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.

Accelerated Wound Healing with a Diminutive Scar through Cocrystal Engineered Curcumin.

Pharmaceutical cocrystals ( Regulatory Classification of Pharmaceutical Co-Crystals Guidance for Industry; Food and Drug Administration, 2018) are crystalline solids produced through supramolecular chemistry to modulate the physicochemical properties of active pharmaceutical ingredients (APIs). Despite their extensive development in interdisciplinary sciences, this is a pioneering study on the efficacy of pharmaceutical cocrystals in wound healing and scar reducing. Curcumin-pyrogallol cocrystal (CUR-PYR) was accordingly cherry-picked since its superior physicochemical properties adequately compensate for limitative drawbacks of curcumin (CUR). CUR-PYR has been synthesized by a liquid-assisted grinding (LAG) method and characterized via FT-IR, DSC, and PXRD analyses. In vitro antibacterial study indicated that CUR-PYR cocrystal, CUR+PYR physical mixture (PM), and PYR are more effective against both Gram-negative (Pseudomonas aeruginosa and Escherichia coli) and Gram-positive (Staphylococcus aureus and Bacillus subtilis) bacteria in comparison with CUR. In vitro results also demonstrated that the viability of HDF and NIH-3T3 cells treated with CUR-PYR were improved more than those received CUR which is attributed to the effect of PYR in the form of cocrystal. The wound healing process has been monitored through a 15 day in vivo experiment on 75 male rats stratified into six groups: five groups treated by CUR-PYR+Vaseline (CUR-PYR.ung), CUR+PYR+Vaseline (CUR+PYR.ung), CUR+Vaseline (CUR.ung), PYR+Vaseline (PYR.ung), and Vaseline (VAS) ointments and a negative control group of 0.9% sodium chloride solution (NS). It was revealed that the wounds under CUR-PYR.ung treatment closed by day 12 postsurgery, while the wounds in other groups failed to reach the complete closure end point until the end of the experiment. Surprisingly, a diminutive scar (3.89 ± 0.97% of initial wound size) was observed in the CUR-PYR.ung treated wounds by day 15 after injury, followed by corresponding values for PYR.ung (12.08 ± 2.75%), CUR+PYR.ung (13.89 ± 5.02%), CUR.ung (16.24 ± 6.39%), VAS (18.97 ± 6.89%), and NS (20.33 ± 5.77%). Besides, investigating histopathological parameters including inflammation, granulation tissue, re-epithelialization, and collagen deposition signified outstandingly higher ability of CUR-PYR cocrystal in wound healing than either of its two constituents separately or their simple PM. It was concluded that desired solubility of the prepared cocrystal was essentially responsible for accelerating wound closure and promoting tissue regeneration which yielded minimal scarring. This prototype research suggests a promising application of pharmaceutical cocrystals for the purpose of wound healing.

A Novel AS1411 Aptamer-Based Three-Way Junction Pocket DNA Nanostructure Loaded with Doxorubicin for Targeting Cancer Cells in Vitro and in Vivo.

Active targeting of nanostructures containing chemotherapeutic agents can improve cancer treatment. Here, a three-way junction pocket DNA nanostructure was developed for efficient doxorubicin (Dox) delivery into cancer cells. The three-way junction pocket DNA nanostructure is composed of three strands of AS1411 aptamer as both a therapeutic aptamer and nucleolin target, the potential biomarker of prostate (PC-3 cells) and breast (4T1 cells) cancers. The properties of the Dox-loaded three-way junction pocket DNA nanostructure were characterized and verified to have several advantages, including high serum stability and a pH-responsive property. Cellular uptake studies showed that the Dox-loaded DNA nanostructure was preferably internalized into target cancer cells (PC-3 and 4T1 cells). MTT cell viability assay demonstrated that the Dox-loaded DNA nanostructure had significantly higher cytotoxicity for PC-3 and 4T1 cells compared to that of nontarget cells (CHO cells, Chinese hamster ovary cell). The in vivo antitumor effect showed that the Dox-loaded DNA nanostructure was more effective in prohibition of the tumor growth compared to free Dox. These findings showed that the Dox-loaded three-way junction pocket DNA nanostructure could significantly reduce the cytotoxic effects of Dox against nontarget cells.

A colorimetric gold nanoparticle aggregation assay for malathion based on target-induced hairpin structure assembly of complementary strands of aptamer.

The authors describe a method for the colorimetric determination of the pesticide malathion. It is based on the use of a hairpin structure consisting of a complementary strand of aptamer and a double-stranded DNA (dsDNA) structure to protect gold nanoparticles (AuNPs) against salt-induced aggregation. In the absence of malathion, the dsDNA structure is preserved on the surface of AuNPs and the color of the AuNPs in solutions containing NaCl remains red. However, in the presence of malathion, a hairpin structure of complementary strand is formed. The Aptamer/Malathion complex and the complementary strand are released from the surface of the AuNPs. As a result, the AuNPs undergo salt-induced aggregation which is accompanied by a color change to blue. The assay allows malathion to be quantified within 35 min (A650/A520 was measured). The detection limit is 1 pM, and response is linear in the 5 pM to 10 nM malathion concentration range. The method is specific and was successfully applied to the determination of malathion in spiked human serum samples. Graphical abstract Schematic representation of detection of malathion based on dsDNA-modified gold nanoparticles (AuNPs) and the hairpin structure of the complementary strand.

A novel chemotherapy drug-free delivery system composed of three therapeutic aptamers for the treatment of prostate and breast cancers in vitro and in vivo.

In this study, a novel chemotherapy drug-free DNA nanocomplex composed of three therapeutic aptamers (IDA, AS1411 and apMNK2F) was designed for treatment of cancer cells. For MTT assay, PC-3 and 4T1 cells (target cells) and CHO cells (nontarget cells) were treated with apMNK2F-AS1411-IDA complex (DNA nanocomplex), as well as AS1411, IDA and apMNK2F alone. Internalization of apMNK2F-AS1411-IDA complex was analyzed by fluorescence imaging and flow cytometry analysis. In the last step, the presented DNA nanocomplex was applied for prohibition of tumor growth in vivo. The results of internalization assay verified that the developed apMNK2F-AS1411-IDA complex was remarkably internalized into PC-3 and 4T1 cells, but not into CHO cells. The results of internalization assay was confirmed by MTT assay. apMNK2F-AS1411-IDA complex was more cytotoxic in PC-3 and 4T1 cells (target) and less cytotoxic in CHO cells (nontarget). Also, the DNA nanocomplex could effectively suppress the growth of tumors in vivo.

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.

Evaluation of the Protective Effects of Lugol's Solution in Rats Poisoned with Aluminum Phosphide (Rice Tablets).

Aluminum phosphide (AlP) is the main component of rice tablets (a pesticide), which produces phosphine gas (PH3) when exposed to stomach acid. The most important symptoms of PH3 toxicity include, lethargy, tachycardia, hypotension, and cardiac shock. It was shown that Iodine can chemically react with PH3, and the purpose of this study is to investigate the protective effects of Lugol solution in poisoning with rice tablets. Five doses (12, 15, 21, 23, and 25 mg/kg) of AlP were selected, for calculating its lethal dose (LD50). Then, the rats were divided into 4 groups: AlP, Lugol, AlP + Lugol, and Almond oil (as a control). After 4 h, the blood pressure and electrocardiogram (ECG) were recorded, and blood samples were obtained for biochemical tests, then liver, lung, kidney, heart, and brain tissues were removed for histopathological examination. The results of the blood pressure showed no significant changes (P > 0.05). In ECG, the PR interval showed a significant decrease in the AlP + Lugol group (P < 0.05). In biochemical tests, LDH, Ca2+, Creatinine, ALP, Mg2+, and K+ represented significant decreases in AlP + Lugol compared to the AlP group (P < 0.05). Also, the administration of Lugol's solution to AlP-poisoned rats resulted in a significant decrease in malondialdehyde levels and a significant increase in catalase activity (P < 0.05). Histopathological evaluation indicates that Lugol improves changes in the lungs, kidneys, brain, and heart. Our results showed that the Lugol solution could reduce tissue damage and oxidative stress in AlP-poisoned rats. We assume that the positive effects of Lugol on pulmonary and cardiac tissues are due to its ability to react directly with PH3.

Pre-concentration of non-steroidal anti-inflammatory drugs in water using dispersive liquid-liquid and single-drop microextraction with high-performance liquid chromatography.

In the present work the combination of two liquid-phase microextraction techniques involving dispersive liquid-liquid microextraction and single-drop microextraction as a new pre-concentration technique is developed for the separation and determination of acidic non-steroidal anti-inflammatory pharmaceutical compounds, namely, naproxen, diclofenac, and ibuprofen, in water samples using high-performance liquid chromatography ultra violet-detection. The extraction conditions were optimized and, under the optimal conditions, the method showed good linearity range of 0.1-1000 µg L(-1) (R > 0.9990), acceptable reproducibilities (relative standard deviation% = 4.5-8.8%), low limits of detection (0.03-0.2 µg L(-1) ), and satisfactory relative recoveries. The developed method was applied for the determination of anti-inflammatory drugs in river and waste water samples.

A highly sensitive fluorescent aptasensor for detection of prostate specific antigen based on the integration of a DNA structure and CRISPR-Cas12a.

Herein, a facile fluorescent CRISPR-Cas12a-based sensing strategy is presented for prostate specific antigen (PSA), as a prostate cancer biomarker, with the assistance of a cruciform DNA nanostructure and PicoGreen (PG) as a fluorochrome. Highly sensitive recognition of PSA is one of the virtues of the proposed method which comes from the use of unique features of both CRISPR-Cas12a and DNA structure in the design of the aptasensor. The presence of PSA creates a cruciform DNA nanostructure in the sample which can be loaded by PG and make sharp fluorescence emission. While, when there is no PSA, the CRISPR-Cas12a digests sequences 1 and 3 as single-stranded DNAs, causing no DNA structure and a negligible fluorescence is detected after addition of PG. This aptasensor presents a sensitive recognition performance with detection limit of 4 pg/mL and a practical use for determination of PSA in serum samples. So, this analytical strategy introduces a convenient and highly sensitive approach for detection of disease biomarkers.

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 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.

Targeted delivery system using silica nanoparticles coated with chitosan and AS1411 for combination therapy of doxorubicin and antimiR-21.

Herein, a novel targeted delivery system was developed for intracellular co-delivery of doxorubicin (DOX) as a chemotherapeutic drug, antimiR-21 as an oncogenic antagomiR. In this system, DOX was loaded into mesoporous silica nanoparticles (MSNs) and chitosan was applied to cover the surface of MSNs. AS1411 aptamer as targeting nucleolin and antimiR-21 were electrostatically attached onto the surface of the chitosan-coated MSNs and formed the final nanocomplex (AACS nanocomplex). The study of drug release was based on DOX release under pH 7.4 and 5.5. Cellular toxicity and cellular uptake assessments of AACS nanocomplex were carried out in nucleolin positive (C26, MCF-7, and 4T1) and nucleolin negative (CHO) cell lines using MTT assay and flow cytometry analysis, respectively. Also, Anti-tumor efficacy of AACS nanocomplex was evaluated in C26 tumor-bearing mice. Overall, the results show that the combination therapy of DOX and antimiR-21, using AACS nanocomplex, could combat the cancer cell growth rate.

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 fluorescent sensing strategy for ultrasensitive detection of oxytetracycline in milk based on aptamer-magnetic bead conjugate, complementary strand of aptamer and PicoGreen.

Misuse of antibiotics in animal husbandry and presence of their residues in animal foods is a serious crisis worldwide and thus, monitoring the level of them in food samples is vital for human health. Herein, a fluorescent aptasensor was developed for highly sensitive quantification of oxytetracycline (OTC) in food samples. This method is based on OTC aptamer conjugated to magnetic beads, functioned as recognition element, complementary strand of OTC aptamer, and PicoGreen (PG) as a sensitive double-stranded DNA (dsDNA) fluorescent dye. Formation of OTC aptamer-magnetic bead conjugate provides the opportunity of sample condensation and separation technology. Additionally, the presence of complementary strand leads to significant fluorescence signal alteration of aptasensor in the presence or absence of target and a noteworthy improvement of the aptasensor sensitivity. In the absence of target, complementary strand could bind to aptamer and form dsDNA on the surface of magnetic bead. As a consequence, adding PG to the sample leads to observation of high fluorescence signal from sample. In contrast, once OTC is added to the sample, it binds to OTC aptamer-magnetic bead complex and prevents hybridization of OTC aptamer and its complementary strand. Hence, after addition of PG to the sample, a weak fluorescence intensity is measured. Under optimized conditions, the linear ranges for OTC detection were 0.2-2 nM and 2-800 nM. The detection limit was calculated to be as low as 0.15 nM for the fabricated aptasensor. Besides the great sensitivity, proposed method demonstrated superior specificity towards OTC once it was used against several antibiotics. More significantly, the recovery rates of OTC in milk ranged from 96.46% to 101.5%, implying the great feasibility of designed sensor as well as its potential to be employed for analysis of OTC in real samples.

An optical aptasensor for aflatoxin M1 detection based on target-induced protection of gold nanoparticles against salt-induced aggregation and silica nanoparticles.

Not only intoxications of aflatoxins are significant risk for human beings, but also; the contamination with these toxins affect the economy. Therefore, developing a rapid, precise and inexpensive determination method is vitally important. Here, a colorimetric aptasensor is introduced for the detection of aflatoxin M1 (AFM1) based on the preservation of gold nanoparticles (AuNPs) against NaCl-induced aggregation by detaching of complementary strand of aptamer (CS) from the aptamer-modified streptavidin coated silica nanoparticles (SNPs) following the addition of target. So, the color of sample remains red. While, in the lack of AFM1, salt-induced aggregation of AuNPs occurs and the color of sample becomes purple as the aptamer/CS (dsDNA)-modified SNPs is stable and CS cannot bind to AuNPs. The proposed aptasensor could detect AFM1 in a linear dynamic range, 300-75,000 ng/L, with a detection limit of 30 ng/L. Also, the sensing method was effectively applied for AFM1 recognition in milk samples.

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.

A smart ATP-responsive chemotherapy drug-free delivery system using a DNA nanostructure for synergistic treatment of breast cancer in vitro and in vivo.

This study demonstrated a chemotherapy drug-free delivery system for breast cancer treatment based on a simple DNA nanostructure composed of sequence 1 containing ATP and AS1411 aptamers and sequence 2 containing antimiR-21. The DNA nanostructure was used for co-delivery of KLA peptide and antimiR-21 as antiapoptotic agents. These therapeutic agents could not be internalised into eukaryotic cells freely which is one of the great features of this targeting platform. The presented delivery system was ATP-responsive, leading to disassembly of the DNA nanostructure in high ATP concentration of cancer cells and restoration of the function of antimiR-21 in these cells. The DNA nanostructure was associated with high cellular uptake by MCF-7 and 4T1 cells due to expression of nucleolin as target of AS1411 on their plasma membranes, while the developed targeting platform could not be internalised into CHO cells because of lack of the active targeting moiety on their surfaces. Furthermore, the results showed that co-delivery of antimiR-21 and KLA peptide using the DNA nanostructure could efficiently prohibit tumour growth in vitro and in vivo and induce a synergistic anticancer activity. Thus, this work provides a new ATP-responsive nanotargeting delivery system and synergistic chemotherapy drug-free regimen for cancer treatment.

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.

A rapid and simple ratiometric fluorescent sensor for patulin detection based on a stabilized DNA duplex probe containing less amount of aptamer-involved base pairs.

In this study, a sensor is described for determination of patulin by using ratiometric fluorescence measurement and strand displacement strategy. In the presence of patulin, the ratiometric fluorescence response decreases, owing to disassembly of DNA duplex structure and target-mediated release of TAMRA-labeled complementary DNA sequence2 (cDNA2). While, in the absence of target, the fluorescence resonance energy transfer (FRET) phenomenon happens between FAM and TAMRA under excitation at 490 nm, resulting in the enhancement of ratiometric signal. The use of ratiometric fluorescence signal with different signal indicators avoids the problem of environmental interference and improves the sensitivity of the aptasensor. Also, the DNA duplex structure contains minimum aptamer-involved base pair sequence, resulting in further improvement of the aptasensor sensitivity. This sensing platform provided a wide linear range from 15 ng/L to 35 µg/L and a detection limit of 6 ng/L for patulin. The aptasensor was used to determine patulin in spiked apple juice samples and showed satisfactory results.

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.