Lin Cheng,Ho Qu,Jun Teng,Li Yo,Feng Xue,Wei Chen,*,2

a School of Food Science and Engineering,Hefei University of Technology,Hefei,230009,China

b College of Veterinary Medicine,Nanjing Agricultural University,Nanjing,210095,China

Abstract We report the design of a sensitive,electrochem ical aptasensor for detection of ochratoxin A (OTA) w ith an extraordinary tunable dynam ic sensing range.This electrochem ical aptasensor is constructed based on the target induced aptamer-folding detection mechanism and the recognition between OTA and its aptamers results in the conformational change of the aptamer probe and thus signal changes for measurement.The dynamic sensing range of the electrochem ical aptasensor is successfully tuned by introduction of free assistant aptamer probes in the sensing system.Our electrochem ical aptasensor shows an extraordinary dynam ic sensing range of 11-order magnitude of OTA concentration from 10-8 to 102 ng/g.Of great significance the signal response in all OTA concentration ranges is at the same current scale,demonstrating that our sensing protocol in this research could be applied for accurate detections of OTA in a broad range w ithout using any complicated treatment of signal amplification Finally,OTA spiked red w ine and maize samples in different dynam ic sensing ranges are determ ined w ith the electrochem ical aptasensor under optim ized sensing conditions.This tuning strategy of dynamic sensing range may offer a promising platform for electrochemical aptasensor optimizations in practical applications.

Keywords: Electrochemical aptasensor;Tunable dynamic detection range;OTA detection;Extraordinary dynamic range

1.Introduction

Presently,extensive studies have been carried out to improve the detection lim it of existing sensing protocols in the fiel of analytical science[1-3].For example,enzymatic catalysis reactions and in vitro nucleic acid amplificatio techniques have been w idely integrated w ith conventional detection protocols to improve the sensitivity [4-7].Meanwhile,due to the intrinsic advantages ofinanomaterials,various functional nanoparticles have also been adopted for signal amplificatio [8-10].As one of the most classic and efficien techniques,electrochemical biosensors have attracted extraordinary attention since the successful commercialization of the personal glucose meter[11-13].Furthermore,w ith the occurrence of aptamers as the recognition probes,the electrochem ical aptasensors have been further w idely utilized for rapid and sensitive detections in many significan field [7,14-16].As mentioned,extensive researches have been executed to improve sensing performances especially detection lim it of electrochemical biosensors[4,7,16-18].However,from the perspective of practical applications,not only the detection lim it,but also the dynam ic sensing range of methods plays equally significan roles.And previous studies have clearly demonstrated that fied dynamic range greatly restricts the performanceof theelectrochemicalbiosensorsinmanyapplications such as the monitoring ofiviral loading,in which the concentration of the target could vary over many orders of magnitude[19].

It is important to note that for the current signal amplification protocols,there is a trade-off between the sensing range and the detection limit.People have to make wise choices of signal amplificatio protocols for achievement of satisfie sensing range or detection limit.Briefl,for signal-off model protocols,the sensing range would be extended toward the lower concentrations with signal amplificatio treatments [7,17];while for signal-on model protocols,the sensing range would be extended toward both high and low concentrations due to the amplifie sensing signals[18,20,21].Because of this dilemma,the sensing signal could not be amplifie unlimitedly even with the most extraordinary amplificatio treatments.In short,for electrochemical biosensors working in either sensing models,the sensing range can only be extended to a certain estimated extent based on the increased signal intensity.

Till now,only few research work on tunable dynamic range of electrochemical biosensors have been reported[3,22-24].For example,Plaxco et al.firs reported the expansion of detection range from the original 81-fold to more than 1000-fold by combining biosensors with identical specificit but differing affinitie [22].Following,Ricci et al.were able to tune the dynamic range of an E-DNA sensor by changing the stability of the stem-loop structured recognition element [19].Besides,Lai et al.achieved the tunable dynamic range of an electrochemical biosensor for gold(III)detection by simply changing the length of the recognition DNA probe[24].Interestingly,the dynamic range modulation in all reported research were successfully carried out with simple protocols.However,all the above reported research took advantage of the stability or rigidity of double stranded DNA and thus the target analytes are only limited to nucleic acids and metal ions.For detection of many important small molecules including toxins,antibiotics and medicine residues et al.,related researches have rarely been reported.Actually,with the continuous screening and reporting of aptamers against common small molecules[25],similar research should be paid further attention due to the easy applications by replacing recognition nucleic acid probes with the aptamers of known sequences.

Herein,in this study,we report an innovative yet straightforward approach to tune the dynamic detection range of an electrochemical aptasensor.Firstly,we make an electrochemical aptasensor for detection of ochratoxin(OTA)as a model target,which has been extensively studied for the excellent recognition between OTA and its aptamer [26,27].This kind of electrochemical aptasensors are mostly based on recognition induced folding of aptamer and subsequent signal variations[14].Compared with traditional signal amplificatio based tune strategy,no amplificatio strategy is needed in our research in order to tune the dynamic detection range.Next based on the aptamer modifie sensing interface of the electrode,we added assistant aptamer recognition probes into the sensing system to act as a tuning component for the tuning of detection range.Through this simple technique,the detection range was precisely controlledandmodulatedfrom10-8ng/gto100 ng/g,over13orders of concentration magnitude.More importantly and of greater significance in such a wide range (13 orders of concentration magnitude),all current response ratios at different analyte concentrations are almost at the same scale (define as the valid signalvariation zone),whichmeansthatthisnon-amplifie sensing current(at the level of traditional biosensors)was also tuned for easy determination of target analyst at wide-range concentrations.To our best knowledge,this study is the firs report about this kind of dynamic sensing range modulation in such a wide range without signal amplifications And this dynamic sensing range modulation strategy could be easily transferred to detection for other analytes by changing the sequence of aptamer probes.

2.Experimental

2.1.Materials and methods

All the single-stranded oligonucleotide probe (ssDNA) and thiol-modifie ssDNA were all synthesized by the Sangon Bioengineering(Shanghai)Co.Ltd.China.The detailed sequences of different oligonucleotide probes were as follows:free aptamer(probe 1),5′-GAT CGG GTG TGG GCG TAA AGG GAG CAT CGG ACA-3′;immobilized aptamer (probe 2),5′-thiol-(CH2)6-AAAGATCGGGTGTGGGCGTAAAGGGAGCAT CGG ACA-Ferrocene-3′.6-Mercapto-1-hexanol(MCH),tris(2-carboxyethyl)phosphine hydrochloride(TCEP),and ochratoxin(OTA) were purchased from J&K Chemical,Shanghai.All otherreagentswereobtainedfromSinopharmChemicalReagent Company,China.Various buffer solutions were prepared from analytical grade chemicals without further purificatio and were prepared with ultrapure water (＞18 MΩ,Milli-Q A10 system,Millipore,U.S.A).Storage buffer used for oligonucleotides was Tris-HCl (10 mM,pH 8.0) containing 1 mM ethylene diamine tetra-acetic acid(EDTA).Detection buffer was 100 mM pH 7.4 phosphate buffer containing 0.5 M sodium chloride.

2.2.Instrumentation

Cyclic voltammetry(CV),electrochemical impedance spectroscopy (EIS),and differential pulse voltammetry (DPV)measurements were carried out on a CHI 660D electrochemical workstation with a conventional three-electrode system composed of a functionalized gold electrode as the working electrode,a platinum wire auxiliary electrode,and a saturated calomel electrode (SCE) as the reference electrode.EIS measurement was performed in 0.1 M KCl solution containing 5 mmol/L[Fe(CN)6]3-/4-,and the frequency range was from 1 to 105Hz at 0.2 V.DPV measurement was carried out in 10 mL detection buffer,and the experiment parameters were as follows:initial potential,0.6 V;fina potential,-0.1 V;pulse amplitude,0.05 V;pulse interval,0.05 s;sampling interval,0.0167 s.

2.3.Fabrication of the sensing interface of OTA

We treated the electrode following the routine protocol of our group as described in [7,16].In more details,before the modificatio of ssDNA probes,the electrode was immersed in freshly prepared piranha solution(H2SO4/H2O23:1 in volume)for 30 min and thoroughly rinsed with ultrapure water.After that,the electrode was sequentially polished carefully to a mirror-like surface with 1,0.3,and 0.05 μm Al2O3powder respectively for 5 min and sonicated in ethanol for 5 min and then in ultrapure water for 5 min to remove the residual Al2O3powder.The electrode was then cleaned by electro-chemical polishing with 30 successive cyclic voltammetry (CV) scans from 1.5 V to-0.35 V in 0.5 mol/L H2SO4at 100 mV/s until a typical stable cyclic voltammogram was obtained.Finally,the electrode was rinsed with ultrapure water and dried in a nitrogen environment.

Scheme 1.The schematic principle diagram of the tunable dynamic sensing range electrochemical aptasensor for OTA detections.

Afterthecleaningoftheelectrode,thethiol-modifie aptamer was firstl treated with TECP solution to activate the thiol groups.Following,a droplet containing the activated aptamer was dropped onto the gold electrode and cultured at 4°C overnight.Then the electrode was rinsed with buffer and further blocked with MCH by immersing the electrode in the blocking reaction at 37°C for 40 min.Finally,the electrode was rinsed using the buffer again and this rinsed sensing interface was ready for OTA detection.

2.4.Detection range modulation research and real sample detection

For the dynamic detection range modulation research,the designed assistant aptamer probes (probe 1) at various concentrations were added into the sensing system and then pre-incubated with OTA samples for different spans of time to achieve different sensing range of OTA.Afterwards,this mixture of the assistant aptamers and the analyte OTA was further loaded and measured by the above-mentioned aptamer functionalized sensor(electrochemical aptasensor).For real spiked red wine sample detections,OTA at different spike concentrations in red wine samples including ultralow,medium and ultrahigh concentrations were measured directly with the above sensing systems respectively with only common filtratio and compared with results measured by conventional instrument.For spiked corn sample detections,the finel grounded negative-confirme corn samples were spiked with OTA at the same concentrations of spiked wine samples and mixed with the vortex mixer for 5 min.The mixture was extracted with the extraction solvent(methanol/water,volume ratio 6:4)and centrifugated at 6000gfor 15 min.Afterward,supernatant was further cleaned with the 0.45 μm filte membrane for detection.

3.Results and discussions

The modulation of the dynamic sensing range of electrochemical aptasensor is of great significanc but facing great challenges.The design and principle of the extraordinary tunable dynamic range of the electrochemical aptasensor is shown in Scheme 1,the key and most important step in this research focused on the modulation of the sensing currents in different concentration ranges into the same equivalent response level or magnitude(signal increase percentage,%).Toward this end,we adopted a very easy strategy by using assistant aptamer probes(probe 1)as shown in Scheme 1 to modulate the response currents in different sensing ranges into the suitable scale in the sensing system.Thereafter,the target analyte OTA at different concentrations ranging from trace amount to extra high abundance could be determined and analyzed in the same reasonable current variation range.

Fig.1.Parameters optimization of the fabricated electrochemical aptasensor with tunable dynamic sensing range.

Firstly,the sensing interface fabrication process and detection conditions of the electrochemical aptasensor were monitored and optimized for the best sensing performance.As a widelyadopted technique [28,29],electric impedance spectrum (EIS)was applied for the characterization of sensing interface fabrication.The results of EIS for the same gold electrode at different stages of the fabrication including initial bare gold electrode,afteraptamerimmobilization,afterblockingandsensing were shown in Fig.1a,indicating that the impedance value(Ret) of each step increased accordingly due to the electron transfer inhibition effect of the immobilized DNA and blocking molecules.Meanwhile,it is also worth mentioning that in Fig.1a the Ret value further increased after the recognition of OTA at different concentrations.This change was attributed to the direct recognition and combination between the aptamer and OTA at the sensing interface,both of which caused the electron transfer inhibition on the electrode surface.Overall,the EIS results in Fig.1a clearly demonstrated the successful fabrication of the sensing interface for subsequent research.Besides,the CV results were also achieved and supported the successful fabrication of the sensing interface (See results in SI).

In order to utilize the assistant aptamer (probe 1) in the research,the OTA recognition time between the immobilized or the free assistant aptamers were both adjusted.Traditionally,detection of OTA is carried out by direct incubating OTA with immobilized recognition aptamers on the sensing interface of electrochemical aptasensors.And it has been found that 60 min is the optimal incubation time for complete recognition of OTA in the sensing system.Herein,with the introduction of the assistant free aptamer(probe 1),another key factor is the incubation time between OTA and the assistant free aptamer before the detection.And as shown in Fig.1b,it is observed that after preincubation of OTA with assistant free aptamer for 20 min,the original current increase did not obviously change compared with that when no assistant free aptamer was added.Further increase ofincubation time induced a distinguished decrease of the sensing current,indicating the occurrence of competitive recognition of OTA with assistant free aptamer.Therefore,to compromise the complete recognition of OTA by both the immobilized aptamer and the assistant free aptamer,60 min was also determined to be the best incubation time for subsequent detections.

Under the optimized conditions,detections of OTA at different concentrations (including the low,medium and high concentration ranges) were carried out.The results shown in Fig.2A indicated that:(1)OTA at all concentrations from 10-8to 102ng/g could be measured directly with all three different sensing systems;(2)the sensing currents of OTA at all concentration ranges with three different sensing systems are all in the scale of 0.1-0.4 μA.Following,the calibration curves for OTA detection in different concentration ranges were all constructed respectively as shown in Fig.2B.As expected,the change trends of OTA measurements in different concentration ranges were the same and the signal increase vs OTA concentration were all in the good linear relationship.Ofinote,the slopes of the calibration curves for OTA detection in different concentration ranges were different.The slope increased as the increase of OTA concentrations in each sensing range(the angel degree of three slopes were 38,44 and 56,respectively).This variation could be attributed to the sequestering effect of the adopted assistant aptamer to the reach of a threshold level of target OTA (fied by the concentration of assistant aptamer) in the sensing system and it was in consistent with the previous report of dynamic range engineering by change the affinit of the recognition probes[9].In detail,in the low sensing range,higher concentration of assistant aptamer probe was used in the sensing system,which induced the obvious screening effect of the signal response and smaller angel of the slope(38°).In the high sensing range,less assistant aptamer probe using gave the small screening effect to target OTA and obvious signal response,which was demonstrated in bigger angel of the slope(56°).Based on these results,the signal increase ratio (%) from 40 to 250 was define as the effective and valid zone for OTA detection.And in this valid sensing zone,even 11-order magnitude of OTA concentration could be well detected by the developed electrochemical aptasensor with an extraordinary tunable dynamic sensing range.

Fig.2.OTA detection results of the dynamic range tunable electrochemical aptasensor(a,low dynamic range;b,medium dynamic range;c,high dynamic range).(A)The DPV signal response of OTA at different concentrations;(B)The calibration curves for OTA detection in different dynamic ranges(angel 1,38°;2,44°;3,56°);(C)Sensing accuracy confirmatio results of the dynamic range tunable electrochemical aptasensor(a,low dynamic range;b,medium dynamic range;c,high dynamic range;transparent zone indicated the valid signal response zone).

Table1 Comparison results of different methods for OTA detections.

It is to be noted that this developed strategy may be with followinglimitations.Morespecificall,atonefiedsignalincrease ratio,there were three possible OTA concentrations without considering the exact sensing system.In order to further confir this potential uncertainty of the developed strategy,we measured three different OTA concentrations (10-5,0.1,and 10 ng/g)with three sensing systems,respectively.The results shown in Fig.2C showed that the fied concentration of OTA could only be measured with the corresponding sensing system so that the measured current variations fell into the valid signal response zone (shown as transparent red in Fig.2C).Incorrect sensing system introduced either under-or over-estimated current signal increase ratio and should not be adopted as a valid index of measurement.In this way by using the correct sensing system,we could ensure good application potentials of the developed electrochemical aptasensor with extra-wide sensing range.Furthermore,thesensingperformanceofthisdynamicrangetunable electrochemical aptasensor was compared with other reported methods for OTA detections.Detailed comparison results were shown in Table1 and demonstrated both the good sensitivity and dynamic range of the electrochemical aptasensor of this research.

Fig.3.Specificit interrogation results of this dynamic range tunable electrochemical aptasensor.

Table2 Determination results of OTA spiked wine and corn samples(n=3).

Besides,we performed specificit measurements of this dynamic range modulated electrochemical aptasensor by applying the analogues of OTA and other common toxins.The results shown in Fig.3 demonstrated that only the target analyte OTA or OTA in the complicated mixture could induce distinguishable current signal responses while other interferent showed negligible effect,indicating that the electrochemical aptasensor has outstanding specificit originated to the excellent property of the adopted OTA aptamer as the recognition probe.

The repeatability of the aptasensor was confirme by measuring the same sample fie times and then the relative standard deviation(RSD)was calculated.A 3.1%of RSD for the repeated measurement of the same sample demonstrated a good repeatability of the electrochemical aptasensor.The reproducibility of this electrochemical aptasensor was further investigated by measuring the same OTA sample in six duplicated times and with six different aptasensors,respectively.Furthermore,inter-and intraprecisions were calculated based on the measurement results.All these results indicated a satisfactory reproducibility (with RSD small than 5%) of the developed range-modulated electrochemical aptasensor.Finally,the designed range-modulated electrochemical aptasensor was applied for detection of OTA spiked wine samples(n=3).Based on the three sequential sensing ranges of the whole dynamic sensing range,four different concentration (0.001,0.01,0.1 and 1 ng/g) were adopted to ensure the recovery research confirme in each sensing range.The results shown in Table2 demonstrated that the recoveries and the relative standard deviations (RSD) were in the range of 92.1%-113.8%and 1.7%-8.3%,respectively.The measurements on OTA spiked wine samples showed the potential for applying the extra-wide dynamic range tunable electrochemical aptasensor to real samples at any levels of concentrations.The stability of this electrochemical aptasensor should be ofino difference with other similar aptasensors.Results indicated that the current response decreased no more than 10% after one month storage at refrigerator,demonstrating the acceptable stability of the electrochemical aptasensor for practical applications.

4.Conclusion

In summary,we reported an easy protocol to tune the dynamic sensing range of traditional electrochemical aptasensors.By using of assistant aptamer probes against target OTA,we dramatically extended the sensing range of the traditional electrochemical aptasensor to 11-order magnitude of OTA concentration from 10-8to 100 ng/g.Such a wide dynamic sensing range had never been reported for OTA detections.And three independent narrow dynamic sensing ranges were achieved for detection of OTA at any concentrations in the whole sensing range.Of great importance,in such wide sensing range and three independent dynamic sensing ranges,the sensing current responses and variation ratio were all at the same scale for different OTA concentrations without any additional signal amplificatio treatments.Besides,we measured OTA spiked real wine samples at each sensing range with the developed electrochemical aptasensor systems and obtained satisfactory results with outstanding reproducibility,recovery and relative standard deviations.We believe that this easy protocol could serve as a platform for aptasensor research on dynamic sensing-range modulation.

Acknowledgements

This work is financiall supported by the NSFC grant of 21475030,the S&T Research Project of Anhui Province15czz03109,the National 10000 Talents-Youth Top-notch Talent Program.