Ultraselective antibiotic sensing with complementary strand DNA assisted aptamer/MoS2 field-effect transistors.

Although aptamer has been demonstrated as an important probe for antibiotic determination, the selective sensing of different antibiotics is still a challenge due to their structure similarities and wide folding degrees of aptamer. Herein, a field-effect transistor using MoS2 nanosheet as the channel and an aptamer DNA (APT) with its configuration shaped by a complementary strand DNA (CS) is employed for kanamycin (KAN) determination. This probe structure contributes to an enhanced selectivity and reliability with reduced device-to-device variations. This MoS2/APT/CS sensor shows time-dependent performance in antibiotic sensing. Prolonged detection time (20 s-300 s) leads to an enhanced sensitivity (1.85-4.43 M-1) and a lower limit of detection (1.06-0.66 nM), while a shorter detection time leads to a broader linear working range. A new sensing mechanism relying on charge release from probe is proposed, which is based on the "replacement reaction" between KAN and APT-CS. This sensor exhibits an extremely high selectivity (selectivity coefficient of 12.8) to kanamycin over other antibiotics including streptomycin, tobramycin, amoxicillin, ciprofloxacin and chloramphenicol. This work demonstrates the merits of probe engineering in label-free antibiotic detection with FET sensor, which presents significant promises in sensitive and selective chemical and biological sensing.

Microchip electrophoresis based aptasensor for multiplexed detection of antibiotics in foods via a stir-bar assisted multi-arm junctions recycling for signal amplification.

Microchip electrophoresis (MCE) was a good available method for high-throughput and rapid detecting chemical pollutants in food samples. However, many of the reported MCE assays involve complex design of microchip, laborious operation and poor universality which limited its promotion in multiple antibiotics' detection. Herein, a multiplexed aptasensor was developed based on a universal double-T type microchip to one-step and simultaneously detect several antibiotics within 3 min using chloramphenicol (CAP) and kanamycin (Kana) as representatives. Besides, a novel stir-bar assisted DNA multi-arm junctions recycling (MAJR) strategy was designed for transducing and amplifying the signal. The brief detection mechanism was as following: the added CAP and Kana can specifically react with their aptamer probes on the stir-bar and produce different single-stranded DNA primer, respectively. Afterwards, the primers can trigger MAJR to form a lot of three- and four-arm DNA junctions corresponding to different targets. The DNA multi-arm junctions can be easily separated and detected by MCE for quantification. Moreover, the stir-bar can facilitate phase separation and obviously eliminate matrix interference in food. The assay was successfully applied in milk and fish samples, showing excellent selectivity and sensitivity with a detection limits of 0.52 pg mL-1 CAP and 0.41 pg mL-1 Kana (S/N = 3). Thus, the assay holds a great potential application for screening of antibiotics in food.

Portable fluoride-selective electrode as signal transducer for sensitive and selective detection of trace antibiotics in complex samples.

Ion-selective electrodes (ISE) can rapidly, sensitively detect their corresponding ions and are suitable for field testing. However, most ISE methods cannot detect other targets directly which limits their practice application. Herein, we established an aptamer-sensing platform to detect organic small molecule using a portable fluoride-selective electrode (FSE). To achieve the purpose, novel signal tags were fabricated based on nano metal-organic frameworks (NMOF) encapsulating F- and labeling aptamers. They were then immobilized on one stir-bar. Subsequently, a double stir-bars (bar-a and b) assisted target recycling strategy was designed to convert organic small molecular target to F- for signal development and amplification. The movement of tags from bar-a to b can be triggered by the analytes. After reaction, the transferred signal tags in bar-b were washed and released F- which can be measured by FSE for qualification of the target. The assay was evaluated to detect kanamycin or chloramphenicol which was employed as the representatives of organic small molecular with a low detection limit of 0.35 nmol L-1 or 0.46 nmol L-1, respectively. Satisfactory performance was observed in complex sample analysis of kanamycin (milk, fish, urine and serum) with a recovery of 91-108% and an RSD (n = 6) <5%. The proposed method broadens the application of traditional FSE to the detection of organic small molecule. And the employment of NMOF which has higher encapsulating capacity of F- for preparing signal tags can be extended to FSE based aptasensors.

Ultratrace antibiotic sensing using aptamer/graphene-based field-effect transistors.

Antibiotic residue, as emerging pollution resulting from antibiotic abuse, poses a serious threat on ecosystem and human health. Conventional methods for antibiotic detection, e.g., liquid/gas chromatography, are based on complicated instruments and time-consuming; therefore, efforts have been made to realize in situ and real-time monitoring of antibiotics. Here, a miniaturized and integratable electronic antibiotic sensor based on field-effect transistor (FET) is reported. The reduced graphene oxide (rGO) nanosheet is used as the channel material and the aptamer RNA for tobramycin is modified onto rGO as the probe. A novel sensor design with 6-mercapto-1-hexanol (MCH)/1-pyrenebutanol (PBA) blocking layer (BL) for structure optimization is applied to enhance the sensor reliability and specificity. This rGO/aptamer/BL sensor shows an ultra-sensitivity to tobramycin with a lower detection limit of 0.3 nM and a quick response within 5 s, as well as a high specificity over other antibiotics such as kanamycin, streptomycin, ciprofloxacin, and tetracycline. The sensing mechanism based on the deformation of the charged aptamer probe is proposed via an in-depth analysis of the interactions between aptamer, tobramycin and rGO. In addition, sensing test performed under controlled microfluidic flow conditions demonstrates a great potential of the sensors in practical applications.

Flexible freestanding graphene paper-based potentiometric enzymatic aptasensor for ultrasensitive wireless detection of kanamycin.

Flexible sensing devices have drawn tremendous attention in the past decades due to their potential applications in future hand-held, potable consumer, and wearable electronics. Here, we firstly developed an ultrasensitive wireless potentiometric aptasensor based on flexible freestanding graphene paper for kanamycin detection. Flexible graphene paper made from a simple vacuum filtration method was used as a biocompatible platform for effective immobilization of aptamer. A nuclease-assisted amplification strategy was introduced into this potentiometric biosensing system in order to significantly improve the detection sensitivity through a classic catalytic recycling reaction of target induced by the nuclease (DNase I). As expected, an ultra-low detection limit of 30.0 fg/mL for kanamycin was achieved. Furthermore, the developed potentiometric enzymatic aptasensor exhibits high selectivity, favorable flexibility, excellent stability and reproducibility, which holds great promising for its routine sensing application.

Enzyme- and label-free electrochemical aptasensor for kanamycin detection based on double stir bar-assisted toehold-mediated strand displacement reaction for dual-signal amplification.

It is critically important to detect antibiotic residues for monitoring food safety. In this study, an enzyme- and label-free electrochemical aptasensor for antibiotics, with kanamycin (Kana) as a typical analyte, was developed based on a double stir bar-assisted toehold-mediated strand displacement reaction (dSB-TMSDR) for dual-signal amplification. First, we modified two gold electrodes (E-1 and E-2) with different DNA probes (S1/S2 hybrid probe in E-1 and DNA fuel strand S3 in E-2). In the presence of Kana, an S1/S2 probe can be disassembled from E-1 to form an S2/Kana complex in supernatant. The S2/Kana could react with S3 on E-2 to form S2/S3 hybrid and release Kana through TMSDR. After then, the target recycling was triggered. Subsequently, the formed S2/S3 hybrid can also trigger a hybridization chain reaction (HCR). Consequently, the dual-signal amplification strategy was established, which resulted in many long dsDNA chains on E-2. The chains can associate with methylene blue (MB) as redox probes to produce a current response for the quantification of Kana. The assay exhibited high sensitivity and specificity with a detection limit at 16 fM Kana due to the dual-signal amplification. The double stir bars system can both increase phase separation and prevent leakage of DNA fuel to reduce background interference. Moreover, it allows flexible sequence design of the TMSDR probes. The assay was successfully employed to detect Kana residues in food and showed potential application value in food safety detection.

A photoelectrochemical aptasensor based on a 3D flower-like TiO2-MoS2-gold nanoparticle heterostructure for detection of kanamycin.

In this work, a sensitive photoelectrochemical aptasensor was developed for kanamycin detection using an enhanced photocurrent response strategy, which is based on the surface plasmon resonance effect of gold nanoparticles deposited on a 3D TiO2-MoS2 flower-like heterostructure. A significant aspect of this development lies in the photoelectrochemical and morphological features of the unique ternary composite, which have contributed to the excellent performance of the sensor. To develop an aptasensor, mercapto-group modified aptamers were immobilised on the photoactive composite as a recognition unit for kanamycin. The TiO2-MoS2-AuNP composite was demonstrated to accelerate the electron transfer, increase the loading of aptamers and improve the visible light excitation of the sensor. Under optimal conditions, the aptasensor exhibited a dynamic range from 0.2 nM to 450 nM of kanamycin with a detection limit of 0.05 nM. Overall, we have successfully synergised both the electrical and the optical merits from individual components to form a ternary composite, which was then demonstrated as an effective scaffold for the development of PEC biosensors.

Evanescent wave aptasensor for continuous and online aminoglycoside antibiotics detection based on target binding facilitated fluorescence quenching.

The biosensors capable for on-site continuous and online monitoring of pollutants in environment are highly desired due to their practical importance and convenience. The group specific detection of pollutants is especially attractive due to the diversity of environmental pollutants. Here we devise an evanescent wave aptasensor based on target binding facilitated fluorescence quenching (FQ-EWA) for the online continuous and group-specific detection of aminoglycoside antibiotics (AMGAs). In FQ-EWA, a fluorophore labeled DNA aptamer selected against kanamycin was used for both the target recognition in solution and signal transduction on optical fiber of EWA. The aptamers form multiple-strand complex (M-Apt) in the absence of AMGAs. The binding between AMGA and the aptamer disrupts M-Apt and leads to the formation of AMGA -aptamer complex (AMGA-Apt). The photo-induced electron transfer between the fluorophore and AMGA partially quenches the fluorescence of AMGA-Apt. The structure-selective absorption of AMGA-Apt over M-Apt on the graphene oxide further quenches the fluorescence of AMGA-Apt. Meanwhile, the unbound aptamers in solution assemble with the unlabeled aptamers immobilized on the fiber to form M-Apt. The amount of M-Apt on the fiber is inversely proportional to the concentration of AMGAs, enabling the signal-off detection of AMGAs from 200nM to 200µM with a detection limit of 26nM. The whole detection process is carried out in an online mode without any offline operation, providing a great benefit for system automation and miniaturization. FQ-EWA also shows great surface regeneration capability and enables the continuous detection more than 60 times.

Superwetting and aptamer functionalized shrink-induced high surface area electrochemical sensors.

Electrochemical sensing is moving to the forefront of point-of-care and wearable molecular sensing technologies due to the ability to miniaturize the required equipment, a critical advantage over optical methods in this field. Electrochemical sensors that employ roughness to increase their microscopic surface area offer a strategy to combatting the loss in signal associated with the loss of macroscopic surface area upon miniaturization. A simple, low-cost method of creating such roughness has emerged with the development of shrink-induced high surface area electrodes. Building on this approach, we demonstrate here a greater than 12-fold enhancement in electrochemically active surface area over conventional electrodes of equivalent on-chip footprint areas. This two-fold improvement on previous performance is obtained via the creation of a superwetting surface condition facilitated by a dissolvable polymer coating. As a test bed to illustrate the utility of this approach, we further show that electrochemical aptamer-based sensors exhibit exceptional signal strength (signal-to-noise) and excellent signal gain (relative change in signal upon target binding) when deployed on these shrink electrodes. Indeed, the observed 330% gain we observe for a kanamycin sensor is 2-fold greater than that seen on planar gold electrodes.

Micellar Electrokinetic Chromatography of Aminoglycosides.

The components of the aminoglycosides, e.g., gentamicin, sisomicin, netilmicin, kanamycin, amikacin, and tobramycin, and related impurities of these antibiotics can be separated by means of micellar electrokinetic chromatography (MEKC). Derivatization with o-phthaldialdehyde and thioglycolic acid is found to be appropriate for these antibiotics. The background electrolyte was composed of sodium tetraborate (100 mM), sodium deoxycholate (20 mM), and ß-cyclodextrin (15 mM) having a pH value of 10.0. This method is valid for evaluation of gentamicin, kanamycin, and tobramycin. It has to be adopted for amikacin, paromomycin, neomycin, and netilmicin.

HPLC-ELSD determination of kanamycin B in the presence of kanamycin A in fermentation broth.

A novel method for the direct determination of kanamycin B in the presence of kanamycin A in fermentation broth using high performance liquid chromatography with evaporative light scattering detector (HPLC-ELSD) was developed. An Agilent Technologies C18 column was utilized, evaporation temperature of 40°C and nitrogen pressure of 3.5 bar, the optimized mobile phase was water-acetonitrile (65:35, v/v), containing 11.6 mm heptafluorobutyric acid (isocratic elution with flow rate of 0.5 mL/min) with the gain 11. Kanamycin B was eluted at 5.6 min with an asymmetry factor of 1.827. The method showed good linearity over the concentration range of 0.05 to 0.80 mg/mL for the kanamycin B (r(2) = 0.9987). The intra-day and inter-day coefficients of variation obtained from kanamycin B were less than 4.3%. Mean recovery of kanamycin B from spiked fermentation broth was 95%. The developed method was applied to the determination of kanamycin B without any interference from other constituents in the fermentation broth. This method offers simple, rapid and quantitative detection of kanamycin B.

Label-free detection of kanamycin based on the aptamer-functionalized conducting polymer/gold nanocomposite.

Highly sensitive label-free detection of kanamycin is achieved with an aptamer sensor based on a conducting polymer/gold self-assembled nanocomposite. The sensor probe is fabricated by covalently immobilizing an in vitro selected DNA aptamer for kanamycin onto gold nanoparticle (AuNP)-comprised conducting polymer, poly-[2, 5-di-(2-thienyl)-1H-pyrrole-1-(p-benzoic acid)] (poly-DPB). The self-assembling of DPB on AuNP is investigated by TEM and UV-vis spectroscopy and the modification of the aptamer sensor is characterized using XPS and electrochemical impedance spectroscopy. The probe is applied to detect kanamycin by using voltammetric techniques. The sensor shows a pair of redox peaks around 0.26/ 0.08 V (vs. Ag/AgCl) for kanamycin captured by the aptamer-immobilized probe. The parameters that can affect the response, such as aptamer concentration, incubation time, temperature, and pH are optimized. The calibration plot shows a linear range from 0.05 µM to 9.0 µM kanamycin with a detection limit of 9.4±0.4 nM. The proposed aptamer sensor is examined with a real sample.

Synthesis, separation, and characterization of amphiphilic sulfated oligosaccharides enabled by reversed-phase ion pairing LC and LC-MS methods.

Synthesis of amphiphilic oligosaccharides is problematic because traditional methods for separating and purifying oligosaccharides, including sulfated oligosaccharides, are generally not applicable to working with amphiphilic sugars. We report here RPIP-LC and LC-MS methods that enable the synthesis, separation, and characterization of amphiphilic N-arylacyl O-sulfonated aminoglycosides, which are being pursued as small-molecule glycosaminoglycan mimics. The methods described in this work for separating and characterizing these amphiphilic saccharides are further applied to a number of uses: monitoring the progression of sulfonation reactions with analytical RP-HPLC, characterizing sulfate content for individual molecules with ESI-MS, determining the degree of sulfation for products having mixed degrees of sulfation with HPLC and LC-MS, and purifying products with benchtop C18 column chromatography. We believe that the methods described here will be broadly applicable to enabling the synthesis, separation, and characterization of amphiphilic, sulfated, and phosphorylated oligosaccharides and other types of molecules substituted to varying degrees with both anionic and hydrophobic groups.

Identification of tobramycin impurities for quality control process monitoring using high-performance anion-exchange chromatography with integrated pulsed amperometric detection.

Commercial-scale fermentation for tobramycin manufacture is carried out with Streptomyces tenebrarius. Impurity profiling during various phases of pharmaceutical production is important for evaluating the effectiveness of a processing step and meeting regulatory requirements. High-performance anion-exchange (HPAE) chromatography with integrated pulsed amperometric detection (HPAE-IPAD) is a highly sensitive method used to assay tobramycin and to assess purity, but no prior publications demonstrated the capability of this technique to monitor purity at various stages of production at either the typical concentrations or in the typical matrices of a manufacturing process. In addition, the identities of the impurity peaks observed in commercial sources of tobramycin when assayed by using HPAE-IPAD are mainly unknown. Regulatory agencies generally require these impurities to be characterized when found above certain limits, and when present at higher levels require toxicological studies. In this paper, we analyze tobramycin samples using HPAE-IPAD at different stages of production and show the impurity profile and concentration changes through the manufacturing process. We successfully identified nearly all the impurity peaks found in commercially available tobramycin, based on known degradation pathways deduced from extreme pH forced degradation studies, which we experimentally reproduced, and based on previously known related substances found in S. tenebrarius fermentation broth. In crude and final tobramycin products, we identified the peaks for neamine, kanamycin B, nebramine, kanosamine, 2-deoxystreptamine. We tentatively identified deoxystreptamine-kanosaminide in crude and final products, and kanamycin A, carbamoyl-kanamycin B and carbamoyl-tobramycin in down stream process intermediates of a S. tenebrarius fermentation culture. Results presented in this paper support the effective use of the HPAE-IPAD method for in-process impurity profiling of tobramycin, and as a stability-indicating technique after product purification.

Rapid and selective micellar electrokinetic chromatography for simultaneous determination of amikacin, kanamycin A, and tobramycin with UV detection and application in drug formulations.

A simple and selective micellar electrokinetic chromatography (MEKC) with UV detection is described for simultaneous determination of amikacin, tobramycin, and kanamycin A, performed in Tris buffer (180 mM; pH 9.1) with 300 mM sodium pentanesulfonate (SPS) as an anionic surfactant. Under this condition, good separation with high efficiency and the required short analysis time is achieved. The linear ranges of the method for the determination of amikacin, tobramycin, and kanamycin A were 0.1-0.5 mg / mL, 0.4-2.0 mg / mL, and 0.4-2.0 mg / mL, respectively; the detection limits (signal-to-noise ratio = 3; injection, 0.5 psi 5 s) were 0.08, 0.2, and 0.2 mg / mL, respectively. The small amount of sample required and the expeditiousness of the procedure allow content uniformity to be determined in individual commercial products.

Development and validation of capillary electrophoresis method for tobramycin with precapillary derivatization and UV detection.

One of the major drawbacks in the analysis of aminoglycoside antibiotics is their lack of UV chromophore and/or fluorophore. Tobramycin, a representative member of this group, was examined in this study. To overcome the detection hurdle, a precapillary derivatization followed by capillary electrophoresis analysis with direct UV detection was investigated. A central composite design was applied to optimize the method and three parameters were selected in this study: buffer pH, temperature and % acetonitrile (ACN). Selectivity between tobramycin main component and its adjacent peaks as well as the peak efficiency and symmetry factors were established as responses. For each response, a model was obtained by a second-order mathematical expression. Successful results were obtained with a simple background electrolyte (BGE) containing 30 mM sodium tetraborate, pH 10.2, and ACN (75:25 v/v). Under these conditions, baseline separation of tobramycin from its adjacent kanamycin B and an unknown peak was achieved. A temperature of 20 degrees C and applied voltage of 28.0 kV were used. The method showed good validation data in terms of precision, limits of quantitation and detection, specificity and linearity and was found to be suitable for analysis of tobramycin bulk pharmaceutical samples.

Regiospecificity assignment for the reaction of kanamycin nucleotidyltransferase from Staphylococcus aureus.

Aminoglycoside nucleotidyltransferases catalyze the transfer of a nucleoside monophosphoryl group from a nucleotide to a hydroxyl group of an aminoglycoside antibiotic. Kanamycin nucleotidyltransferase [ANT (4',4' ')-I] from Staphylococcus aureus confers resistance to numerous aminoglycosides with a 4' or 4' ' hydroxyl group in the equatorial position. The synthesis of m-nitrobenzyl triphosphate, a new substrate of kanamycin nucleotidyltransferase, is reported. The kanamycin nucleotidyltransferase catalyzed reaction of kanamycin A with m-nitrobenzyl triphosphate is 2 orders of magnitude slower than that with ATP. The MALDI-TOF spectra of the purified products of both reactions revealed that kanamycin A was modified only at one position. The regiospecificity of the reaction catalyzed by kanamycin nucleotidyltransferase of kanamycin A with either ATP or m-nitrobenzyl triphosphate was determined directly by one- and two-dimensional hetero- and homonuclear NMR techniques. The site of the modification was unambiguously assigned to the 4' hydroxyl of kanamycin A; thus, the products formed are 4'-(adenosine-5'-phosphoryl)-kanamycin A and 4'-(m-nitrobenzyl phosphoryl)-kanamycin A. This eliminates the uncertainty concerning the point of modification since this could not be determined from the crystal structure of the enzyme with bound MgAMPCPP and kanamycin A [Pedersen, L. C., Benninig, M. M., and Holden, H. M. (1995) Biochemistry 34, 13305-13311].