Ultrasensitive Ochratoxin A Detection in Cereal Products Using a Fluorescent Aptasensor Based on RecJf Exonuclease-Assisted Target Recycling.

Ochratoxin A (OTA) is a mycotoxin widely found in foodstuffs such as cereal grains. It greatly threatens human health owing to its strong toxicity and high stability. Aptasensors have emerged as promising tools for the analysis of small molecule contaminants. Nucleic-acid-based signal amplification enables detectable signals to be obtained from aptasensors. However, this strategy often requires the use of complex primers or multiple enzymes, entailing problems such as complex system instability. Herein, we propose a fluorescent aptasensor for the ultrasensitive detection of OTA in cereal products, with signal amplification through RecJf exonuclease-assisted target recycling. The aptamer/fluorescein-labeled complementary DNA (cDNA-FAM) duplex was effectively used as the target-recognition unit as well as the potential substrate for RecJf exonuclease cleavage. When the target invaded the aptamer-cDNA-FAM duplex to release cDNA-FAM, RecJf exonuclease could cleave the aptamer bonded with the target and release the target. Thus, the target-triggered cleavage cycling would continuously generate cDNA-FAM as a signaling group, specifically amplifying the response signal. The proposed exonuclease-assisted fluorescent aptasensor exhibited a good linear relationship with OTA concentration in the range from 1 pg/mL to 10 ng/mL with an ultralow limit of detection (6.2 ng/kg of cereal). The analytical method showed that recoveries of the cereal samples ranged from 83.7 to 109.3% with a repeatability relative standard deviation below 8%. Importantly, the proposed strategy is expected to become a common detection model because it can be adapted for other targets by replacing the aptamer. Thus, this model can guide the development of facile approaches for point-of-care testing applications.

Ultrasensitive Detection Strategy of Norovirus Based on a Dual Enhancement Strategy: CRISPR-Responsive Self-Assembled SNA and Isothermal Amplification.

Spherical nucleic acids (SNAs) have been used to construct various nanobiosensors with gold nanoparticles (AuNPs) as nuclei. The SNAs play a critical role in biosensing due to their various physical and chemical properties, programmability, and specificity recognition ability. In this study, CRISPR-responsive self-assembled spherical nucleic acid (CRISPR-rsSNA) detection probes were constructed by conjugating fluorescein-labeled probes to the surface of AuNPs to improve the sensing performance. Also, the mechanism of ssDNA and the role of different fluorescent groups in the self-assembly process of CRISPR-rsSNA were explored. Then, CRISPR-rsSNA and reverse transcription-recombinase polymerase amplification (RT-RPA) were combined to develop an ultrasensitive fluorescence-detection strategy for norovirus. In the presence of the virus, the target RNA sequence of the virus was transformed and amplified by RT-RPA. The resulting dsDNA activated the trans-cleavage activity of CRISPR cas12a, resulting in disintegrating the outer nucleic acid structure of the CRISPR-rsSNA at a diffusible rate, which released reporter molecules. Norovirus was quantitated by fluorescence detection. This strategy facilitated the detection of the norovirus at the attomolar level. An RT-RPA kit for norovirus detected would be developed based on this method. The proposed method would be used for the detection of different viruses just by changing the target RNA and crRNA of the CRISPR cas12a system which provided a foundation for high-throughput detection of various substances.

A novel universal small-molecule detection platform based on antibody-controlled Cas12a switching.

Molecular diagnostics play an important role in illness detection, prevention, and treatment, and are vital in point-of-care test. In this investigation, a novel CRISPR/Cas12a based small-molecule detection platform was developed using Antibody-Controlled Cas12a Biosensor (ACCBOR), in which antibody would control the trans-cleavage activity of CRISPR/Cas12a. In this system, small-molecule was labeled around the PAM sites of no target sequence(NTS), and antibody would bind on the labeled molecule to prevent the combination of CRISPR/Cas12a, resulting the decrease of trans-cleavage activity. Biotin-, digoxin-, 25-hydroxyvitamin D3 (25-OH-VD3)-labeled NTS and corresponding binding protein were separately used to verify its preformance, showing great universality. Finally, one-pot detection of 25-OH-VD3 was developed, exhibiting high sensitivity and excellent specificity. The limit of detection could be 259.86 pg/mL in serum within 30 min. This assay platform also has the advantages of low cost, easy operation (one-pot method), and fast detection (∼30 min), would be a new possibilities for the highly sensitive detection of other small-molecule targets.

N-doped molybdenum oxide quantum dots as fluorescent probes for the quantitative detection of copper ions in environmental samples.

A novel, sensitive, and selective fluorescence sensor based on N-doped Mo oxide quantum dots (N-MoOx QDs) was fabricated for the detection of Cu2+ ions in water. The presence of Cu2+ induced dynamic fluorescence quenching of the N-MoOx QDs. The sensing conditions were optimized to enhance selectivity and sensitivity. Under optimal conditions, the linear relationship between fluorescence response at 408 nm and Cu2+ concentration was determined. The linear range of this relationship was 1-100 µM. The limits of detection (LOD) and quantitation (LOQ) for Cu2+ were 0.78 µM and 2.34 µM, respectively. The method was successfully applied to detect Cu2+ in water samples with satisfactory sample recovery rates from 91.7 to 116.4%. The sensor exhibits high selectivity toward Cu2+, making it useful for environmental sample monitoring.

Determination of Lincomycin in Milk Using Cu-Based Metal-Organic Framework Adsorbent and Liquid Chromatography-Tandem Mass Spectrometry.

Antibiotic drug residues can adversely affect the human body. Lincomycin is a common veterinary drug that can form residues in foods of animal origin. However, the detection of trace residue levels of lincomycin residues in real samples is challenging. Here, a simple solid phase extraction (SPE) method was developed for the enrichment of lincomycin from cow milk samples before its detection by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The adsorbent used in the SPE was a Cu-based metal-organic framework (Cu-MOF) prepared by the solvothermal synthesis approach. The prepared MOFs were characterized using scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR), X-ray diffractometry (XRD), differential thermogravimetric analysis (TG-DTA), and N2 adsorption-desorption experiments. The adsorption capacity (adsorption equilibrium, extraction time, pH), and elution solvent parameters were investigated. Under the optimized conditions of the HPLC-MS/MS method, lincomycin was detected in the linear range of 10-200 g/L with a detection limit of 0.013 ng/mL. Commercial milk samples were spiked with lincomycin, and a recovery rate between 92.3% and 97.2% was achieved. Therefore, the current method can be successfully applied for the enrichment and determination of lincomycin from milk samples.

Ultrasensitive Automatic Detection of Small Molecules by Membrane Imaging of Single Molecule Assays.

Determination of trace amounts of targets or even a single molecule target has always been a challenge in the detection field. Digital measurement methods established for single molecule counting of proteins, such as single molecule arrays (Simoa) or dropcast single molecule assays (dSimoa), are not suitable for detecting small molecule, because of the limited category of small molecule antibodies and the weak signal that can be captured. To address this issue, we have developed a strategy for single molecule detection of small molecules, called small molecule detection with single molecule assays (smSimoa). In this strategy, an aptamer is used as a recognition element, and an addressable DNA Nanoflower (DNF) attached on the magnetic beads surface, which exhibit fluorescence imaging, is employed as the output signal. Accompanied by digital imaging and automated counting analysis, E2 at the attomolar level can be measured. The smSimoa breaks the barrier of small molecule detection concentration and provides a basis for high throughput detection of multiple substances with fluorescence encoded magnetic beads.

Design and synthesis of DNA hydrogel based on EXPAR and CRISPR/Cas14a for ultrasensitive detection of creatine kinase MB.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems exhibit significant potential in developing biosensing technology due to their collateral cleavage capabilities. Herein, we introduced the collateral cleavage activity of CRISPR/Cas14a to activate DNA hydrogel for ultrasensitive detection of the myocardial infarction biomarker creatine kinase MB (CK-MB). In this strategy, the designed CRISPR/Cas14a system can be activated by introducing complementary DNA (cDNA) derived from competitive dissociation and exponential amplification (EXPAR), which is positively correlated with creatine kinase isoenzyme (CK-MB) concentration. Then the activated Cas14a protein can be utilized to indiscriminately cleave the DNA hydrogel cross-linker strand, leading to the degradation of the gel matrix and thus releasing the pre-encapsulated PtNPs/Cu-TCPP(Fe). PtNPs/Cu-TCPP(Fe) can trigger the TMB reaction, leading to an increase in absorbance value at 450 nm, thus enabling the quantitative detection of CK-MB. The proposed strategy combines CRISPR/Cas14a with DNA hydrogel for the first time, improving the programmability of DNA hydrogel and providing a reliable, sensitive, and versatile detection platform for trace non-nucleic acid targets.

Detection of ochratoxin A by fluorescence sensing based on mesoporous materials.

We developed a new ochratoxin A (OTA) aptamer biosensor to promptly detect OTA in food. Mesoporous silica nanoparticles were used as carriers, and aptamers were used as recognition probes and gating molecules. The fluorescent dye rhodamine 6G was loaded into mesoporous silica, and through electrostatic contact, the OTA aptamer was adsorbed on amino-modified mesoporous silica. The fluorescent dye released from the mesopore in the presence of OTA because of the conformational change induced in the aptamer by the target. The amount of ochratoxin was determined by measuring the fluorescence intensity. Our findings revealed a positive relationship between the fluorescence intensity and OTA concentration, with a limit of detection of 0.28 ng mL-1, and the detection range was 0.05-200 ng mL-1. The recovery rate was 80.7%-110.8% in real samples. The proposed approach is suitable for the quantification of other toxins.

Aptamer-based photonic crystals enable ultra-trace detection of staphylococcal enterotoxin B without labels.

Staphylococcal enterotoxin B (SEB) is one of the most common serotypes in staphylococcal food-poisoning cases. A rapid, sensitive, and simple method for SEB detection is crucial for public health. A photonic crystal (PC) sensing material for label-free detection of ultra-trace SEB was proposed in this study. Gold nanoparticle-doped silica microspheres were stacked to form an opal PC through self-assembly, and SEB aptamers, as the recognition element, were modified onto the PC. When the target protein of SEB came in contact with the PC sensing material, the reflection peak intensity of PCs decreased accordingly. The detection range was 1 × 10-6 to 1 ng mL-1, and the detection limit was 0.103 × 10-6 ng mL-1. Furthermore, the PC sensing material had great specificity and accuracy, which can be used for real sample monitoring. This PC sensing material achieved ultra-sensitive detection, which did not involve complicated preparation processes and reporter labelling.

A highly sensitive fluorometric biosensor for Fumonisin B1 detection based on upconversion nanoparticles-graphene oxide and catalytic hairpin assembly.

Fumonisin (FB) is a common mycotoxin in corn, wheat, oats, and their related products. FB1 is the most predominant among fumonisins and is responsible for severe food contamination that may have deleterious effects on public health. Therefore, the demand for achieving sensitive detection of FB1 is becoming more and more pressing. In the present study, a creative biosensor for FB1 detection was developed based on fluorescence resonance energy transfer between upconversion nanoparticles (UCNPs) and graphene oxide (GO) with catalytic hairpin assembly (CHA) target recycling and amplification. In the presence of FB1, UCNPs were bound to the CHA amplification products away from the GO surface. Simultaneously, a strong fluorescence signal was produced at 980 nm (near-infrared light). The correlation between the concentration of FB1 and the fluorescence intensity exhibited a high relevance (R2 = 0.9971) ranging from 0.032 to 500 ng mL-1, and the method reached a considerably low limit of detection (0.0121 ng mL-1). It could be applied for the detection of FB1 in corn, oats, and infant supplements. The sensitive detection of FB1 proves the application potential of this method on food safety detection. This biosensor can enable high-throughput fungal toxin detection by allowing the use of various aptamers.

A novel biosensor based on antibody controlled isothermal strand displacement amplification (ACISDA) system.

The overuse of antibiotics has aroused widespread concern in recent decades. Their residues in food and environment may pose potential risks to human health. Therefore, highly sensitive and rapid detection methods of antibiotics are urgently needed. Inspired by allosteric transcription factors (aTFs), we proposed a novel strategy for small molecules detection based on antibody controlled isothermal chain displacement amplification (ACISDA). A combination of nicking endonuclease, Klenow Fragment polymerase, specific antibody and a pair of antigen-labeled DNA regulate the synthesis of a G-quadruplex by isothermal chain displacement amplification. The presence of a target induces the antibody dissociation from the antigen-labeled DNA, which induces the synthesis of a G-quadruplex, and a fluorescent signal is produced by thioflavine T (ThT) binding to G-quadruplex. To test this notion, norfloxacin-conjugated DNA (named Primer-NOR) was prepared and ACISDA system was established combining with anti-norfloxacin antibody. This system could detect norfloxacin in a linear range of 0.1 ∼ 500 ng/mL with detection limit of 0.04 ng/mL, and this system could be applied to the detection of norfloxacin in real samples with good performance. Meanwhile, this system could also realize washing-free, immobilization-free and "ready-to-use", and could be used for other small molecules quickly by replacing the antigen-labeled DNA and specific antibody.

Target-responsive DNA hydrogel with microfluidic chip smart readout for quantitative point-of-care testing of creatine kinase MB.

Swift and effective diagnosis of acute myocardial infarction (AMI) is critical to patient survival due to its serious life-threatening effects and increasing incidence. Creatine kinase MB (CK-MB) is one of the markers of AMI. In this work, we enabled a portable visual quantitative assay of CK-MB by incorporating target-responsive DNA hydrogel with a microfluidic chip. The CK-MB aptamer and the complementary short DNA strand were grafted onto the polyacrylamide strand separately and formed the hydrogel by base-paired linkage. Upon introduction of CK-MB, the aptamer bound to CK-MB. This led to hydrogel dissociation and subsequent release of pre-trapped gold nanoparticles (AuNPs), which is proportional to the concentration of CK-MB. To achieve portable on-site detection, we further combined the hydrogel with a microfluidic chip and utilized the color change caused by the released AuNPs to take picture and analyze the average gray value. Then, as low as 0.027 nM CK-MB could be detected by cell phone. With good portability, visualization, and simple sample handling, this method has great potential for quantitative point-of-care testing (POCT) of targets in resource-constrained settings.

Bifunctional ligand-mediated amplification of polydiacetylene response to biorecognition of diethylstilbestrol for on-site smartphone detection.

Polydiacetylene (PDA) is very suited for sensitively detecting large biomolecules, and its unique chromatic properties enable visual read-out. However, application to the selective detection of small molecules remains challenging. Here, bifunctional ligands are studied to amplify the color change of PDA for biorecognition of small molecules for the smartphone-based detection of diethylstilbestrol (DES). PDA is decorated with streptavidin (PDA-SA, blue), and biotin-modified DES (bio-DES) is prepared as a bifunctional ligand to couple with PDA-SA and DES antibody. Since multiple bio-DES can bind to a single SA, then multiple SAs on PDA lead to an increased surface coverage of the vesicle. In samples without DES, PDA-SA-bio-DES-DES antibody complexes will form, leading to a color transition (blue to red); this color transition is greatly amplified by antibody-induced aggregation of the complexes. When DES is present, aggregation is inhibited due to competition for the antibody and PDA-SA-bio-DES retains its blue color. A linear relationship (0.4-1250 ng mL-1) is found between the colorimetric response and the logarithmic DES concentration, with adequate selectivity, accuracy (82.24-118.64%), and precision (below 8.24%). Finally, a paper-based DES PDA biosensor is developed with visual and smartphone-based detection limits of 10 ng mL-1 and 0.85 ng mL-1 in water, respectively.

Highly Ordered, Plasmonic Enhanced Inverse Opal Photonic Crystal for Ultrasensitive Detection of Staphylococcal Enterotoxin B.

Although there is considerable interest in self-assembly of ordered, porous "inverse opal" structures for optical, electronic, and chemical applications, uncontrolled defect formation limits the usefulness of such materials. Herein, we develop a highly ordered and plasmonic enhanced sensing inverse opal photonic crystal (IOPC) material. The co-assembly of the colloidal template with the matrix material avoids the need for liquid penetration into the preassembled colloidal crystals and minimizes the associated rupture and inhomogeneity of the resulting IOPC. Au nanoparticles (Au NPs) not only act as a "bridge" between recognition elements (aptamers) and IOPCs, but also can amplify optical signals. Furthermore, the enhancement mechanism of Au NPs is simulated by COMSOL. During the detection process, the optical signal of the sensing Au-Apt IOPC responds to the Staphylococcal enterotoxin B with a concentration ranging from 10-2 to 103 pg mL-1, and the limit of detection is 2.820 fg mL-1. Spiked real sample detection indicates that the as-proposed method possessed good accuracy. The sensing Au-Apt IOPC provides an extensive biosensor platform to detect a variety of toxic and harmful substances through replacing the aptamer by other recognition elements, such as antibodies or receptors.

Dual Sensitization Smartphone Colorimetric Strategy Based on RCA Coils Gathering Au Tetrahedra and Its Application in the Detection of CK-MB.

In recent years, the combination of DNA nanotechnology and biosensing has been extensively reported. Herein, we attempted to develop a dual sensitization smartphone colorimetric strategy based on rolling circle amplification (RCA) coils gathering Au tetrahedra and explore its application. The dual sensitization effect of this strategy was achieved by rolling circle amplification (RCA) and Au tetrahedra. Under the initiation of the complementary DNA, a large number of ssDNA were generated, achieving amplification of the reaction signal. At the same time, due to the formation of Au tetrahedra, more gold nanoparticles could be gathered under the same conditions, and the signal would be amplified again. Using software ImageJ, the gray value of the reaction solution can be analyzed, detecting the target timely under the practical conditions of lack of equipment. By selecting aptamers with strong binding affinity, we applied this strategy to detect creatine kinase isoenzymes (CK-MB), showing a limit of detection of 0.8 pM, which performed well in actual detection and can meet the needs for real-time detection of CK-MB. Therefore, a universal detection platform was developed, which has broad application prospects in biosensing, clinical diagnosis, food detection, and other fields.

A highly sensitive method for simultaneous detection of hAAG and UDG activity based on multifunctional dsDNA probes mediated exponential rolling circle amplification.

DNA glycosylase is an indispensable DNA damage repair enzyme which can recognize and excise the damaged bases in the DNA base excision-repair pathway. The dysregulation of DNA glycosylase activity will give rise to the dysfunction of base excision-repair and lead to abnormalities and diseases. The simultaneous detection of multiple DNA glycosylases can help to fully understand the normal physiological functions of cells, and determine whether the cells are abnormal in pre-disease. Regrettably, the synchronous detection of functionally similar DNA glycosylases is a great challenge. Herein, we developed a multifunctional dsDNA probe mediated exponential rolling circle amplification (E-RCA) method for the simultaneously sensitive detection of human alkyladenine DNA glycosylase (hAAG) and uracil-DNA glycosylase (UDG). The multifunctional dsDNA probe contains the hypoxanthine sites and the uracil sites which can be recognized by hAAG and UDG respectively to generate apyrimidinic (AP) sites in the dsDNA probe. Then the AP sites will be recognized and cut by endonuclease Ⅳ (Endo IV) to release corresponding single-stranded primer probes. Subsequently, two padlock DNA templates are added to initiate E-RCA to generate multitudinous G-quadruplexes and/or double-stranded dumbbell lock structures, which can combine N-methyl mesoporphyrin IX (NMM) and SYBR Green Ⅰ (SGI) for the generation of respective fluorescent signals. The detection limits are obtained as low as 0.0002 U mL-1 and 0.00001 U mL-1 for hAAG and UDG, respectively. Notably, this method can realize the simultaneous detection of two DNA glycosylases without the use of specially labeled probes. Finally, this method is successfully applied to detect hAAG and UDG activities in the lysates of HeLa cells and Endo1617 cells at single-cell level, and to detect the inhibitors of DNA glycosylases.

A fluorescence aptasensor based on controlled zirconium-based MOFs for the highly sensitive detection of T-2 toxin.

T-2 toxin is one of class A trichothecene mycotoxins produced by Fusarium, presenting genotoxic, cytotoxicity, and immunotoxicity for animals and humans. Therefore, It is urgent to establish a rapid test method with high sensitivity, good selectivity and reliability. In this research, by adjusting the synthesis conditions, a kind of NH2-UiO-66 with high quenching efficiency was screened out. On this basis, we constructed a novel fluorescence sensor via Cy3-labeled aptamer (Cy3-aptamer). With the help of π-π interaction, hydrogen bond and coordination, NH2-UiO-66 could adsorb and quench the fluorescence of Cy3-aptamer based on FRET and PET. In the presence of T-2 toxin, it recognized and bound to Cy3-aptamer, leading to the disintegration of the NH2-UiO-66/Cy3-aptamer compound. As the energy transfer process was blocked, the fluorescence intensity was restored, enabling a highly sensitive response to T-2 toxin. There was a good linear correlation between fluorescence intensity and T-2 toxin concentration in the range of 0.5-100 ng ml -1. The LOD of this fluorescence aptasensor was 0.239 ng ml-1 (S/N = 3). Besides, the recoveries of milk and beer were 89.86-108.99% (RSD = 2.0-2.6%) and 92.31-111.51% (RSD = 2.3-2.9%), respectively. The fluorescence aptasensor exhibited advantages of excellent analytical performance, convenient operation procedure and good selectivity. Predictably, the aptasensor was supposed to detect antibiotics and other pollutants, describing an intriguing blueprint and potential application prospect in food safety, biochemical sensing and environmental conservation.

A fluorescence aptasensor for the sensitive detection of T-2 toxin based on FRET by adjusting the surface electric potentials of UCNPs and MIL-101.

T-2 toxin is a class A trichothecene mycotoxin produced by Fusarium, which exhibits genotoxic, cytotoxic, and immunotoxic effects in animals and humans. In this study, we developed an aptasensor for the sensitive detection of T-2 toxin, which was based on fluorescence resonance energy transfer (FRET), and acted by adjusting the electric potential on the surface of upconversion nanoparticles (UCNPs) and MIL-101(Cr). In addition, it combined the excellent spectral properties of UCNPs with the good adsorption quenching abilities of metal organic frameworks (MOFs). Under the action of π-π stacking interactions, the UCNPs-aptamer was adsorbed onto the surface of MIL-101, leading to fluorescence quenching due to the occurrence of FRET. After the addition of T-2 toxin, owing to its selective binding to the UCNPs-aptamer, the UCNPs-aptamer moved away from MIL-101(Cr), resulting in fluorescence recovery. Moreover, the extent of fluorescence recovery was positively correlated with the concentration of T-2 toxin. The limit of detection (LOD) of this sensor was 0.087 ng mL-1 (S/N = 3), and a good linear correlation was observed between the fluorescence intensity and the T-2 toxin concentration in the range of 0.1-100 ng mL-1. Moreover, the recovery of this method was 97.52-109.53% for corn meal samples (relative standard deviation, RSD = 1.7-2.4%) and 90.81-100.02% for beer samples (RSD = 2.4-2.7%). By adjusting the surface electric potentials, the efficient fluorescence aptasensor combined the advantages of UCNPs and MIL-101(Cr) and allowed the first application of such a system in toxin detection, thereby indicating its potential food sample analysis and biochemical sensing.

Ultrasensitive Detection of 17ß-Estradiol (E2) Based on Multistep Isothermal Amplification.

17ß-Estradiol (E2) can cause an adverse effect on the human endocrine system even at the nanomolar level. Measurements of very low levels of E2 remain a critical challenge due to insufficient sensitivity. In this study, a multistep isothermal amplification fluorescence strategy was constructed, which could realize the exponential amplification of target E2. Specifically, strand displacement reaction (SDA), rolling circle amplification (RCA), and multiprimed rolling circle amplification (MRCA) were combined in a series to quantify trace complementary strand of E2 (cDNA). The E2 aptamer and cDNA were hybridized and modified on the magnetic beads. E2 could bind to its aptamer and cause the release of the cDNA. Then, cDNA would combine with the template DNA, initiating the SDA-RCA-MRCA. The molecular beacons, possessing low background signal, whose fluorescence was quenched in the state of chain folding, could be specifically recognized by the long single-stranded DNA (L-ssDNA) generated by the multistep isothermal amplification triggered by cDNA, and then the fluorescence of the molecular beacons could be restored. Therefore, the E2 could be quantitatively detected by the recovery fluorescence intensity. The fluorescence value showed a good linear relationship with the concentration of E2 in the range of 0.001836-183.6 nM, and the limit of detection (LOD) was as low as 63.09 fM. In addition, the recovery rates of this method spiked in milk and water were 80.8-107.0%, respectively. This method has the advantage of multistep isothermal amplification to obtain abundant fluorescence signals, which may provide a new possibility for highly sensitive detection of other small-molecule targets.