toxins Article
Label-Free Optical Detection of Mycotoxins Using Specific Aptamers Immobilized on Gold Nanostructures Ali Ghanim Al-Rubaye 1,2, * ID , Alexei Nabok 1 Eszter Takács 4 and András Székács 4 ID 1 2 3 4
*
ID
, Gaelle Catanante 3 , Jean-Louis Marty 3 ,
Materials and Engineering Research Institute, Sheffield Hallam University, Sheffield S1 1WB, UK;
[email protected] Basra Technical Institute, Southern Technical University, 61002 Basra, Iraq Department of Biochemistry and Molecular Biology, University of Perpignan, 66100 Perpignan, France;
[email protected] (G.C.);
[email protected] (J.-L.M.) Agro-Environmental Research Institute, NARIC, 1011-1239 Budapest, Hungary;
[email protected] (E.T.);
[email protected] (A.S.) Correspondence:
[email protected]
Received: 26 June 2018; Accepted: 13 July 2018; Published: 16 July 2018
Abstract: This work focuses on the development of the novel label-free optical apta-sensors for detection of mycotoxins. A highly sensitive analytical method of total internal reflection ellipsometry (TIRE) combined with Localized Surface Plasmon Resonance (LSPR) phenomenon in nano-structured gold films was exploited here for the first time for detection of aflatoxin B1 and M1 in direct assay with specific aptamers immobilized on the surface of gold. The achieved detection of low molecular weight molecules, such as aflatoxin B1 and M1, in a wide range of concentrations from 100 ng/mL down to 0.01 ng/mL is remarkable for the LSPR method. The study of binding kinetics of aflatoxin molecules to their respective aptamers using dynamic TIRE measurements yielded the values of affinity constants in the range of 10−8 –10−7 mol, which is characteristic for highly specific aptamer/target interactions similar to that for monoclonal antibodies. The effect of aptamers’ DNA chain length on their binding characteristics was analyzed. Keywords: optical label-free biosensor; mycotoxins; aptamer; LSPR; gold nanostructures; total internal reflection ellipsometery Key Contribution: The development of label-free optical aptasensor based on the LSPR phenomenon in nano-structured gold films suitable for detection of low molecular weight analytessuch as mycotoxins (aftatoxin B1 and M1).
1. Introduction This work is dedicated to the detection of mycotoxins produced by different fungi species that may grow on various agricultural products, such as grains, nuts, coffee beans, spices, fruits, and associated food and animal feed, stored at inappropriate conditions, i.e., elevated temperatures and high humidity [1]. Because of the toxic, carcinogenic, and endocrine disruptive properties of mycotoxins health and environmental control organizations worldwide established very low limits (typically in sub-ppb level of concentrations) for mycotoxins in food and feed, which makes their detection extremely difficult, especially considering their low molecular weight [2]. Although the modern analytical methods such as HPLS or mass spectroscopy can cope with these demands, the above mentioned methods are expensive and time consuming. Therefore the development of biosensors Toxins 2018, 10, 291; doi:10.3390/toxins10070291
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suitable for express and cost-effective detection of mycotoxins is of great demand nowadays [3]. Optical biosensors lead this development with a number of biosensors based on the different instrumentation and principles (i.e., Surface Plasmon Resonance (SPR), Total Internal Reflection Ellipsometry (TIRE), Optical Waveguide Lightmode Spectroscopy (OWLS), Mach–Zehnder (MZ) interferometers, etc.) [3]. We would like to highlight the method of total internal reflection ellipsometry (TIRE) which was developed in the last 10–15 years, as a highly sensitive analytical tool in bio-sensing, particularly attractive for the detection of low molecular weight analytes, and mycotoxins in particular [4–8]. Another interesting optical phenomenon of localized surface plasmon resonance (LSPR) [9] has recently attracted the attention of researches because of its potentially high sensitivity allowing the detection of single molecules [10]. Localized surface plasmon resonance (LSPR) is the phenomenon related to the collective oscillation of conducting electrons in resonance with incident an electromagnetic field that typically appear in metal nanostructures with dimensions comparable to the wavelength of light. LSPR is typically manifested as an absorption band in the middle of visible spectral range [11], position of which is affected by refractive index of the medium; the latter fact actually constitutes the principle of LSPR sensing since small changes of local refractive index due to absorption of molecules cause a shift of LSPR band to higher wavelengths [12]. The sensitivity of LSPR sensors based on UV-vis absorption spectroscopy is not very high and limit the use of LSPR for the detection of rather large protein molecules. A combination of LSPR and TIRE methods increases the sensitivity further and allows the detection of small molecules such as mycotoxins. The detection of aflatoxin B1 in concentrations down to 0.1 ng/mL was achieved recently in immunoassay with specific monoclonal antibodies using a combination of LSPR and TIRE [13]. One of the obstacles to achieving a high sensitivity of LSPR via immunosensors lies in much smaller evanescent field decay length as compared to traditional SPR [14,15]. From that point of view, the use of artificial bio-receptors, such as aptamers having much smaller dimensions than antibodies could be beneficial. Aptamers are short single strand DNA or RNA oligomers synthesized to accommodate particular target molecules of inorganic, organic, and bio-organic origin by selecting nucleotides from a large random sequence pool using SELEX process [16,17]. Generally, aptamers are getting increasingly common in biosensing, effectively replacing antibodies [18]. Aptamers can be immobilised on the surface via functional groups, such as thiols, on one end and may contain either fluorescent or redox labels on the other end, and are therefore used in optical or electro-chemical biosensors. We recently reported on the use of aptamers in conjunction with the TIRE method for the detection of ochratoxin A. Where the achieved low detection limit (LDL) was in the range of 0.01 ng/mL [19]. The novel and important part of that work was the use of unlabeled aptamers. One of the advantages of our sensing technology, e.g., ellipsometry, is in the high accuracy of measurements of the thin films’ thickness. Where the process of aptamers wrapping round the target molecules is accompanied by reduction in the aptamer layer thickness, which was actually recorded in our experiments [19]. In this work, we attempted the detection of aflatoxins B1 and M1 in assay with specific aptamers using a combination of the Localized Surface Plasmon Resonances phenomenon in nanostructured gold films with the method of TIRE. 2. Results and Discussion 2.1. Study of Aflatoxin/Aptamer Binding Using LSPR/TIRE Figure 1a shows a set of ∆ spectra recorded on our LSPR transducers functionalized with anti- aftatoxin (anti-AFT) B1 after consecutive binding steps of aflatoxin B1 starting from small concentrations. A progressive “blue” (to shorter wavelength) spectral shift was observed, which is typically associated with the reduction in the aptamer layer thickness due to aptamers coiling around the target molecules. Similar results were obtained for binding aflatoxin M1 to its specific aptamer (see Figure 1b), however the saturation of the response was observed at the much smaller concentrations of AFT M1 of 1 ng/mL, which could be caused by a smaller concentration of active
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aptamers immobilized the was surface. In addition, the cross-sensitivity of the aptamers tested, tested, and no spectralonshift recorded when attempted binding AFT B1two to an aptamerwas specific to and no spectral shift was recorded when attempted binding AFT B1 to an aptamer specific to AFT M1, tested, and no shift Also, was recorded attempted AFT to an aptamer to AFT M1, andspectral vice versa. both thewhen anti-AFT B1andbinding anti-AFT M1B1aptamers do notspecific bind OTA and vice versa. Also, bothAlso, the anti-AFT anti-AFT M1anti-AFT aptamersM1 do aptamers not bind OTA (ochratoxin A). AFT M1, and A). vice versa. both theB1and anti-AFT B1and do not bind OTA (ochratoxin
(ochratoxin A). Au-5nm-nano. detection of aflatoxin b1 300
Au-5nm-nano. detection of aflatoxin b1
300
Δ in degrees Δ in degrees
200 200 100
Aptamer Aflatoxin b1 0.01ng-ml Aflatoxin b1 0.1ng-ml Aptamer Aflatoxin b1 1ng-ml Aflatoxin b1 0.01ng-ml Aflatoxin b1 10ng-ml Aflatoxin b1 0.1ng-ml Aflatoxin b1 100ng-ml Aflatoxin b1 1ng-ml Aflatoxin b1 10ng-ml Aflatoxin b1 100ng-ml
100 0 0
-100 504 -100 504
(a) (a)
523 523
542 562 Wavelength (nm) 542 562 Wavelength (nm)
581 581
600 600
(b) (b)
Figure1.1.Series SeriesofofTIRE TIRE∆∆spectra spectrarecorded recordedupon uponbiding bidingAFT AFTB1 B1(a) (a)and andAFT AFTM1 M1(b) (b)totorespective respective Figure Figure 1. Series of TIRE ∆ spectra recorded upon biding AFT B1 (a) and AFT M1 (b) to respective specific aptamers. specific aptamers. specific aptamers.
The fitting of the TIRE spectra to a three layer model [13], allowed the evaluation of the aptamer Thefitting fitting ofthe theTIRE TIREspectra spectratotoaathree threelayer layermodel model[13], [13],allowed allowedthe theevaluation evaluationofofthe theaptamer aptamer The layer thicknessofthat is shown in Figure 2. The absolute values of the aptamer layer thickness, which layer thickness that is shown in Figure 2. The absolute values of the aptamer layer thickness, which layer thickness that is shown Figure 2. The absolute values of the aptamer layer thickness, whichof depends on several factors,in i.e., aptamer chain length, concentration of aptamers, concentration dependson onseveral severalfactors, factors, i.e.,aptamer aptamerchain chainlength, length,concentration concentrationof ofaptamers, aptamers,concentration concentrationofof depends buffer, etc., arei.e., not informative actually. The main interest was in the changes of aptamer MgCl2 in the MgCl2 2ininthe thebuffer, buffer,etc., etc.,are arenot notinformative informativeactually. actually.The Themain maininterest interestwas wasininthe thechanges changesofofaptamer aptamer MgCl layer thickness upon binding the target molecules. The decrease in the anti-AFT B1 aptamer layer layerthickness thicknessupon uponbinding bindingthe thetarget targetmolecules. molecules.The Thedecrease decreaseininthe the anti-AFTB1 B1aptamer aptamerlayer layer layer thickness (14.2%), upon increasing the concentration of AFT B1, is muchanti-AFT more noticeable (Figure 2a) thickness (14.2%), upon increasing the concentration of AFT B1, is much more noticeable (Figure 2a) thickness upon the concentration of the AFTrespective B1, is much more noticeable than that(14.2%), (2.6%) for theincreasing layer of anti-AFT M1 binding target (Figure 2b). (Figure 2a) than that (2.6%) for the layer of anti-AFT M1 binding the respective target (Figure 2b). than that (2.6%) for the layer of anti-AFT M1 binding the respective target (Figure 2b).
Figure 2. Dependences of the aptamer layer thickness vs concentration of AFT B1 (a) and AFT M1 (b). Figure Dependences theaptamer aptamer layer thickness concentration AFTB1 B1 (a)and andAFT AFTM1 M1(b). (b). Figure 2.2.Dependences ofofthe layer thickness vsvsconcentration ofofAFT (a) The processes of aptamer/target binding are illustrated by the corresponding insets. Theprocesses processesofofaptamer/target aptamer/targetbinding bindingare areillustrated illustratedby bythe thecorresponding correspondinginsets. insets. The
As was suggested, the decrease in the aptamer layer thickness upon binding aflatoxins is caused suggested, the ininthe layer thickness binding isiscaused by As coiling around the target molecules, this processupon is schematically illustrated on inset Aswas wasaptamers suggested, thedecrease decrease theaptamer aptamerand layer thickness upon bindingaflatoxins aflatoxins caused by coiling aptamers around the target molecules, and this process is schematically illustrated on in Figure 2a. The differences in the binding abilities of anti-AFT B1 and anti-AFT M1 are most-likely by coiling aptamers around the target molecules, and this process is schematically illustrated oninset inset ininrelated Figure 2a. The differences binding abilities ofofanti-AFT B1 M1 are their chain length (50 and 21, respectively). The much shorter aptamer to Figureto 2a. Theoligonucleotides differencesininthe the binding abilities anti-AFT B1and andanti-AFT anti-AFT M1 aremost-likely most-likely related tototheir chain length (50(50 and 21, respectively). TheThe much aptamer to AFT M1 may not fully wrap the target molecule, but rather have a small bend inshorter the chain (see inset related theiroligonucleotides oligonucleotides chain length and 21, respectively). much shorter aptamer AFT M1 may not fully wrap the target molecule, but rather have a small bend in the chain (see inset in Figure 2b), which is still detectable using a sensitive ellipsometry instrument. The spectral to AFT M1 may not fully wrap the target molecule, but rather have a small bend in the chain (see ininset Figure 2b), which is still detectable using a sensitive instrument. The spectral resolution of our instrument did not the ellipsometry detection of aflatoxins below 0.01 ng/mL. in Figure 2b),ellipsometry which is still detectable using aallow sensitive ellipsometry instrument. The spectral resolution of our ellipsometry instrument did not allow the detection of aflatoxins below 0.01 ng/mL. Nevertheless, the minimal detected concentration of 0.01 ng/mL for both aflatoxins corresponded to resolution of our ellipsometry instrument did not allow the detection of aflatoxins below 0.01 ng/mL. Nevertheless, the minimal detected concentration of 0.01 ng/mL for both aflatoxins corresponded to part per billion, is adetected remarkable result for direct format. Nevertheless, thewhich minimal concentration of 0.01aptamer ng/mL assay for both aflatoxins corresponded to part per billion, which is a remarkable result for direct aptamer assay format. part per billion, which is a remarkable result for direct aptamer assay format. 2.2. Study of Aflatoxin-Aptamer Binding Kinetics 2.2. Study of Aflatoxin-Aptamer Binding Kinetics Following the procedure described earlier [6–8,13], the study of the kinetics of binding AFT B1 the procedure described [6–8,13], the study of the kinetics of binding B1 to aFollowing specific aptamer was carried out byearlier recording a number of TIRE spectra during bindingAFT reaction, toand a specific aptamer was carried out by recording a number of TIRE spectra during binding reaction, then plotting the time dependences of the values of Ψ or Δ at the particular wavelength. Figure and then plotting the time dependences of the values of Ψ or Δ at the particular wavelength. Figure
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2.2. Study of Aflatoxin-Aptamer Binding Kinetics Following the procedure described earlier [6–8,13], the study of the kinetics of binding AFT B1 to a specific aptamer was carried out by recording a number of TIRE spectra during binding reaction, and Toxins then 2018, plotting thePEER timeREVIEW dependences of the values of Ψ or ∆ at the particular wavelength.4Figure 3a 10, x FOR of 8 shows a typical time dependence of ∆ at 600 nm during binding of 10 ng/mL of AFT B1 to specific 3a shows a typical time dependence of Δ at 600 by nm fitting during the binding 10the ng/mL of AFT B1 to specific aptamer. The time constant τ was evaluated dataofto rising exponential function. aptamer. The time constant τ was evaluated by fitting the data to the rising exponential function. The The same procedure was repeated for different concentrations of AFT B1, and the obtained values of τ same procedure was repeated for different concentrations of AFT B1, and the obtained values of τ were plotted as 1/τ against AFT B1 concentration (C) in Figure 3b. were plotted as 1/τ against AFT B1 concentration (C) in Figure 3b.
Figure 3. Typical time dependence of ∆ during binding AFT B1 of 1 ng/mL to aptamers (a); evaluation
Figure 3. Typical time dependence of ∆ during binding AFT B1 of 1 ng/mL to aptamers (a); evaluation of the association constant KA (b). of the association constant KA (b).
The absorption rate ( ) and desorption rate ( ) were found, respectively, from the gradient and intercept of the linear 1/τ = rate in(kFigure 3b.found, Then, respectively, the associationfrom and affinity The absorption rate (k a )graph and desorption the gradient d ) were constants were found as K A = ka/kd and KD = 1/KA. The obtained values of KA = 3.12·107 (mol−1) and KD and intercept of the linear graph 1/τ = k a C + k d in Figure 3b. Then, the association and affinity −8 (mol) shows high specificity of AFT B1/aptamer interaction, similar to that of monoclonal = 3.2 × 10 constants were found as KA = ka /kd and KD = 1/KA . The obtained values of KA = 3.12·107 (mol−1 ) antibodies to AFT B1 [8]. and KD = 3.2 × 10−8 (mol) shows high specificity of AFT B1/aptamer interaction, similar to that of Asimilar analysis was carried out for binding the kinetics of AFT M1 to its respective aptamer monoclonal antibodies to AFT B1 [8]. 6 and yielded the values of KA = 4.64 × 10 (mol−1) and KD = 2.1 × 10−7 (mol). The obtained values of the Asimilar analysis carried for binding thereported kineticsinof[17,20]. AFT M1 its respective aptamer affinity coefficient forwas anti-AFT B1 out are similar to those Thetoaffinity of a shorter 6 − 1 − 7 and anti-AFT yielded the KA = 4.64 (mol ) and × 10 (mol). Thea obtained values of D = 2.1 M1 values aptamerofappeared to × be 10 much smaller thanKthat of anti-AFT B1 having longer chain. the affinity coefficient forsuggestion anti-AFT B1 areeffect similar to those reported in [17,20]. The affinity of a shorter This proved our initial of the of the aptamer nucleotide chain length on its affinity.
anti-AFT M1 aptamer appeared to be much smaller than that of anti-AFT B1 having a longer chain. Conclusions This3.proved our initial suggestion of the effect of the aptamer nucleotide chain length on its affinity. The combination of a simple LSPR transducer based on nanostructured gold films and the
3. Conclusions optical method of total internal reflection ellipsometry (TIRE) allowed for label-free detection of small
molecules of aflatoxinof in adirect assay with specific aptamers immobilized on the surface of gold. The combination simple LSPR transducer based on nanostructured gold filmsThe and the observed decrease in the aptamer layer thickness upon the binding of their specific target molecules optical method of total internal reflection ellipsometry (TIRE) allowed for label-free detection of directly confirms the quite obvious model of aptamers wrapping round the target. The concentration small molecules of aflatoxin in direct assay with specific aptamers immobilized on the surface of gold. range for aflatoxin B1 detection stretches from 0.01 ng/mL to 100 ng/mL, where the minimal detected The concentration observed decrease in the aptamer layer to thickness the binding specific biosensors, target molecules of aflatoxins corresponding 0.01 ppbupon is remarkably lowof fortheir LSPR-based directly confirms the quite obvious model of aptamers wrapping round the target. The concentration especially considering the small molecular weight of aflatoxin toxin molecules and direct aptamer range for format. aflatoxin B1 sensitivity, detection stretches from ng/mL to as 100 ng/mL, wheremethods the minimal detected assay Such although it may0.01 not be as good that for advance of HPLC or mass spectroscopy, is corresponding suitable for the to detection of ismycotoxins in low concentrations close to the concentration of aflatoxins 0.01 ppb remarkably for LSPR-based biosensors, legislated limits [2], but costmolecular and time of the analysis is definitely lower.and Thedirect study aptamer of especially considering the the small weight of aflatoxin toxin much molecules binding kinetics of AFT B1 and AFT B1 to specific aptamers confirmed the high specificity of the assay format. Such sensitivity, although it may not be as good as that for advance methods of HPLC or withfor thethe affinity constants found to be tens of nano-moles, which are massaptamer/target spectroscopy,reaction is suitable detection of mycotoxins inin concentrations close to the legislated similar to the values reported earlier [17,20] and similar to those of antigen/antibody reactions [5–8]. limits [2], but the cost and time of the analysis is definitely much lower. The study of binding kinetics The shorter-chain aptamer to AFT M1 appeared to be less specific as compared to longer-chain of AFT B1 and AFT B1 to specific aptamers confirmed the high specificity of the aptamer/target aptamer to AFT B1. To our knowledge, this is the first report on the experimental evaluation of reaction withaffinity. the affinity constants found to be in tens of nano-moles, which are similar to the values aptamers
reported earlier [17,20] and similar to those of antigen/antibody reactions [5–8]. The shorter-chain
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aptamer to AFT M1 appeared to be less specific as compared to longer-chain aptamer to AFT B1. To our knowledge, this is the first report on the experimental evaluation of aptamers affinity. Toxins 2018, 10, x FOR PEER REVIEW 4. Experimental Details
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4. Experimental Details 4.1. TIRE Experimental Set-Up 4.1. TIRE Set-Up The TIREExperimental experimental set-up shown schematically in Figure 4a–c is based on M2000, J.A. Woollam, The spectroscopic ellipsometer (a) shown operating in the 370–1680 nm wavelength range. The principles TIRE experimental set-up schematically in Figure 4a–c is based on M2000, J.A. of TIRE were described in detail previously Briefly, the 370–1680 method of TIRE combinesrange. the advanced Woollam, spectroscopic ellipsometer (a) [4–8]. operating in the nm wavelength The spectroscopic with traditional Kretschmann SPR geometry. The addition principlesellipsometry of TIRE wereinstrumentation described in detail previously [4–8]. Briefly, the method of TIRE combines ◦ glass the advanced ellipsometry traditional SPR into of a 68 prism spectroscopic (b) provides the conditionsinstrumentation of total internalwith reflection for theKretschmann coupling of light geometry. The addition of a 68° glass prism (b) provides the conditions of total internal reflection for(c) is thin gold film (either continuous or nano-structured) deposited on glass. A 0.2 mL PTFE cell the coupling of light into thin gold film (either continuous or nano-structured) deposited on glass. A attached to the sample and allows the injection of different solutions into the cell to study different 0.2 mL PTFE cell (c) is attached to the sample and allows the injection of different solutions into the biochemical reactions. The TIRE method has an advantage of recoding two ellipsometric parameters Ψ cell to study different biochemical reactions. The TIRE method has an advantage of recoding two and ∆, which represent, respectively, the amplitude ratio and phase shift between p- and s-components ellipsometric parameters Ψ and Δ, which represent, respectively, the amplitude ratio and phase shift of polarized betweenlight. p- and s-components of polarized light.
Figure 4. Schematic diagram of TIRE experimental set-up: spectroscopic ellipsometer M2000 (a), 68°
Figure 4. Schematic diagram of TIRE experimental set-up: spectroscopic ellipsometer M2000 (a), prism (b), reaction cell (c). Typical TIRE spectra of 25 nm thick Au film deposited on glass (d). 68◦ prism (b), reaction cell (c). Typical TIRE spectra of 25 nm thick Au film deposited on glass (d). Reproduced from [5], Copyright 2008, Elsevier, New York, NY, USA. Reproduced from [5], Copyright 2008, Elsevier, New York, NY, USA.
Figure 4d shows typical TIRE spectra of Ψ and Δ recorded on continuous 25 nm thick gold film deposited glass,typical where TIRE Ψ spectrum curve, while25Δnm spectrum Figure 4d on shows spectra resembles of Ψ and a∆traditional recorded SPR on continuous thick gold represents a phase drop near the resonance [5]. The parameter of Δ is particularly sensitive to film deposited on glass, where Ψ spectrum resembles a traditional SPR curve, while changes ∆ spectrum in thearefractive index of the a medium, and[5]. thus, molecular of adsorption. Small changes in to either represents phase drop near resonance Thetoparameter ∆ is particularly sensitive changes thickness or refractive index of the molecular layer deposited on gold cause the shift of the Δ in the refractive index of a medium, and thus, to molecular adsorption. Small changes in either spectrum; the values of thickness can be obtained by the data fitting and can be subsequently used thickness or refractive index of the molecular layer deposited on gold cause the shift of the ∆ spectrum; as the TIRE sensor output [5]. In addition to standard TIRE spectroscopic measurements, we can the values can be obtained the data fitting and cantobestudy subsequently as the TIRE recordof thethickness time dependencies of Ψ andby Δ at particular wavelengths the kineticsused of molecular sensor output [5]. In addition adsorption or binding [5–8].to standard TIRE spectroscopic measurements, we can record the time
dependencies of Ψ and ∆ at particular wavelengths to study the kinetics of molecular adsorption or 4.2. [5–8]. Preparation of LSPR Transducers binding The fabrication of nanostructured gold films for LSPR experiments is schematically outlined in
4.2. Preparation of LSPR Transducers Figure 5. Standard microscopic glass slides are cleaned with hot “piranha” solution (1:3 H2O2/H2SO4)
for 1fabrication h, then rinsed deionized water dried stream of nitrogen gas. A layer of gold in The of with nanostructured goldand films forunder LSPRaexperiments is schematically outlined −7 Torr vacuum using Edwards 360 metal was thermally evaporated on clean glass slides in 10 Figure 5. Standard microscopic glass slides are cleaned with hot “piranha” solution (1:3 H2 O2 /H2 SO4 ) evaporation unit. The thickness of Au layers was in the range from 5 nm to 10 nm and controlled by for 1 h, then rinsed with deionized water and dried under a stream of nitrogen gas. A layer of gold was Quartz Crystal Microbalance QCM sensor during evaporation. The gold coated slides were then thermally evaporated on clean glass slides in 10−7 Torr vacuum using Edwards 360 metal evaporation annealed in air at 550–580 °C for 10 h as described in [10], which transforms the continuous gold films unit. into The nanostructures thickness of Au layers was in the range from 5gold nm to 10 nm andthe controlled by Quartz consisting of randomly distributed islands with average lateral Crystal Microbalance QCM sensor during evaporation. The gold coated slides were then annealed dimensions of 100 to 200 nm and exhibiting a distinctive LSPR band in visible spectral range ◦ in air[10,13,15]. at 550–580 C for 10 h as described in [10], which transforms the continuous gold films into
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nanostructures consisting of randomly distributed gold islands with the average lateral dimensions of 100Toxins to 200 nm and exhibiting a distinctive LSPR band in visible spectral range [10,13,15]. 2018, 10, x FOR PEER REVIEW 6 of 8
Figure 5. The sequence of steps of samples preparation. Figure 5. The sequence of steps of samples preparation.
4.3. Aptamers and other Chemicals 4.3. Aptamers and other Chemicals The aptamers used in our work were synthesized and purified by Microsynth (Schutzenstrasse, The aptamers used in our work were synthesized purified by (Schutzenstrasse, Balgach, Switzerland). The sequence of nucleotides for and anti-aflatoxin B1 Microsynth (AFT B1) and anti-aflatoxin Balgach, Switzerland). The sequence of nucleotides for anti-aflatoxin B1 (AFT B1) and M1 (AFT M1) aptamers, which were established previously in [17,20] are shown below: anti-aflatoxin M1 (AFT M1) aptamers, which were established previously in [17,20] are shown below: for AFT 5′SH-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3′ 0 SH-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-30 for AFT B1,5 and 5′SH-ACTGCTAGAGATTTTCCACAT-3′ for AFT M1. 0 B1, andFor 5 SH-ACTGCTAGAGATTTTCCACAT-3 for AFT M1. our purposes, we use aptamers with no 0labels on 3′ termini, but only SH groups at 5′ termini forFor immobilization onwe theuse surface of gold. our purposes, aptamers with no labels on 30 termini, but only SH groups at 50 termini Aflatoxin B1on and were of purchased from Sigma-Aldrich. The other chemicals used were for immobilization theM1 surface gold. magnesium chloride (MgCl 2 ), Dithiothreitol (DTT) from Sigma-Aldrich. Allchemicals reagents used were were of Aflatoxin B1 and M1 were purchased from Sigma-Aldrich. The other analytical grade. Deionized Milli-Q water was used for preparation of reagents throughout the magnesium chloride (MgCl2 ), Dithiothreitol (DTT) from Sigma-Aldrich. All reagents were of analytical experiments. grade. Deionized Milli-Q water was used for preparation of reagents throughout the experiments. Hepes binding buffer (HBB, mM) was prepared dissolving mM Hepes sodium salt Hepes binding buffer (HBB, 5050 mM) was prepared byby dissolving 5050 mM Hepes sodium salt (3 (3 mM mM MgCl 2, 120 mM NaCl, and 5 mM KCl) in deionized Milli-Q water. The pH of the buffer was MgCl2 , 120 mM NaCl, and 5 mM KCl) in deionized Milli-Q water. The pH of the buffer was adjusted to to 7.4. Similarly, phosphate binding buffer (PBB, 100 mM) was prepared by dissolving 10 7.4.adjusted Similarly, phosphate binding buffer (PBB, 100 mM) was prepared by dissolving 10 mM Na2 HPO4 , mM Na2HPO4, 1.76 mM KH2PO4, 3 mM MgCl2, 2.7 mM KCl, and 137 mM NaCl in deionized water. 1.76 mM KH2 PO4 , 3 mM MgCl2 , 2.7 mM KCl, and 137 mM NaCl in deionized water. The pH of the The pH of the buffer was adjusted to 7.4. The presence of MgCl2 in both HBB and PBB is crucial for buffer was adjusted to 7.4. The presence of MgCl2 in both HBB and PBB is crucial for preventing preventing self-coiling of aptamers. self-coiling of aptamers. The aptamers are delivered in lyophilized form. Therefore, aptamer stock solution is prepared The aptamers are delivered in lyophilized form. Therefore, aptamer stock solution is prepared at 100 µM by adding an appropriate volume of de-ionized water. For long-term storage, as-received at 100 µM bywere adding an appropriate volume of de-ionized water.water For long-term as-received aptamers prepared in small 100 µM aliquots in deionized and storedstorage, frozen. The stock aptamers prepared smallat100 aliquots in deionized and stored frozen. The stock solutionwere of aptamer wasin diluted theµM desired concentration withwater HBB or PBB supplemented with 1 solution of aptamer was diluted at the desired concentration with HBB or PBB supplemented with mM of DTT and then subjected to heating 90 °C for 5 min and cooling 4 °C for 5 min cycle in PCR 1 mM of DTT anduse. then subjected to heating 90 ◦ C for 5 min and cooling 4 ◦ C for 5 min cycle in PCR machine before machine before use. Aflatoxin B1 and M1 stock solution (1 mg mL−1) were prepared in acetonitrile. In addition, −1 ) were prepared in acetonitrile. In addition, Aflatoxin B1 and M1were stockprepared solutionin(1 mgor ·mL further diluted solutions HBB PBS. We maintained the percentage of acetonitrile further were prepared in HBB or PBS. We maintained percentage of acetonitrile at lessdiluted than 2%solutions in the final assay throughout the experimental procedure. the All of the working solutions werethan prepared before and storedthe at 4experimental °C when not procedure. in use. at less 2% infreshly the final assayuse throughout All of the working solutions were prepared freshly before use and stored at 4 ◦ C when not in use. 4.4. Immobilization of Aptamers on Gold 4.4. Immobilization of Aptamers on Gold Immobilization of anti-AFT B1 and anti-AFT M1 aptamers on nanostructured gold films was carried out following protocols in detail [18,19,21]. shown in Figure 5, afilms 100 µL Immobilization of the anti-AFT B1 described and anti-AFT M1 in aptamers onAs nanostructured gold was of respective aptamer ofdescribed optimizedin concentration 5 µM orAs 2 µM in PBB or HBB carried out following thesolutions protocols detail in [18,19,21]. shown in Figure 5, abuffer 100 µL with 1 mM of DTTofwas pipetted onto prepared glass slides of supplemented respective aptamer solutions optimized concentration 5 µM or 2with µM nano-structured in PBB or HBBgold. buffer After incubation for 4 h in a humidity chamber, the slides were rinsed with PBB remove unbound supplemented with 1 mM of DTT was pipetted onto prepared glass slides with to nano-structured gold. aptamers. The resultant aptamer modified slides were either used directly as aptasensor or stored in After incubation for 4 h in a humidity chamber, the slides were rinsed with PBB to remove unbound binding buffer at 4 °C for several days without any noticeable decrease in their functionality.
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aptamers. The resultant aptamer modified slides were either used directly as aptasensor or stored in binding buffer at 4 ◦ C for several days without any noticeable decrease in their functionality. Author Contributions: A.G.A.-R. (experimental work, data analysis, writing), A.N. (supervision, data analysis, writing), G.C. and J.-L.M. (aptamer assay, discussion of results), E.T. (experimental work, data analysis), A.S. (discussion of results, funding). Funding: This work was funded by NATO SPS program, project NUKR.SFPP 984637. Acknowledgments: This work was funded by NATO SPS program, project NUKR.SFPP 984637, Ali Ghanim Al Rubaye wishes to acknowledge the financial support from the Ministry of Higher Education and Scientific research in Iraq. Conflicts of Interest: The authors declare no conflict of interest.
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