.
Cloning and sequencing
According to the flow cytometry results from enrichment assay, the purified pool of 11th round was cloned into pt257r/t vector and transformed into Escherichia coli Top10 by using InsTAclone PCR Cloning kit. The forty positive colonies were selected to extract plasmids by GeneJET Plasmid Miniprep kit (Life Technologies, Lithuania, www. fishersci.com) and sequenced (Bioneer, South Korea). The sequences were analysed using the DNAMAN and CLUSTALX softwares and their secondary structures were predicted by Mfold server (http://unafold.rna.albany.edu/?q=mfold) . According to homology and secondary structure analysis of sequences, two representative aptamer candidates (Apt22 and Apt8) were synthesized with ATTO647N fluorescence label at the 5′-end (Microsynthcompany, Switzerland) for binding assays.
Determination of the dissociation constants of selected aptamers
ATTO647N-labeled Apt22 and Apt8 were selected and synthesized. The binding affinities of the selected aptamers were assessed by incubating FMBs (300 ng FbpA) with different concentrations of ATTO647N-labeled aptamers (0–160 nM) in 250 μL of binding buffer at 22 °C for 30 min. The beads were washed twice with 500 μL of washing buffer, suspended in 300 μL of washing buffer and then subjected to flow cytometry analysis. The equilibrium dissociation constants (Kd) of the aptamers were obtained by fitting the dependence of fluorescence intensity of specific binding (Y) on the concentration of the aptamer (X) to the eq. Y = B max X/(Kd + X) using Sigmaplot.
Specificity evaluation of the selected aptamer
The ATTO647N-labeled Apt22 was tested for specificity by evaluation of its ability to bind FbpA, CD25, PD-L1 and PD1 proteins-coated magnetic beads. All steps were carried out as previously described for Kd calculation, except the concentration of aptamer was fixed at 100 nM and then analyzed by flow cytometry.
Fluorometric aptamer based assay based on the use of graphene oxide (GO)
Quantification of FbpA
For FbpA assay, the purchased GO (Sigma # 794341) was diluted to a concentration of 60 μg.mL−1 using phosphate buffered saline. Different concentrations of FbpA (0–200 nM), and a fixed concentration of ATTO647Nlabeled Apt22 (100 nM) were separately added and mixed. The mixture was incubated for 30 min at room temperature with mild shaking, followed by the addition of GO (30 μg.mL−1 final concentration) and mild shaking for 15 min at room temperature. Finally, the mixtures were added into 96-well black plates and the fluorescence intensities were measured, λEx = 600 nm and λEm = 665 nm, by a Synergy H4 microplate reader (BioTeK, USA). All experiments were repeated three times.
Analysis of FbpA in human serum samples
For analysis of FbpA in human serum samples, the working solutions containing different concentrations of FbpA (0–100 nM) and a fixed concentration of ATTO647N-labeled Apt22 (100 nM) were prepared in serum (healthy adult human serum) samples (50-fold diluted with phosphate buffered saline). The mixtures were incubated for 30 min at room temperature with mild shaking, followed by the addition of GO (90 μg.mL−1 final concentration) and shaking mildly for 15 min at room temperature. Finally, the fluorescence intensities were recorded.
Results and discussion
In vitro selection of aptamers, their cloning and clustering
The purified recombinant FbpA protein was proposed as target for antigen detection because it is highly immunogenic and extremely secreted by M.tuberculosis . In this study, the recombinant FbpA (His Tag) protein was attached to Ni-NTA magnetic agarose beads via His6 tag/Ni-NTA interaction. ssDNA sequences binding to FbpA were isolated simultaneously from a random ssDNA library consisted of 1015 oligonucleotides. The amount of ssDNA binding to target was significantly increased with increasing the SELEX round (Fig. 1). In the aim of selecting aptamers with high affinity for FbpA, the selection pressure was gradually increased, including the reduction of FbpA concentration during SELEX rounds, increase of beads concentration and washes steps during counter rounds, also using salmon sperm DNA as an appropriate competitor.
During enrichment assay, after 11 rounds of screening, no further increase in affinity between ssDNA and FbpA occurred based on the flow cytometry results (Fig. 1). So, this round was chosen for cloning and sequencing. The results showed that the FL4 log intensity increased from 2976.5 to 1,155,000 during 11 rounds of selection and no more increase was observed in further selection round, which may be attributed to the saturation of the binding sites on FbpA-coated beads. So, the pool of 11th round was amplified by PCR and then cloned.
Forty individual clones of 11th round were sequenced and analyzed by DNAMAN software based on their homology and phylogenetic tree. All sequences were classified in seven clusters. According to homology and secondary structures analysis, two representative aptamer candidates (Apt22 and Apt8) were synthesized with ATTO647N fluorescence label and used for the next experiments. The sequences of the Apt22 and Apt8 have been shown in Table S1.
Characterization of the selected aptamers
ATTO647N-labeled Apt22 and Apt8 showed high binding affinity to the target, FbpA protein (Fig. S1). Therefore, these selected aptamers were chosen for further characterization and their secondary structures were analyzed using Mfold server (http://unafold.rna.albany.edu/?q=mfold)  (Fig. 2(a)). To quantitatively evaluate the binding affinity of the selected aptamers, FMBs were incubated with different concentrations of ATTO647N-labeled Apt22 and Apt8. Using non-linear regression analysis, the Kd of Apt22 and Apt8 aptamers were found to be 62.95 nM and 202 nM, respectively (Fig. 2(b)). Apt22 was used for the subsequent experiments, owing to its better binding affinity towards FbpA.
Specificity is important for the evaluation of aptamer performance. The binding of Apt22 for CD25, PD1, PDL1 proteins was investigated using flow cytometry assay. The results indicated that Apt22 had high selectivity towards FbpA protein in comparison with PD1, PDL1 and CD25 proteins (Fig. 3).
Principle of the designed aptasensor
The development of aptamer to FbpA was successful and the aptamer (Apt22) was tested in a novel sensor. Scheme 1 illustrates the sensing platform based on turn-off/on fluorescence for detection of FbpA. In the absence of target molecule (FbpA), ATTO647N-Apt22 binds onto the GO via hydrophobic and π-interaction. So, the fluorescence of ATTO is quenched by GO as a fluorescence quencher. In the presence of target (FbpA), the aptamer interacts with its target and forms FbpA/Apt 22 complex and does not bind onto the GO. It has been shown that GO hardly interacts with rigid double-stranded DNA (dsDNA) or aptamer-target complexes . So, in this condition, the fluorophore (ATTO) is far away from GO and a strong fluorescence intensity is observed. This turn-on/off assay is beneficial to the access of rapid target detection.
The fluorescence spectrum of ATTO647N-Apt22 in the absence of GO represented a strong fluorescence intensity (Fig. S2(A)). However, in the presence of GO, more than 90% quenching of the fluorescence emission was observed (Fig. S2(B)). This result revealed high fluorescence quenching efficiency of GO. For the best function of the presented sensor, it is important to obtain the optimal concentration of GO. Fig. S2 indicates the fluorescence quenching of ATTO647N-Apt22 at different concentrations of GO (0–60 μg.mL−1). When GO was introduced, the relative fluorescence intensity of Apt22 was gradually reduced and reached the minimum at 30 μg.mL−1 GO. Hence, 30 μg.mL−1 GO was used as the optimized concentration in the next experiments.
Characterization of the aptasensor
Under the optimal condition, the sensing platform was used for FbpA detection
. The relative fluorescence intensity of the sensor evidently increased with the increasing concentration of FbpA (Fig. S3). The linear range was from 5 to 200 nM with linear equation y = 1.012× + 0.3598 (Fig. 4). The limit of detection (LOD) was measured to be 1.5 nM based on three times standard deviation of the blank signal (3σ)/Slope. The results can be attributed to the quenching effect of GO . Also, The high affinity of Apt22 for FbpA is making this fluorescent aptasensor a promising candidate for the rapid detection of protein markers.
Different studies have reported new approaches for the detection of M. tuberculosis. These analytical techniques were summarized in Table S2 [21, 22, 30,31,32,33]. Compared to the aptasensor, some of these techniques need costly and complicated equipments. Also, the aptasensor showed an acceptable LOD compared to these approaches.
Analysis of spiked serum samples
In order to confirm the applicability of this analytical method for real sample analysis, it was used to determinate the FbpA in serum samples. 90 μg.mL−1 was taken as the optimized concentration of GO in this assay. In this GO concentration, the fluorescence intensity of the sensor was quenched down to 23% of its original fluorescence signal. Fig. S4 indicates the fluorescence intensity change of the aptasensor in different concentrations of FbpA in serum samples. The relative fluorescence intensity of the aptasensor increased with the increasing concentration of FbpA. The linear equation was y = 0.3534× + 1.3956, and regression coefficient was 0.9994. The detection limit was determined to be 2.1 nM which is lower than the LOD of conventional plate based ELISA assay for the detection of Ag85 in serum samples (183 nM) . Also recovery assay was performed to investigate the suitability of the reported sensing method for actual applications. Recoveries of the spiked concentrations of FbpA ranged from 91.8% to 97% (Table 1). This shows the GO-based aptasensor to be well suited for the determination of FbpA in serum sample.
Tuberculosis (TB) is still the major global health infectious diseases and has been associated with high morbidity, especially in developing countries. Early diagnosis of active TB has remained an elusive challenge for efficiently controlling the epidemic disease. Therefore, it is needed to develop rapid and sensitive methods for TB detection.
This study is the first report of screening aptamers with high affinity and specificity for Ag85A (FbpA) protein of M. tuberculosis. Among the aptamer candidates, Apt22 was identified as the aptamer which had the best dissociation constant (Kd 62.95 nM) and high specificity towards FbpA. In addition, ATTO647N-labeled Apt22 was used for development of a fluorescent aptasensor based on graphene oxide (GO).
The designed aptasensor was extremely sensitive for detection of FbpA in both buffer and blood serum samples. This aptasensor can detect the target in PBS and serum with low detection limits of 1.5 nM and 2.1 nM, respectively. However, construction of GO needs substantial skills. Also, interaction of other proteins with GO in serum samples can influence the sensitivity of the aptasensor. On the basis of its performance, the GO-based fluorescent aptasensor revealed a promising future for the detection of protein biomarkers of M. tuberculosis.
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Financial support of this study was provided by Mashhad University of Medical Sciences. This report has been extracted from the Ph.D. thesis of Najmeh Ansari.
The author(s) declare that they have no competing interests.
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Ansari, N., Ghazvini, K., Ramezani, M. et al. Selection of DNA aptamers against Mycobacterium tuberculosis Ag85A, and its application in a graphene oxide-based fluorometric assay. Microchim Acta 185, 21 (2018). https://doi.org/10.1007/s00604-017-2550-3
- Fluorescent assay
- Limit of detection
- Secretory protein