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A novel colorimetric competitive aptamer assay for lysozyme detection based on superparamagnetic nanobeads
Author’s Accepted Manuscript A novel colorimetric competitive aptamer assay for lysozyme detection based on superparamagnetic nanobeads Rupesh K. Mishra, Akhtar Hayat, Geetesh K. Mishra, Gaëlle Catanante, Vinay Sharma, JeanLouis Marty www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(16)31035-9 http://dx.doi.org/10.1016/j.talanta.2016.12.083 TAL17173
To appear in: Talanta Received date: 9 October 2016 Revised date: 27 December 2016 Accepted date: 29 December 2016 Cite this article as: Rupesh K. Mishra, Akhtar Hayat, Geetesh K. Mishra, Gaëlle Catanante, Vinay Sharma and Jean-Louis Marty, A novel colorimetric competitive aptamer assay for lysozyme detection based on superparamagnetic nanobeads, Talanta, http://dx.doi.org/10.1016/j.talanta.2016.12.083 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A novel colorimetric competitive aptamer assay for lysozyme detection based on superparamagnetic nanobeads Rupesh K. Mishrac,a, Akhtar Hayat#b,a, Geetesh K. Mishrad, Gaëlle Catanantea, Vinay Sharmac, Jean-Louis Marty#a
a
Université de Perpignan via Domitia, Laboratoire BAE, Building S 52 Av. Paul Alduy,
66860 Perpignan Cedex, France b
Interdisciplinary Research Centre in Biomedical Materials (IRCBM), COMSATS
Institute of Information Technology (CIIT), Lahore 54000, Pakistan c
Department of Biosciences and Biotechnology, Banasthali University, Rajasthan,
304022 India d
Sabanci University Nanotechnology Research and Application Center, Orta Mahalle
34956, Tuzla, Istanbul, Turkey
Abstract Lysozyme (Lys) commonly presents in wines and are known to cause toxicological impact on human health. The need of highly sensitive and reliable detection methods are evident in such matrix. In this work, we developed a competitive aptamer based assay for detection of Lys by employing carboxylated magnetic beads as a support to immobilize the target molecule Lys. The used aptamer sequence was biotinylated which further binds with Streptavidin-Alkaline phosphatase (Stp-ALP) in the micro wells. Colorimetric tests were performed in order to optimize different experimental parameters. The Lys assay showed a good linearity in the range of 5-140 nM with a limit of detection (LOD) 10 nM. The mid-point value (IC50) 110 nM and the analysis time (60 min) validated the developed aptasensor as a promising tool for routine use. The assay displayed good recoveries of Lys in the range 99.00-99.27% and was demonstrated for the detection of Lys in wine samples.
1. Introduction Lysozyme (Lys) is a comparatively small protein (14.3 kDa) containing of merely 129 amino acid residues, and is widely distributed in nature[1, 2]. It has an isoelectric point of 11 and constitutes 3.5% of egg white protein. It is clear that lysozyme's relatively small size and simplicity makes it an excellent model agent for novel methods in protein detection. This protein is also known as N-acetylmuramide glycan hydrolase due to its properties to destroy bacterial cellular membranes by catalyzing the hydrolysis of glycosidic
bonds
between
N-acetylmuramic
acid
and
N-acetylglucosamine
peptidoglycan residues of gram-positive bacterial cell walls[3]. Moreover, the monitoring of the lysozyme level is used as a marker for some health problems such as bronchopulmonary dysplasia in newborns[4], conjunctivitis, kidney problems[5] and leukemia[5, 6]. In addition, Lys has been widely used as an antimicrobial agent in the production of wine[7, 8] cheese[9] beers as well as to prolong the shelf-life of shrimp, surimi products and sausages[10]. In particular, in wine-making, Lys has been used since 1990 to prevent or mitigate heterolactic fermentation[7]. The maximum permitted level of lysozyme in wine samples is 500 mg L−1 (35 μM)[2]. Being an egg-protein, lysozyme is considered as an allergen; therefore developing new, rapid, cheap and sensitive methods for the detection of Lys is of great significance. Currently, the analytical methods developed for the determination of Lys in wine are either
based
on
chromatographical
techniques or
on
immunosensing
using
ELISA[11] [12]. Numerous sensors have been designated also as alternatives to these classic
analytical
methods,
mainly
based
on
electrochemical
and
optical
methods[13], [14], [15], [16], [17], [11] [13]. Among the different types of transducers, surface
plasmon
resonance
(SPR)
[18]and
electrochemical
methods
[19],
[17],[20],[2]could offer few important advantages over classical methods. But still needs innovation to improve the sensing abilities and accuracy for these contaminants. Among different types of bioassays developments, optical ones are in demand because of remarkable sensitivity, simple instrumentation, rapid response, low cost and their ability to be portable easily. Various optical methods have been used for Lys detection for instance a recent work of Yao et al.,[21] on Lys detection in egg white using gold nanoparticles aggregations using cationic polymer and aptamer. Similarly, a luminescence based label free detection of Lys in buffer was developed using Gquadruplex[22].
Recently, there is a great attention in the development of aptasensors[23, 24]. Aptamers are artificial DNA or RNA oligonucleotides screened in vitro which have the capability to bind to small molecules (proteins) or even whole cells. Aptamers are capable of recognizing their targets with affinities and specificities frequently similar or even beyond those of antibodies[25, 26]. Ever then Cox and Ellington found the first Lysbinding Apt in 2001, several Lys-Apts have been stated in the literature with erratic binding affinities to their target analyte[27, 28]. Their advantages are enormous over antibodies and enzymes as they are relatively easy to synthesized, offer better affinity and specificity, facile chemical modification and long term stability. In the same context, magnetic beads (MBs) were also appeared as authoritative and adaptable tool for the expansion of numerous biosensing stage for numerous contaminants [29-32]. The recognition methods include the analysis of electroactive species amplified by enzyme labels [33] or carbon based labels containing various enzymes[34, 35]. It has been reported that MBs based aptamer-assays have many advantages over conventional ones: (1) short interaction time between biological component and the coated nanobeads because of increase in surface area as well as decrease of matrix effect [36, 37] (2) more possibilities for manipulations[31] (3) less time consuming due to decreased coating, competition and blocking times[38]; and (4) modification of the MBs in numerous ways, permitting different strategies of immobilization[39]. To the best of our knowledge, aptasensor based on MBs approach for Lys detection is not yet reported. Herein, we have demonstrated a sensitive, accurate and reliable novel strategy to quantify Lys in wine samples. The aim was to combining the advantages of MBs and optical techniques. To develop the assay, MBs were chosen to immobilize the Lys and coated in 96 microtiter plate. The assay is based on the competition between MBs coupled Lys and free Lys to bind with the biotin labeled aptamers. This strategy was further employed for the first time to detect Lys quantities in wine samples. The Lys recoveries (99.00-99.27%) were recorded in a good agreement with the spiked Lys concentrations. The assay demonstrates good stability with RSD of 3.87%. The advantages of the developed assay are their possible miniaturization, following portability and the use of minimal volume of samples and reagents with added advantages of high throughput sample analysis.
2. Experimental 2.1. Materials Potassium dihydrogen phosphate, sodium monophosphate, bovine serum alumina (BSA), lysozyme (Lys), casein, magnesium chloride, potassium chloride, sodium chloride, diethanolamine (DEA), N-hydroxysuccinimide (NHS), streptavidin-alkaline phosphatase
(Strep-ALP),N-(3-dimethylaminopropyle)-N’-ethyle-carbodiimide
hydrochloride (EDC) were purchased from Sigma (St. Louis, MO, USA). The whole milk was purchased from European Reference Material (Geel, Belgium). The carboxylic-functionalized Dynabeads (MBs) (10 mg/mL) were purchased from Invitrogen (USA). All reagents were analytical reagent grade. The aptamer was procured from Eurogenetic (France) with following sequences with modifications at the 3’ terminals: AptLysBio 5’- GCA GCT AAG CAG GCG GCT CAC AAA ACC ATT CGC ATG CGG C-Bio-3’ All solutions were prepared in MilliQ water. The buffers used were: binding buffer (BB) (1mM MgCl2, 2.7mM KCl, 140mM NaCl, 0.1mM Na2HPO4 and 1.8mM, KH2PO4 pH 7.4), MES buffer (100mM MES and 0.09%NaCl pH 6) and DEA buffer (10% DEA pH 9.5).
2.2. Apparatus The colorimetric measurements were performed with a Labsystems Multiskan EX microtiter plate reader (Thermo Life Sciences, Cergy Pontoise, France). Maxisorp 96microtiter plates were obtained from Nunc (Roskilde, Denmark). A horizontal shaker (IKA, Vibrax-VXR) was also used. 96 well microplates, PS, U-bottom were obtained from Greiner bio-one (France). Adem-Mag 96 (adapted for 96-well micro titer plates) and Adem-MagSV (single magnet position adapted for 1.5 mL micro tubes) were from Ademtech S.A (France). 2.3. Procedures 2.3.1 Immobilization of Lys on MBs
As described in section 2.1, the carboxylic-functionalized magnetic beads (MBs), Dynabeads were mixed properly to ensure their homogenous dispersions before taken out of the stock vial. A volume of 50 µL of MBs (10 mg/mL) were washed three times using 100 µL of BB to remove the preservatives. Then, 100µL of 100 mM EDC and 25 mM NHS were added and the mixture was incubated at room temperature for 1 h with gentle shaking. Later, the MBs were washed three times with 100 µL of BB and further incubated with 100µL of 1µM Lys for 1 h at room temperature with mixing in shaker. After incubation with Lys, the modified MBs were rinsed three times using BB and resuspended in it to store at 4 ºC. For control experiments, MBs were prepared by following the same procedure without addition of Lys solutions.
2.3.2 Optimization of various parameters Various assay parameters were optimized before proceeding for real tests. Optimization of aptamer concentrations for Lys detection was accomplished using 100 µL different concentrations as 0.312, 0.625, 1.25, 2.5 µg/ml (see conditions in section 2.3.1). Then incubation time between Lys and aptamer was investigated from 0-90 mins in each 15 mins interval. Similarly, optimization of Strep-ALP dilution for the accessibility and interaction with AptLysBio was tested using 100 µL of streptavidin-ALP added at dilutions ratios of 1: 1000, 1:10000, 1: 12500, 1:50000, 1:75000 and 1:100000 with in BB for 1h with shaking. Finally, Stp-ALP incubation with biotinylated aptamer for facile detection of Lysozyme was optimized. Various time durations such as 0, 20, 40, 60, 80 and 100 min was tested using 100 µL of p-NPP (4 mg.mL-1 in DEA buffer).
2.3.3 Colorimetric-based competitive aptasensor for Lys The modified MBs with Lys (1.5/100 of stock solution, 20 µL) were coated in the wells of microtiter plate. Further, the microtiter plate containing MBs were blocked with a 50 µL solution of 5% whole milk in BB for 1h by applying a gentle shaking to avoid nonspecific adsorption following the washing step. The competition step was performed using 50µL of Lys solution at different concentrations and 50µL of ApLysBio for 1h. Subsequently, 100 µL of streptavidin-ALP was added at dilution of 1/12500 in BB for 1h with shaking. 100 µL of p-NPP (4 mg.mL-1 in DEA buffer) were added with incubation period of 30 min at room temperature. The Lys detection strategy using aptasensor is illustrated in schematics 1. The absorbance was measured at λ=405 nm
using the microtiter plate reader. Based on the absorbance values, the % binding of Lys was calculated.
2.3.4 Preparation of wine samples Wine samples were prepared by following our previously reported work [2]. Briefly, 1 mL of wine sample was spiked with the desired concentration of Lys and allowed to stand for 3 min. Next, 200μL of 5M NaCl solution containing 5% Tween-20 surfactant were added to 200μL of Lys-wine mixture and diluted to a final volume of 1mL using 20 mM MES buffer pH 6 with 1mM MgCl2. This mixture was further centrifuged at 5000 rpm for 5 min and diluted 20 times using the buffer aforesaid. All washing steps were performed by adding three times 100µL of BB between steps and the supernatants were removed using a magnet.
3. Results and Discussions 3.1. Optimizations of Aptamer assay parameters The immobilization of Lys on MBs was very imperative to develop the aptamer assay. The colorimetric test was performed to authenticate the surface modification of MBs with Lys to evaluate the capability of MBs-Lys for development of competition tests, and also to optimize the different investigational parameters. The Lys coupled MBs were magnetically immobilized in to the micro-wells using Adem-Mag 96 magnet. The immobilized Lys and the free Lys in the solution competed for the anti AptLysTRANBio in solution. Further, the colorimetric detection was based on the hydrolysis of p-NP in the presence of Strep-ALP to the colored product p-nitrophenol (p-NP). First to augment various experimental conditions and parameters, initial assays were carried out with MBs-Lys. The coupling of MBs-Lys in to the micro wells was optimized by stimulating coating times ranging from 0 to 25 min. No significant change was observed in signal intensities; consequently the coating time was reduced to 5 min. The blocking step was also studied, to avoid non specific adsorption of aptamers. Various proteins such as casein (1%), BSA (1%) and whole milk (5%) were tried as blocking agents. The whole milk (5%) was chosen as the best among the three tested blocking agents. In order to enhance the sensitivity, optimization of other significant experimental
conditions such aptamer concentrations, as appropriate binding time of biotinylated aptamer (AptLysBio) and MBs-Lys, Str-ALP and AptLysBio and ALP -pNPP interaction are also very vital for aptamer assay development. Moreover, the optimized concentrations of AptLysBio and ALP-pNPP would improve the analytical features such as LOD, linear range, and sensitivity of the method. The attachments of Lys-MBs to aptamer certainly influence the attachment of Str-ALP to it, consequently enrichment in the signal. As represented in the figure 1, various aptamer concentrations were tested to choose an optimal quantity for the assay and 1µg/ml was found to be the best for Lys quantitation accurately. The suitable interaction time between Lys and aptamer was also assured while recording the response in different time duration starting from 0-90 min at the intervals of 15 min. It is revealed from the shown figure 2a that a time of 60 min was good enough to perform the assays and to establish a calibration curve. To find out the best enzyme concentrations in the assay, experiments with different dilutions of Str-ALP were sequentially performed to see the signal dynamics with respect to the dilutions. Figure 2b shows the dilution effects on absorbance. To access the stability of Lys-MBs, the modified beads were stored at 4 ºC for two weeks. The obtained results were similar to those obtained with freshly prepared Lys-MBs, indicating no detectable loss of the activity. The Stp-ALP incubation with biotinylated aptamer was also evaluated to increase the sensitivity of the assay. Figure 2c shows that among tested time durations, 60 min was turning out to be the best duration for Str-ALP coupling with ALP biotin.
3.2 Lys detection using Aptamer assay Using the optimized experimental parameters, the calibration curve was performed with free Lys solution. The calibration curve was constructed based on the % binding of Lys on to the aptamer vs Lys concentration. The percentage binding was calculated based on the absorbance recorded of each individual concentrations of Lys. The curve obtained with colorimetric assays for the Lys-MBs is shown in figure 3, which showed a broad dynamic range from 5-220 nM with an excellent linearity (Y= 0.00494x+0.02212, R2=0.9999) ranging from 5-140 nM. Due to the percentage error (5%), the LOD was defined as the toxin concentration corresponds to the 80% of anti Lys-Apt binding depending upon the maximum value of standard deviation. The developed Lys-Apt assay demonstrates a good possibility for real tests in Lys contaminated samples. We obtained a good LOD of Lys upto 10 nM as this concentration may easily be distinct to
10 nM and 20 nM with good sensitivity of 0.0050 nM. Table 1 summarizes the analytical values of the developed assay consisting of correlation coefficient R, midpoint (IC) and LOD values derived from the regression equation, obtained for Lys-Apt colorimetric assay with MBs.
3.3 Application of developed assay in wine sample analysis To inspect the viability and assay accuracy of Lys-Apt colorimetric autosensing platform, spiked wine samples with various concentrations of target molecule Lys were analyzed by developed strategy to see the interferences from the extract. The recovery test also signifies the suitability of the biomolecule towards the matrix (wine). Initially, wine samples were diluted to different concentrations with 5M NaCl solution containing 5% Tween-20 surfactant which were added to 200 μL of Lys-wine mixture and diluted to a final volume of 1mL using 20 mM MES buffer pH 6 with 1mM MgCl2. Ultimately, 3 different Lys concentrations, 10, 50 and 110 nM were spiked and tested in wine. The concentrations were chosen from the linear range (LOD, and mid-point value). No countable difference was observed with respect to linear working range hence, observed optical data was compared to the calibration curves obtained for the aptamer based assay. The relatively stumpy matrix effect could be explicated by the specificity of the aptamers towards Lys and also the blocking proteins. This inordinate advantage validated our developed assay for real sample analysis. Moreover, all agreements with calibration, assay demonstrated a good recovery range from 99% to 99.27% represented in Table 2. Consequently, the Lys-Apt-based colorimetric autosensing could be applied for quantitative monitoring of Lys in the complex system. The precision and stability of the assays was calculated by the %RSD for the triplicate measurements.
3.4 Aptamer assay selectivity The specificity of the developed assay was assessed by employing Lys-Apt biorecognition event. For assay specificity to Lys, control experiments were performed using three most common proteins, Casein, BSA and Cyt C present in one solution as well as individually. It was noticed from figure 4 that, when BSA (250nM), Casein (250 nM) and Cyt C (250 nM) were incubated and detected individually with the aptasensor, only Lys induced a significant response, while the others had very less responses when they are present in higher concentration level (5 times more than lysozyme). As
demonstrated, no obvious interference was obtained for other three proteins and a good specificity of the aptasensor to Lys could be obtained. These results are strong proof of the highly selective detection of Lys. No cross reactions were observed from similar interfering proteins, even in the case of cytochrome c, which is structurally similar to Lys. The response of cytochrome c was negligible in the presence of AptLysTRAN.
4. Conclusions This work presents for the first time an MB mediated aptamer assay to detect Lys in wine samples. The assay preparation is simple including the interaction of biotinylated Lys-apt with Lys couples on MBs. During the procedure of detection, the aptamer interacts Lys and could be able to provide signals. Under the optimized experimental parameters, the optical signals were proportional to the varied Lys concentrations thus, obtained a good calibration data for Lys. The LOD was 10 nM with excellent linearity 5-140 nM for Lys. The developed assay showed potential application for small target molecules and protein quantitation using colorimetry with possible extension to similar molecules detections. The assay is rapid, simple, sensitive and selective route for such analytes detection in real samples.
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Figure Legends
Scheme 1: Figure illustrating the strategies and steps of Lysozyme detection using aptamer and MBs combination.
Figure 1: Optimization of aptamer concentration for Lys detection assay. Uncertainty values corresponding to replicated experiments (n = 3). Figure 2a: The suitable interaction time between Lys and aptamer under the optimized parameters. Figure 2b: Optimization of Strep-ALP dilution for the accessibility and interaction with AptLysBio. Figure 2c: Representation of Stp-ALP incubation with biotinylated aptamer for facile detection of Lysozyme. Figure 3: Calibration curve of lysozyme using colorimetry based on competition assay and binding to lysozyme. Error bars represents standard deviation of the mean, n=3. Figure 4: Specificity of the developed aptasensor for Lys detection in the presence of other co-contaminants (Casein, BSA and Cyt C).
Table Legends
Table 1: Analytical features of developed aptasensor for Lys.
Analytical parameters
Experimental findings
Dynamic range
5-220 nM
Linearity
5-140 nM
LOD
10 nM
Specificity
Yes (Lys)
Mid point
110 nM
2
R
0.9999
Sensitivity
0.005 nM
Table 2: Recovery percentage for spiked Lys in wine samples using colorimetric test.
Spiked Lys (nM)
Found Lys (nM)
R.S.D. %
Recovery %
A.E.%
10
9.9
3.82
99.00
15.6
50
49.6
3.60
99.20
16.2
110
109.2
3.75
99.27
15.9
Highlights · · ·
Highly sensitive lysozyme detection route. First report on MB coupled aptamer assay. Assay based on unique combination of biotinylated aptamer and Streptavidin interaction.
Lys-Apt binding %
100 80 60 40 20 0 10
[Lys] nM
100
Scheme 1
Scheme 1
Figure 1
Fig 1
Absl = 405 nm 0.1
0.2
0.3
0.4
0.5
0.6
0.0
0.5
1.5
2.0
[Apt-Lys-Bio] (mg/ml)
1.0
2.5
Fig 2a
Figure 2a
Absl = 405 nm
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0
20
60
Time (min)
40
80
100
Fig. 2b
Figure 2b
Absl = 405 nm
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0
1/1000
1/12500
1/50000
1/75000
Dilutions of strep-ALP
1/10000
1/100000
Fig. 2c
Figure 2c
Absl = 405 nm
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0
20
60
Time (min)
40
80
100
Fig. 3
Figure 3
Lys-Apt binding % 0
20
40
60
80
100
10
[Lys] nM
100
Fig. 4
Figure 4
Absl = 405 nm
0.0
0.2
0.4
0.6
0.8
1.0
Lys
Casein
BSA
Cyt C
Table 1
Table 1
Experimental findings 5-220 nM 5-140 nM 10 nM Yes (Lys) 110 nM 0.9999
0.005 nM
Analytical parameters Dynamic range Linearity LOD Specificity Mid point R2
Sensitivity
PII: DOI: Reference:
S0039-9140(16)31035-9 http://dx.doi.org/10.1016/j.talanta.2016.12.083 TAL17173
To appear in: Talanta Received date: 9 October 2016 Revised date: 27 December 2016 Accepted date: 29 December 2016 Cite this article as: Rupesh K. Mishra, Akhtar Hayat, Geetesh K. Mishra, Gaëlle Catanante, Vinay Sharma and Jean-Louis Marty, A novel colorimetric competitive aptamer assay for lysozyme detection based on superparamagnetic nanobeads, Talanta, http://dx.doi.org/10.1016/j.talanta.2016.12.083 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A novel colorimetric competitive aptamer assay for lysozyme detection based on superparamagnetic nanobeads Rupesh K. Mishrac,a, Akhtar Hayat#b,a, Geetesh K. Mishrad, Gaëlle Catanantea, Vinay Sharmac, Jean-Louis Marty#a
a
Université de Perpignan via Domitia, Laboratoire BAE, Building S 52 Av. Paul Alduy,
66860 Perpignan Cedex, France b
Interdisciplinary Research Centre in Biomedical Materials (IRCBM), COMSATS
Institute of Information Technology (CIIT), Lahore 54000, Pakistan c
Department of Biosciences and Biotechnology, Banasthali University, Rajasthan,
304022 India d
Sabanci University Nanotechnology Research and Application Center, Orta Mahalle
34956, Tuzla, Istanbul, Turkey
Abstract Lysozyme (Lys) commonly presents in wines and are known to cause toxicological impact on human health. The need of highly sensitive and reliable detection methods are evident in such matrix. In this work, we developed a competitive aptamer based assay for detection of Lys by employing carboxylated magnetic beads as a support to immobilize the target molecule Lys. The used aptamer sequence was biotinylated which further binds with Streptavidin-Alkaline phosphatase (Stp-ALP) in the micro wells. Colorimetric tests were performed in order to optimize different experimental parameters. The Lys assay showed a good linearity in the range of 5-140 nM with a limit of detection (LOD) 10 nM. The mid-point value (IC50) 110 nM and the analysis time (60 min) validated the developed aptasensor as a promising tool for routine use. The assay displayed good recoveries of Lys in the range 99.00-99.27% and was demonstrated for the detection of Lys in wine samples.
1. Introduction Lysozyme (Lys) is a comparatively small protein (14.3 kDa) containing of merely 129 amino acid residues, and is widely distributed in nature[1, 2]. It has an isoelectric point of 11 and constitutes 3.5% of egg white protein. It is clear that lysozyme's relatively small size and simplicity makes it an excellent model agent for novel methods in protein detection. This protein is also known as N-acetylmuramide glycan hydrolase due to its properties to destroy bacterial cellular membranes by catalyzing the hydrolysis of glycosidic
bonds
between
N-acetylmuramic
acid
and
N-acetylglucosamine
peptidoglycan residues of gram-positive bacterial cell walls[3]. Moreover, the monitoring of the lysozyme level is used as a marker for some health problems such as bronchopulmonary dysplasia in newborns[4], conjunctivitis, kidney problems[5] and leukemia[5, 6]. In addition, Lys has been widely used as an antimicrobial agent in the production of wine[7, 8] cheese[9] beers as well as to prolong the shelf-life of shrimp, surimi products and sausages[10]. In particular, in wine-making, Lys has been used since 1990 to prevent or mitigate heterolactic fermentation[7]. The maximum permitted level of lysozyme in wine samples is 500 mg L−1 (35 μM)[2]. Being an egg-protein, lysozyme is considered as an allergen; therefore developing new, rapid, cheap and sensitive methods for the detection of Lys is of great significance. Currently, the analytical methods developed for the determination of Lys in wine are either
based
on
chromatographical
techniques or
on
immunosensing
using
ELISA[11] [12]. Numerous sensors have been designated also as alternatives to these classic
analytical
methods,
mainly
based
on
electrochemical
and
optical
methods[13], [14], [15], [16], [17], [11] [13]. Among the different types of transducers, surface
plasmon
resonance
(SPR)
[18]and
electrochemical
methods
[19],
[17],[20],[2]could offer few important advantages over classical methods. But still needs innovation to improve the sensing abilities and accuracy for these contaminants. Among different types of bioassays developments, optical ones are in demand because of remarkable sensitivity, simple instrumentation, rapid response, low cost and their ability to be portable easily. Various optical methods have been used for Lys detection for instance a recent work of Yao et al.,[21] on Lys detection in egg white using gold nanoparticles aggregations using cationic polymer and aptamer. Similarly, a luminescence based label free detection of Lys in buffer was developed using Gquadruplex[22].
Recently, there is a great attention in the development of aptasensors[23, 24]. Aptamers are artificial DNA or RNA oligonucleotides screened in vitro which have the capability to bind to small molecules (proteins) or even whole cells. Aptamers are capable of recognizing their targets with affinities and specificities frequently similar or even beyond those of antibodies[25, 26]. Ever then Cox and Ellington found the first Lysbinding Apt in 2001, several Lys-Apts have been stated in the literature with erratic binding affinities to their target analyte[27, 28]. Their advantages are enormous over antibodies and enzymes as they are relatively easy to synthesized, offer better affinity and specificity, facile chemical modification and long term stability. In the same context, magnetic beads (MBs) were also appeared as authoritative and adaptable tool for the expansion of numerous biosensing stage for numerous contaminants [29-32]. The recognition methods include the analysis of electroactive species amplified by enzyme labels [33] or carbon based labels containing various enzymes[34, 35]. It has been reported that MBs based aptamer-assays have many advantages over conventional ones: (1) short interaction time between biological component and the coated nanobeads because of increase in surface area as well as decrease of matrix effect [36, 37] (2) more possibilities for manipulations[31] (3) less time consuming due to decreased coating, competition and blocking times[38]; and (4) modification of the MBs in numerous ways, permitting different strategies of immobilization[39]. To the best of our knowledge, aptasensor based on MBs approach for Lys detection is not yet reported. Herein, we have demonstrated a sensitive, accurate and reliable novel strategy to quantify Lys in wine samples. The aim was to combining the advantages of MBs and optical techniques. To develop the assay, MBs were chosen to immobilize the Lys and coated in 96 microtiter plate. The assay is based on the competition between MBs coupled Lys and free Lys to bind with the biotin labeled aptamers. This strategy was further employed for the first time to detect Lys quantities in wine samples. The Lys recoveries (99.00-99.27%) were recorded in a good agreement with the spiked Lys concentrations. The assay demonstrates good stability with RSD of 3.87%. The advantages of the developed assay are their possible miniaturization, following portability and the use of minimal volume of samples and reagents with added advantages of high throughput sample analysis.
2. Experimental 2.1. Materials Potassium dihydrogen phosphate, sodium monophosphate, bovine serum alumina (BSA), lysozyme (Lys), casein, magnesium chloride, potassium chloride, sodium chloride, diethanolamine (DEA), N-hydroxysuccinimide (NHS), streptavidin-alkaline phosphatase
(Strep-ALP),N-(3-dimethylaminopropyle)-N’-ethyle-carbodiimide
hydrochloride (EDC) were purchased from Sigma (St. Louis, MO, USA). The whole milk was purchased from European Reference Material (Geel, Belgium). The carboxylic-functionalized Dynabeads (MBs) (10 mg/mL) were purchased from Invitrogen (USA). All reagents were analytical reagent grade. The aptamer was procured from Eurogenetic (France) with following sequences with modifications at the 3’ terminals: AptLysBio 5’- GCA GCT AAG CAG GCG GCT CAC AAA ACC ATT CGC ATG CGG C-Bio-3’ All solutions were prepared in MilliQ water. The buffers used were: binding buffer (BB) (1mM MgCl2, 2.7mM KCl, 140mM NaCl, 0.1mM Na2HPO4 and 1.8mM, KH2PO4 pH 7.4), MES buffer (100mM MES and 0.09%NaCl pH 6) and DEA buffer (10% DEA pH 9.5).
2.2. Apparatus The colorimetric measurements were performed with a Labsystems Multiskan EX microtiter plate reader (Thermo Life Sciences, Cergy Pontoise, France). Maxisorp 96microtiter plates were obtained from Nunc (Roskilde, Denmark). A horizontal shaker (IKA, Vibrax-VXR) was also used. 96 well microplates, PS, U-bottom were obtained from Greiner bio-one (France). Adem-Mag 96 (adapted for 96-well micro titer plates) and Adem-MagSV (single magnet position adapted for 1.5 mL micro tubes) were from Ademtech S.A (France). 2.3. Procedures 2.3.1 Immobilization of Lys on MBs
As described in section 2.1, the carboxylic-functionalized magnetic beads (MBs), Dynabeads were mixed properly to ensure their homogenous dispersions before taken out of the stock vial. A volume of 50 µL of MBs (10 mg/mL) were washed three times using 100 µL of BB to remove the preservatives. Then, 100µL of 100 mM EDC and 25 mM NHS were added and the mixture was incubated at room temperature for 1 h with gentle shaking. Later, the MBs were washed three times with 100 µL of BB and further incubated with 100µL of 1µM Lys for 1 h at room temperature with mixing in shaker. After incubation with Lys, the modified MBs were rinsed three times using BB and resuspended in it to store at 4 ºC. For control experiments, MBs were prepared by following the same procedure without addition of Lys solutions.
2.3.2 Optimization of various parameters Various assay parameters were optimized before proceeding for real tests. Optimization of aptamer concentrations for Lys detection was accomplished using 100 µL different concentrations as 0.312, 0.625, 1.25, 2.5 µg/ml (see conditions in section 2.3.1). Then incubation time between Lys and aptamer was investigated from 0-90 mins in each 15 mins interval. Similarly, optimization of Strep-ALP dilution for the accessibility and interaction with AptLysBio was tested using 100 µL of streptavidin-ALP added at dilutions ratios of 1: 1000, 1:10000, 1: 12500, 1:50000, 1:75000 and 1:100000 with in BB for 1h with shaking. Finally, Stp-ALP incubation with biotinylated aptamer for facile detection of Lysozyme was optimized. Various time durations such as 0, 20, 40, 60, 80 and 100 min was tested using 100 µL of p-NPP (4 mg.mL-1 in DEA buffer).
2.3.3 Colorimetric-based competitive aptasensor for Lys The modified MBs with Lys (1.5/100 of stock solution, 20 µL) were coated in the wells of microtiter plate. Further, the microtiter plate containing MBs were blocked with a 50 µL solution of 5% whole milk in BB for 1h by applying a gentle shaking to avoid nonspecific adsorption following the washing step. The competition step was performed using 50µL of Lys solution at different concentrations and 50µL of ApLysBio for 1h. Subsequently, 100 µL of streptavidin-ALP was added at dilution of 1/12500 in BB for 1h with shaking. 100 µL of p-NPP (4 mg.mL-1 in DEA buffer) were added with incubation period of 30 min at room temperature. The Lys detection strategy using aptasensor is illustrated in schematics 1. The absorbance was measured at λ=405 nm
using the microtiter plate reader. Based on the absorbance values, the % binding of Lys was calculated.
2.3.4 Preparation of wine samples Wine samples were prepared by following our previously reported work [2]. Briefly, 1 mL of wine sample was spiked with the desired concentration of Lys and allowed to stand for 3 min. Next, 200μL of 5M NaCl solution containing 5% Tween-20 surfactant were added to 200μL of Lys-wine mixture and diluted to a final volume of 1mL using 20 mM MES buffer pH 6 with 1mM MgCl2. This mixture was further centrifuged at 5000 rpm for 5 min and diluted 20 times using the buffer aforesaid. All washing steps were performed by adding three times 100µL of BB between steps and the supernatants were removed using a magnet.
3. Results and Discussions 3.1. Optimizations of Aptamer assay parameters The immobilization of Lys on MBs was very imperative to develop the aptamer assay. The colorimetric test was performed to authenticate the surface modification of MBs with Lys to evaluate the capability of MBs-Lys for development of competition tests, and also to optimize the different investigational parameters. The Lys coupled MBs were magnetically immobilized in to the micro-wells using Adem-Mag 96 magnet. The immobilized Lys and the free Lys in the solution competed for the anti AptLysTRANBio in solution. Further, the colorimetric detection was based on the hydrolysis of p-NP in the presence of Strep-ALP to the colored product p-nitrophenol (p-NP). First to augment various experimental conditions and parameters, initial assays were carried out with MBs-Lys. The coupling of MBs-Lys in to the micro wells was optimized by stimulating coating times ranging from 0 to 25 min. No significant change was observed in signal intensities; consequently the coating time was reduced to 5 min. The blocking step was also studied, to avoid non specific adsorption of aptamers. Various proteins such as casein (1%), BSA (1%) and whole milk (5%) were tried as blocking agents. The whole milk (5%) was chosen as the best among the three tested blocking agents. In order to enhance the sensitivity, optimization of other significant experimental
conditions such aptamer concentrations, as appropriate binding time of biotinylated aptamer (AptLysBio) and MBs-Lys, Str-ALP and AptLysBio and ALP -pNPP interaction are also very vital for aptamer assay development. Moreover, the optimized concentrations of AptLysBio and ALP-pNPP would improve the analytical features such as LOD, linear range, and sensitivity of the method. The attachments of Lys-MBs to aptamer certainly influence the attachment of Str-ALP to it, consequently enrichment in the signal. As represented in the figure 1, various aptamer concentrations were tested to choose an optimal quantity for the assay and 1µg/ml was found to be the best for Lys quantitation accurately. The suitable interaction time between Lys and aptamer was also assured while recording the response in different time duration starting from 0-90 min at the intervals of 15 min. It is revealed from the shown figure 2a that a time of 60 min was good enough to perform the assays and to establish a calibration curve. To find out the best enzyme concentrations in the assay, experiments with different dilutions of Str-ALP were sequentially performed to see the signal dynamics with respect to the dilutions. Figure 2b shows the dilution effects on absorbance. To access the stability of Lys-MBs, the modified beads were stored at 4 ºC for two weeks. The obtained results were similar to those obtained with freshly prepared Lys-MBs, indicating no detectable loss of the activity. The Stp-ALP incubation with biotinylated aptamer was also evaluated to increase the sensitivity of the assay. Figure 2c shows that among tested time durations, 60 min was turning out to be the best duration for Str-ALP coupling with ALP biotin.
3.2 Lys detection using Aptamer assay Using the optimized experimental parameters, the calibration curve was performed with free Lys solution. The calibration curve was constructed based on the % binding of Lys on to the aptamer vs Lys concentration. The percentage binding was calculated based on the absorbance recorded of each individual concentrations of Lys. The curve obtained with colorimetric assays for the Lys-MBs is shown in figure 3, which showed a broad dynamic range from 5-220 nM with an excellent linearity (Y= 0.00494x+0.02212, R2=0.9999) ranging from 5-140 nM. Due to the percentage error (5%), the LOD was defined as the toxin concentration corresponds to the 80% of anti Lys-Apt binding depending upon the maximum value of standard deviation. The developed Lys-Apt assay demonstrates a good possibility for real tests in Lys contaminated samples. We obtained a good LOD of Lys upto 10 nM as this concentration may easily be distinct to
10 nM and 20 nM with good sensitivity of 0.0050 nM. Table 1 summarizes the analytical values of the developed assay consisting of correlation coefficient R, midpoint (IC) and LOD values derived from the regression equation, obtained for Lys-Apt colorimetric assay with MBs.
3.3 Application of developed assay in wine sample analysis To inspect the viability and assay accuracy of Lys-Apt colorimetric autosensing platform, spiked wine samples with various concentrations of target molecule Lys were analyzed by developed strategy to see the interferences from the extract. The recovery test also signifies the suitability of the biomolecule towards the matrix (wine). Initially, wine samples were diluted to different concentrations with 5M NaCl solution containing 5% Tween-20 surfactant which were added to 200 μL of Lys-wine mixture and diluted to a final volume of 1mL using 20 mM MES buffer pH 6 with 1mM MgCl2. Ultimately, 3 different Lys concentrations, 10, 50 and 110 nM were spiked and tested in wine. The concentrations were chosen from the linear range (LOD, and mid-point value). No countable difference was observed with respect to linear working range hence, observed optical data was compared to the calibration curves obtained for the aptamer based assay. The relatively stumpy matrix effect could be explicated by the specificity of the aptamers towards Lys and also the blocking proteins. This inordinate advantage validated our developed assay for real sample analysis. Moreover, all agreements with calibration, assay demonstrated a good recovery range from 99% to 99.27% represented in Table 2. Consequently, the Lys-Apt-based colorimetric autosensing could be applied for quantitative monitoring of Lys in the complex system. The precision and stability of the assays was calculated by the %RSD for the triplicate measurements.
3.4 Aptamer assay selectivity The specificity of the developed assay was assessed by employing Lys-Apt biorecognition event. For assay specificity to Lys, control experiments were performed using three most common proteins, Casein, BSA and Cyt C present in one solution as well as individually. It was noticed from figure 4 that, when BSA (250nM), Casein (250 nM) and Cyt C (250 nM) were incubated and detected individually with the aptasensor, only Lys induced a significant response, while the others had very less responses when they are present in higher concentration level (5 times more than lysozyme). As
demonstrated, no obvious interference was obtained for other three proteins and a good specificity of the aptasensor to Lys could be obtained. These results are strong proof of the highly selective detection of Lys. No cross reactions were observed from similar interfering proteins, even in the case of cytochrome c, which is structurally similar to Lys. The response of cytochrome c was negligible in the presence of AptLysTRAN.
4. Conclusions This work presents for the first time an MB mediated aptamer assay to detect Lys in wine samples. The assay preparation is simple including the interaction of biotinylated Lys-apt with Lys couples on MBs. During the procedure of detection, the aptamer interacts Lys and could be able to provide signals. Under the optimized experimental parameters, the optical signals were proportional to the varied Lys concentrations thus, obtained a good calibration data for Lys. The LOD was 10 nM with excellent linearity 5-140 nM for Lys. The developed assay showed potential application for small target molecules and protein quantitation using colorimetry with possible extension to similar molecules detections. The assay is rapid, simple, sensitive and selective route for such analytes detection in real samples.
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Figure Legends
Scheme 1: Figure illustrating the strategies and steps of Lysozyme detection using aptamer and MBs combination.
Figure 1: Optimization of aptamer concentration for Lys detection assay. Uncertainty values corresponding to replicated experiments (n = 3). Figure 2a: The suitable interaction time between Lys and aptamer under the optimized parameters. Figure 2b: Optimization of Strep-ALP dilution for the accessibility and interaction with AptLysBio. Figure 2c: Representation of Stp-ALP incubation with biotinylated aptamer for facile detection of Lysozyme. Figure 3: Calibration curve of lysozyme using colorimetry based on competition assay and binding to lysozyme. Error bars represents standard deviation of the mean, n=3. Figure 4: Specificity of the developed aptasensor for Lys detection in the presence of other co-contaminants (Casein, BSA and Cyt C).
Table Legends
Table 1: Analytical features of developed aptasensor for Lys.
Analytical parameters
Experimental findings
Dynamic range
5-220 nM
Linearity
5-140 nM
LOD
10 nM
Specificity
Yes (Lys)
Mid point
110 nM
2
R
0.9999
Sensitivity
0.005 nM
Table 2: Recovery percentage for spiked Lys in wine samples using colorimetric test.
Spiked Lys (nM)
Found Lys (nM)
R.S.D. %
Recovery %
A.E.%
10
9.9
3.82
99.00
15.6
50
49.6
3.60
99.20
16.2
110
109.2
3.75
99.27
15.9
Highlights · · ·
Highly sensitive lysozyme detection route. First report on MB coupled aptamer assay. Assay based on unique combination of biotinylated aptamer and Streptavidin interaction.
Lys-Apt binding %
100 80 60 40 20 0 10
[Lys] nM
100
Scheme 1
Scheme 1
Figure 1
Fig 1
Absl = 405 nm 0.1
0.2
0.3
0.4
0.5
0.6
0.0
0.5
1.5
2.0
[Apt-Lys-Bio] (mg/ml)
1.0
2.5
Fig 2a
Figure 2a
Absl = 405 nm
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0
20
60
Time (min)
40
80
100
Fig. 2b
Figure 2b
Absl = 405 nm
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0
1/1000
1/12500
1/50000
1/75000
Dilutions of strep-ALP
1/10000
1/100000
Fig. 2c
Figure 2c
Absl = 405 nm
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0
20
60
Time (min)
40
80
100
Fig. 3
Figure 3
Lys-Apt binding % 0
20
40
60
80
100
10
[Lys] nM
100
Fig. 4
Figure 4
Absl = 405 nm
0.0
0.2
0.4
0.6
0.8
1.0
Lys
Casein
BSA
Cyt C
Table 1
Table 1
Experimental findings 5-220 nM 5-140 nM 10 nM Yes (Lys) 110 nM 0.9999
0.005 nM
Analytical parameters Dynamic range Linearity LOD Specificity Mid point R2
Sensitivity