Robust tag-free aptasensor for monitoring of tobramycin: Architecting of rolling circle amplification and fluorescence synergism.

With the unpredictable risks on human health and ecological safety, tobramycin (TOB) as an extensively applied antibiotic has embraced global concern. Herein, a label-free fluorescent aptasensor was developed that opened up an innovative sensing strategy for monitoring trace TOB levels. Based on the rolling circle amplification (RCA) process, a giant DNA building was established by the catalytic action of T4 DNA ligase and Phi 29 DNA polymerase with the cooperation of the specific aptamer as a primer skeleton. By having the role of signal amplifier template, the RCA product with the G-quadruplex sequence duplications was decorated by a high number of the thioflavin T (ThT) fluorescent dyes. The aptasensor with good selectivity toward TOB achieved a detection limit as low as 150 pM. Thanks to its accurate target quantification, ease of operation, economic manufacture, as well as high potency for real-time and point-of-care testing, the represented aptasensor is superb for clinical application and food safety control.

A highly sensitive electrochemical aptasensor for cocaine detection based on CRISPR-Cas12a and terminal deoxynucleotidyl transferase as signal amplifiers.

Cocaine is one of the mainly used illegal drugs in the world. Using the signal amplification elements of terminal deoxynucleotidyl transferase (TdT) and CRISPR-Cas12a, a highly sensitive and simple electrochemical aptasensor was introduced for cocaine quantification. When, no cocaine existed in the sample, the 3'-end of complementary strand of aptamer (CS) was extended by TdT, leading to the activation of CRISPR-Cas12a and remaining of very short oligonucleotides on the working electrode. So, the current signal was remarkably promoted. With the presence of cocaine, CS left the electrode surface. Thus, nothing changed following the incubation of TdT and CRISPR-Cas12a and the Aptamer/Cocaine complex presented on the electrode. Consequently, the [Fe(CN)6]3-/4- could not freely reach the electrode surface and the signal response was weak. Under optimal situations, the biosensor revealed a wide linear relation from 40 pM to 150 nM with detection limit of 15 pM for cocaine. The sensitivity of the analytical system was comparable and even better than other reported methods for cocaine detection. The designed method displayed excellent cocaine selectivity. The aptasensor could work well for cocaine assay in serum samples. So, the aptasensor is expected to be an efficient analytical method with broad applications in the determination of diverse analytes.

A novel colorimetric aptasensor for ultrasensitive detection of aflatoxin M1 based on the combination of CRISPR-Cas12a, rolling circle amplification and catalytic activity of gold nanoparticles.

Colorimetric approaches have received noticeable attention among sensing methods in view of simplicity and watching the color change of sample by the naked eyes. However, developing colorimetric sensing methods which show high sensitivity is still problematic. Herein, based on CRISPR-Cas12a, rolling circle amplification (RCA) and catalytic activity of gold nanoparticles (AuNPs), a colorimetric aptasensor was introduced for highly sensitive detection of aflatoxin M1 (AFM1). In the presence of AFM1, the CRISPR-Cas12a is inactivated and large single-stranded DNA (ssDNA) structures are formed on the surface of AuNPs following the addition of T4 DNA ligase and phi29 DNA polymerase. So, the sample color remains yellow after addition of 4-nitrophenol. However, no huge DNA structure is observed on the surface of AuNPs in the absence of target because of activation of CRISPR-Cas12a and digestion of primer. So, the color of sample switches to colorless. The results indicated that the biosensor had high selectivity toward AFM1 and the approach achieved a detection limit as low as 0.05 ng/L. In addition, it could sensitively identify AFM1 in the spiked milk samples. Overall, this approach is highly sensitive and does not require sophisticated equipment. Therefore, it maintains promising potential for other mycotoxins detection in real samples by simply replacing the applied sequences.

A novel electrochemical aptasensor for ochratoxin a sensing in spiked food using strand-displacement polymerase reaction.

Herein, an aptasensor is presented for electrochemical determination of ochratoxin A (OTA) based on nontarget-triggered production of rolling circular amplification (RCA). The surface of gold electrode is modified with thiolated complementary strand of aptamer (CS) as both capture probe and primer and OTA aptamer (Apt) as both sensing molecule and padlock probe (PLP). Following the addition of OTA, Apt/OTA conjugate is formed and detached from the electrode surface. Therefore, no RCA is produced after incubation of the modified electrode with T4 DNA ligase and phi29 DNA polymerase and a sharp current signal occurs. The analytical response ranged from 30 pM to 120 nM with detection limit of 5 pM. The designed aptasensor showed superior analytical performance in comparison with other approaches for OTA detection. Also, the approach exhibited good performance for OTA determination in spiked grape juice samples. The technique presented in this study, can be applied to develop sensors for detecting different toxins by replacing the relevant aptamers and complementary strands.

A DNA triangular prism-based fluorescent aptasensor for ultrasensitive detection of prostate-specific antigen.

In this study, a fluorescent aptasensor is described for ultrasensitive detection of prostate-specific antigen (PSA) using DNA triangular prism as a platform for attachment of fluorophore (PicoGreen, PG), streptavidin magnetic beads (SMBs) and RecJf exonuclease as enhancers of fluorescence difference between presence and absence of target. Presence of PSA leads to the formation of the DNA origami. So, a strong fluorescence is observed following the addition of PG. while, the DNA triangular prism cannot be formed in the lack of target. Thus, a very weak fluorescence can be measured after addition of PG. The proposed biosensor indicated high selectivity, a broad linear range from 200 pg/mL to 300 ng/mL and a very low detection limit of 30 pg/mL for PSA. Applying the designed aptasensor, PSA was successfully detected in human serum samples. This work provides a new way for detection of biomarkers in clinical samples.

An ultrasensitive electrochemical sensing method for detection of microcystin-LR based on infinity-shaped DNA structure using double aptamer and terminal deoxynucleotidyl transferase.

This study develops a novel electrochemical sensing platform for microcystin-LR (MC-LR) detection. This aptasensor comprises the hybridization of double aptamer to its complementary strand (CS) on the surface of electrode and generation of an Infinity-shaped DNA structure in the absence of target by terminal deoxynucleotidyl transferase (TdT). The formation of Infinity-shaped construction leads to the development of an ultrasensitive aptasensor for MC-LR detection. In the presence of MC-LR, double aptamer is dissociated from its CS because of its high affinity for MC-LR and leaves the surface of electrode. Subsequently, no Infinity-shaped structure is formed following the introduction of TdT and a strong current signal is observed. The proposed method was employed for specific detection of MC-LR in the range from 60 pM to 1000 nM with a detection limit of 15 pM. The credibility of the approach was confirmed by detection of MC-LR in real samples like serum and tap water samples. This study provides a new aptasensor for detection of MC-LR as well as other toxin analysis.

An electrochemical sensing platform based on ladder-shaped DNA structure and label-free aptamer for ultrasensitive detection of ampicillin.

Herein, an electrochemical aptasensor is described for detection of ampicillin (Ampi). The sensing strategy is based on the application of a ladder-shaped DNA structure as a multi-layer physical block on the surface of gold electrode. Attributing to the electrostatic repulsion and physical prevention of the ladder-shaped DNA structure, ultrasensitive detection of Ampi was achieved with a detection limit as low as 1 pM. In the presence of Ampi, the ladder-shaped DNA structure is disassembled and detached from the electrode surface. This leads to the high access of [Fe(CN)6]3-/4- as a redox indicator to the electrode surface and a strong redox peak. The aptasensor response for Ampi detection was in the linear range from 7 pM to 100 nM with the detection limit of 1 pM. The presented analytical strategy showed its application in detecting Ampi in the spiked milk samples with satisfactory performance. This work can be easily expanded for different targets by alternating the corresponding aptamers.

An ultrasensitive electrochemical sensor for 17ß-estradiol using split aptamers.

Herein, a simple electrochemical sensor is proposed for 17ß-estradiol (E2) determination. In this sensing platform, split DNA aptamers for E2 were applied as recognizing agents. In the presence of E2, split aptamers are bound to E2 and establish split1-E2-split2 complex as a bridge on the surface of electrode. This physical bar leads to ultrasensitive detection of E2. This aptasensor is capable of recognizing E2 within 30 min without complicated procedures and expensive equipment. This sensing approach indicated a wide linear range with detection limits of 0.5 pM and 0.7 pM for E2 in tap water and milk samples, respectively. Simplicity and high specificity of the proposed electrochemical aptasensor make it an ideal sensing platform for ultra-low detection of other targets in complicated samples by replacement of split DNA aptamers.

A novel electrochemical aptasensor based on nontarget-induced high accumulation of methylene blue on the surface of electrode for sensing of α-synuclein oligomer.

This study describes a novel electrochemical aptasensor for detection of α-synuclein (α-syn) oligomer, an important biomarker related to Parkinson's and Alzheimer's diseases. The sensing platform is based on exonuclease I (Exo I), terminal deoxynucleotidyl transferase (TdT) and methylene blue. The aptasensor exploits the improved sensitivity because of applications of TdT and Exo I and also a label-free aptamer (Apt). Furthermore, direct immobilization of complementary strand of aptamer (CS) instead of Apt on the surface of electrode prohibits Apt self-assembled monolayer aggregation and keeps the function of the Apt. In the absence of α-syn oligomer, TdT enhances lengths of Apt and CS and so, increases accumulation of methylene blue as redox agent on the surface of electrode, leading to a strong current signal. While in the presence of α-syn oligomer, Exo I digests CS on the electrode surface, resulting in less accumulation of methylene blue on the electrode surface and a weak current signal. The relative electrochemical signal of the aptasensor increased linearly with the logarithm of α-syn oligomer concentration in the range from 60 pM to 150 nM. The detection limit was 10 pM. Furthermore, the sensor showed high precision and repeatability for detection of α-syn oligomer in serum samples.

A fluorescent aptasensor based on a DNA pyramid nanostructure for ultrasensitive detection of ochratoxin A.

Analytical techniques for detection of ochratoxin A (OTA) in food products and blood serum are of great significance. In this study, a fluorescent aptasensor was developed for sensitive and specific detection of OTA, based on a DNA pyramid nanostructure (DPN) and PicoGreen (PG) dye. The designed aptasensor inherits characteristics of DPN, such as high stability and capacity for PG loading. PG, as a fluorescent dye, could bind to double-stranded DNA (dsDNA). In the absence of OTA, the pyramid structure of DPN remains intact, leading to a very strong fluorescence emission. Because of higher affinity of aptamer for its target relative to its complementary strand, upon addition of target, the pyramid structure of DPN is disassembled, leading to a weak fluorescence emission. The presented aptasensor showed high specificity toward OTA with a limit of detection (LOD) as low as 0.135 nM. Besides, the designed sensing strategy was successfully utilized to recognize OTA in serum and grape juice with LODs of 0.184 and 0.149 nM, respectively.

A label-free fluorescent aptasensor for selective and sensitive detection of streptomycin in milk and blood serum.

Sensitive and fast detection of antibiotic residues in animal derived foods and blood serum is of great interest. In this study a fluorescent aptasensor was designed for selective and sensitive detection of streptomycin (STR) based on Exonuclease III (Exo III), SYBR Gold and aptamer complimentary strand. In the absence of STR, the fluorescence intensity is weak. Upon addition of STR, the aptamer binds to its target, leading to release of complementary strand from aptamer and more protection against Exo III function. Following addition of SYBR Gold, a strong fluorescence intensity is obtained. This aptasensor showed a high selectivity toward STR with a limit of detection (LOD) as low as 54.5 nM. The validity of the procedure and applicability of the aptasensor were successfully assessed by detection of STR in a spiked milk and blood serum without interference from the sample matrix.