Label-free Aptasensor Research Articles

A label-free colorimetric aptasensor based on an engineered chimeric aptamer and Au@FeP nanocomposites for the detection of kanamycin

The abuse of antibiotics such as kanamycin (KAN) has caused severe environmental pollution and health threat, attracting considerable attention for the monitoring of antibiotic residues. Here, a novel bivalent anti-KAN chimeric aptamer (Chi-Apt) was rationally designed through an efficient post-screening strategy that connected two homogenous anti-KAN aptamers with a poly-T linker. The engineered Chi-Apt achieved 34-fold enhanced binding affinity for KAN that was revealed by biolayer interferometry (BLI) measurement compared with the original aptamer. Meanwhile, molecular docking simulation and isothermal titration calorimetry (ITC) assay demonstrated that the engineered Chi-Apt has lower binding free energies and stronger non-covalent binding forces for KAN than the original aptamer. The engineered Chi-Apt was further evaluated by developing a label-free colorimetric aptasensor based on [email protected] nanocomposites for KAN detection. The aptasensor exhibited a lower limit of detection (LOD) than that achieved by the colorimetric aptasensor based on the original aptamer, with a LOD of 0.98 nM and a dynamic response range of 2.4–150 nM. Moreover, good recoveries of 103.3–110.1% in tap water and 107.6–112.7% in milk samples were obtained by the proposed colorimetric aptasensor. The recoveries detected by the proposed colorimetric aptasensor in real samples were consistent with that obtained by HPLC analysis, suggesting the good practicality and accuracy of the colorimetric aptasensor. This study may provide an efficient strategy for engineering high affinity aptamers to construct sensitive biosensing platforms against other targets.

An enzyme-free and label-free fluorescent aptasensor for sensitive detection of kanamycin in milk samples based on hybridization chain reaction

An enzyme-free and label-free fluorescent aptasensor for sensitive detection of kanamycin in milk samples based on hybridization chain reaction

Molecular Docking Insight into the Label-Free Fluorescence Aptasensor for Ochratoxin A Detection.

Ochratoxin A (OTA) is the most common mycotoxin and can be found in wheat, corn and other grain products. As OTA pollution in these grain products is gaining prominence as a global issue, the demand to develop OTA detection technology has attracted increasing attention. Recently, a variety of label-free fluorescence biosensors based on aptamer have been established. However, the binding mechanisms of some aptasensors are still unclear. Herein, a label-free fluorescent aptasensor employing Thioflavin T (ThT) as donor for OTA detection was constructed based on the G-quadruplex aptamer of the OTA aptamer itself. The key binding region of aptamer was revealed by using molecular docking technology. In the absence of the OTA target, ThT fluorescent dye binds with the OTA aptamer to form an aptamer/ThT complex, and results in the fluorescence intensity being obviously enhanced. In the presence of OTA, the OTA aptamer binds to OTA because of its high affinity and specificity to form an aptamer/OTA complex, and the ThT fluorescent dye is released from the OTA aptamer into the solution. Therefore, the fluorescence intensity is significantly decreased. Molecular docking results revealed that OTA is binding to the pocket-like structure and surrounded by the A29-T3 base pair and C4, T30, G6 and G7 of the aptamer. Meanwhile, this aptasensor shows good selectivity, sensitivity and an excellent recovery rate of the wheat flour spiked experiment.

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.

Screening and identification of a DNA aptamer to construct the label-free fluorescent aptasensor for ultrasensitive and selective detection of clothianidin residue in agricultural products

Clothianidin pesticide not only pollutes the ecological environment, but also poses a potential threat to human health. Thus, it is of great importance to develop efficient and accurate methods to recognize and detect clothianidin residues in agricultural products. Aptamer has the advantages of easy modification, high affinity and good stability, which is particularly suitable as a recognition biomolecule for pesticide detection. However, the aptamer against clothianidin has not been reported. Herein, the aptamer (named CLO-1) had good selectivity and strong affinity (Kd = 40.66 ± 3.47 nM) to clothianidin pesticide, which was screened for the first time by Capture-SELEX strategy. The binding effect of CLO-1 aptamer to clothianidin was further studied by circular dichroism (CD) spectroscopy and molecular docking technique. Finally, the CLO-1 aptamer was used as the recognition molecule to construct a label-free fluorescent aptasensor using GeneGreen dye as sensing signal for the highly sensitive detection of clothianidin pesticide. The constructed fluorescent aptasensor had the limit of detection (LOD) as low as 5.527 μg L−1 for clothianidin, and displayed good selectivity against other competitive pesticides. The aptasensor was applied to detect the clothianidin spiked in tomatoes, pears and cabbages, and the recovery rate was good in the range of 81.99%–106.64%. This study provides a good application prospect for the recognition and detection of clothianidin.

A new portable toluidine blue/aptamer complex-on-polyethyleneimine-coated gold nanoparticles-based sensor for label-free electrochemical detection of alpha-fetoprotein.

The quantification of alpha-fetoprotein (AFP) as a potential liver cancer biomarker which is generally found in ultratrace level is of significance in biomedical diagnostics. Therefore, it is challenging to find a strategy to fabricate a highly sensitive electrochemical device towards AFP detection through electrode modification for signal generation and amplification. This work shows the construction of a simple, reliable, highly sensitive, and label-free aptasensor based on polyethyleneimine-coated gold nanoparticles (PEI-AuNPs). A disposable ItalSens screen-printed electrode (SPE) is employed for fabricating the sensor by successive modifying with PEI-AuNPs, aptamer, bovine serum albumin (BSA), and toluidine blue (TB), respectively. The AFP assay is easily performed when the electrode is inserted into a small Sensit/Smart potentiostat connected to a smartphone. The readout signal of the aptasensor derives from the electrochemical response of TB intercalating into the aptamer-modified electrode after binding with the target. The decrease in current response of the proposed sensor is proportional to the AFP concentration due to the restriction of the electron transfer pathway of TB by a number of insulating AFP/aptamer complexes on the electrode surface. PEI-AuNPs improve SPE's reactivity and provide a large surface area for aptamer immobilization whereas aptamer provides selectivity to the target AFP. Consequently, this electrochemical biosensor is highly sensitive and selective for AFP analysis. The developed assay reveals a linear range of detection from 10 to 50000pgmL-1 with R 2 = 0.9977 and provided a limit of detection (LOD) of 9.5pgmL-1 in human serum. With its simplicity and robustness, it is anticipated that this electrochemical-based aptasensor will be a benefit for the clinical diagnosis of liver cancer and further developed for other biomarkers analysis.

One-step fabrication of the novel electrochemical sensing platform for the ultrasensitive determination of Microcystin-LR

To ensure the safety of the natural ecosystem, it is critical to develop portable, easy-to-operate and environmentally friendly quantitative analytical electrodes for the determination of microcystin-LR (MC-LR) in environmental water. Herein, a simple, mask-free, and one-step method based on laser-induced graphene (LIG) technology is designed for the fabrication of (3D) graphene electrodes integrated with gold nanoparticles (AuNPs). The 3-electrode system with AuNPs-modified graphene was prepared directly by overlaying gold leaf on the polyimide (PI) film followed by patterned CO2 laser irradiation. The reference electrode region was then uniformly coated with Ag/AgCl paste, and the working electrode was functionalized with the aptamer through Au-S self-assembly and bonding to obtain a label-free MC-LR aptasensor. The developed MC-LR aptasensor has a detection limit of 0.053 pM and a linear range from 0.1 pM to 10 nM. Furthermore, the fabricated aptasensor presents good stability, reproducibility, specificity and detection in real samples. These sensors show great promise as a low-cost, simple and portable approve for label-free detection MC-LR in environmental samples.

Generation of a high-affinity DNA aptamer for on-site screening of toxic aristolochic acid I in herbal medicines and botanical products

Aristolochic Acid I (AAI) is an environmental and foodborne toxin found in the Aristolochia and Asarum species of plants that are widespread all over the world. Therefore, there is an urgent need to develop a sensitive and specific biosensor for identifying AAI. Aptamers as a powerful biorecognition element provide the most viable options for solving this problem. In this study, we used library-immobilized SELEX to isolate an AAI-specific aptamer with a KD value of 86 ± 13 nM. To verify the practicability of the selected aptamer, a label-free colorimetric aptasensor was designed. This aptasensor exhibited a low detection limit of 225 nM. Besides, it had been further applied for the determination of AAI in real samples and the recoveries ranged from 97.9% to 102.4%. In the future, AAI aptamer will provide a promising tool for safety evaluation in various fields of agriculture, food, and medication.

Methamphetamine determination using label-free impedimetric aptasensor based on ceria nanocomposite

Methamphetamine determination using label-free impedimetric aptasensor based on ceria nanocomposite

Split aptamer-based fluorescent biosensor for ultrasensitive detection of cocaine using N-methyl mesoporphyrin IX as fluorophore

Cocaine is an extensively used illicit drug worldwide. So, the development of a sensing method for cocaine detection is necessary for examination and lawful action against drug trafficking and abuse. A label-free and simple split aptamer-based fluorescent aptasensor was proposed for cocaine detection using the N-methyl mesoporphyrin IX (NMM)/G-quadruplex system as a fluorescent indicator. The working principle of the developed aptasensor is an increase in the emission intensity of fluorescence of the NMM in the attendance of cocaine due to the formation of split G-quadruplex structure as a suitable platform for attachment of NMM. Under optimized conditions, the designed fluorescent aptasensor exhibited a useful linear concentration range from 50 pM to 25 nM and a limit of detection (LOD) of 16 pM. The applied potential of the designed fluorescent aptasensor was demonstrated by cocaine detection in spiked human serum samples with satisfying recoveries.

Ultra-sensitive label-free SERS biosensor with high-throughput screened DNA aptamer for universal detection of SARS-CoV-2 variants from clinical samples

COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused an ongoing global pandemic with economic and social disruption. Moreover, the virus has persistently and rapidly evolved into novel lineages with mutations. The most effective strategy to control the pandemic is suppressing virus spread through early detection of infections. Therefore, developing a rapid, accurate, easy-to-use diagnostic platform against SARS-CoV-2 variants of concern remains necessary. Here, we developed an ultra-sensitive label-free surface-enhanced Raman scattering-based aptasensor as a countermeasure for the universal detection of SARS-CoV-2 variants of concern. In this aptasensor platform, we discovered two DNA aptamers that enable binding to SARS-CoV-2 spike protein via the Particle Display, a high-throughput screening approach. These showed high affinity that exhibited dissociation constants of 1.47 ± 0.30 nM and 1.81 ± 0.39 nM. We designed a combination with the aptamers and silver nanoforest for developing an ultra-sensitive SERS platform and achieved an attomolar (10−18 M) level detection limit with a recombinant trimeric spike protein. Furthermore, using the intrinsic properties of the aptamer signal, we demonstrated a label-free aptasensor approach, enabling use without the Raman tag. Finally, our label-free SERS-combined aptasensor succeeded in detecting SARS-CoV-2 with excellent accuracy, even in clinical samples with variants of concern, including the wild-type, delta, and omicron variants.

Electrochemical aptasensor based on exonuclease III-mediated signal amplification for sensitive detection of vomitoxin in cornmeal

Vomitoxin (DON) residues in grains are of great concern to public health. Herein, a label-free aptasensor was constructed to detect DON distributed in grains. Cerium-based metal-organic framework composite gold nanoparticles (CeMOF@Au) were used as substrate materials to facilitate electron transfer and provided more binding sites for DNA. The separation of DON-aptamer (Apt) complex and cDNA was achieved by magnetic separation technique based on magnetic beads (MBs), ensuring the specificity of the aptasensor. Exonuclease III (Exo III)-assisted cDNA cycling process strategy would be triggered when cDNA was separated and introduced to the sensing interface for further signal amplification. Under optimal conditions, the constructed aptasensor presented a wide detection range from 1 × 10−8 mg·mL−1 to 5 × 10−4 mg·mL−1 for DON, and the detection limit was 1.79 × 10−9 mg·mL−1, including a satisfactory recovery in cornmeal sample spiked with DON. The results showed that the proposed aptasensor had high reliability and promising application potential in detecting DON.

Design of a novel aptamer/molecularly imprinted polymer hybrid modified Ag–Au@Insulin nanoclusters/Au-gate-based MoS2 nanosheet field-effect transistor for attomolar detection of BRCA1 gene

Early detection of breast cancer, the first main cause of death in women, with robust assay platforms using appropriate biomarkers is of great importance for diagnosis and follow-up of the disease progression. This paper introduces an extra selective and sensitive label-free aptasensor for the screening of BRCA1 gene biomarker by taking advantage of a gate modified with aptamer and molecularly imprinted polymer hybrid (MIP) as a new synthetic receptor film coupled with an electrolyte-gated molybdenum disulfide (MoS2) field-effect transistor (FET). The Au gate surface of FET was modified with insulin stabilized bimetallic Ag–Au@nanoclusters (Ag–Au@InsNCs), after which, the immobilization of the hybridized aptamer and o-phenylenediamine was electropolymerized to form an aptamer-MIP hybrid receptor. The output characteristics of Apta-MIP hybrid modified Au gate MoS2 FET device were followed as a result of change in electrical double layer capacitance of electrolye-gate interface. The magnitude of decrease in the drain current showed a linear response over a wide concentration range of 10 aM to 1 nM of BRCA1 ssDNA with a sensitivity as high as 0.4851 μA/decade of concentration and a limit of detection (LOD) of 3.0 aM while very low responses observed for non-imprinted polymer. The devised aptasensor not only was capable to the discrimination of the complementary versus one-base mismatch BRCA1 ssDNA sequence, but also it could detect the complementary BRCA1 ssDNA in spiked human serum samples over a wide concentration range of 10 aM to 1.0 nM with a low LOD of 6.4 aM and a high sensitivity 0.3718 μA/decade.

A novel label-free dual-mode aptasensor based on the mutual regulation of silver nanoclusters and MoSe2 nanosheets for reliable detection of ampicillin

Current methods for the rapid detection of trace antibiotics in the environment remains problems of low accuracy and false negative or false positive, making the development of fast, and accurate, and reliable methods for antibiotic testing a major challenge that needs to be addressed. Herein, we developed a novel label-free colorimetric and fluorescent dual-mode aptasensor assembled by the strong interaction of layered MoSe2 nanosheets (MoSe2 NSs) with ampicillin (AMP) aptamer functionalized silver nanoclusters (Apt-AgNCs) that specifically bind AMP to allow the sensitive and selective detection of AMP. Apt-AgNCs could be adsorbed on the surface of MoSe2 NSs via van der Waals force to form a nanocomposite, Apt-AgNCs/MoSe2 NSs. Interestingly, Apt-AgNCs/MoSe2 NSs act together to construct dual mode aptasensor through modulation of the intrinsic peroxidase activity of MoSe2 NSs and the fluorescence of Apt-AgNCs. In the presence of AMP, Apt-AgNCs could specifically bind AMP, triggering desorption from the MoSe2 NSs surface, leading to a decrease in the peroxidase activity of the system with the recovery in Apt-AgNCs fluorescence. The dual-signal aptasensor exhibited good linear colorimetric and fluorescence responses in the AMP concentration ranges of 0.115–2.00 μM and 6–100 nM, respectively. Furthermore, the aptasensor was successfully measured AMP levels in commercially-bought milk and lake water with satisfactory results. Unlike single-signal aptasensors, the constructed dual-signal aptasensor could not only improve the detection precision, but also reduce the false positive or false negative results. These promising results suggest that the dual-readout strategy as demonstrated is general mode for the detection of other antibiotics or compounds using various aptamers functionalized AgNCs in concert with MoSe2 NSs.

Discovery of DNA aptamers targeting SARS-CoV-2 nucleocapsid protein and protein-binding epitopes for label-free COVID-19 diagnostics

The spread of COVID-19 has affected billions of people across the globe, and the diagnosis of viral infection still needs improvement. Because of high immunogenicity and abundant expression during viral infection, SARS-CoV-2 nucleocapsid (N)protein could be an important diagnostic marker. This study aimed to develop a label-free optical aptasensor fabricated with a novel single-stranded DNA aptamer to detect the N protein. The N-binding aptamers selected using asymmetric-emulsion PCR-SELEX and their binding affinity and cross-reactivity were characterized by biolayer interferometry. The tNSP3 aptamer (44 nt) was identified to bind the N protein of wild type and Delta and Omicron variants with high affinity (KD in the range of 0.6-3.5nM). Utilizing tNSP3 to detect the N protein spiked in human saliva evinced the potential of this aptamer with a limit of detection of 4.5nM. Mass spectrometry analysis was performed along with molecular dynamics simulation to obtain an insight into how tNSP3 binds to the N protein. The identified epitope peptides are localized within the RNA-binding domain and C terminus of the N protein. Hence, we confirmed the performance of this aptamer as an analytical tool for COVID-19 diagnosis.

CMWCNT/CoHCF/AuNPs nanocomposites aptasensor for electrochemical detection of interleukin-6

In this paper, a novel electrochemical sensing platform for the sensitive detection of interleukin 6 (IL-6) was designed and fabricated. A label-free aptasensor was fabricated by modifying the glassy carbon electrode (GCE) using a nanocomposite composed of carboxylated multi-walled carbon nanotubes (cMWCNTs), cobalt hexacyanoferrate (CoHCF) and gold nanoparticles (AuNPs). The nanocomposite improved electron transfer rate and increased intercalation of signaling molecules. Moreover, it also offers desirable capabilities for the immobilization of biomolecules. The cMWCNTs/CoHCF/AuNPs modified electrode showed good electrochemical characteristics and biocompatibility, thus achieving excellent sensor performance. The differential pulse voltammetry (DPV) was used to detect the peak current of CoHCF in the redox process. The peak current is proportional to the concentration of IL-6 on the electrode surface. Under optimal conditions, the proposed aptamer sensor exhibits a linear range from 0.5 pg/mL to 1000 pg/mL and has a low detection limit of 0.17 pg/mL. In addition, the IL-6 aptamer sensor has good selectivity, high stability, and satisfactory reproducibility, showing broad potential in clinical research and diagnostic applications.

Docking-aided rational tailoring of a fluorescence- and affinity-enhancing aptamer for a label-free ratiometric malachite green point-of-care aptasensor

Although nucleic acid aptasensors are increasingly applied in the detection of environmentally hazardous biomolecules, several formidable challenges remain with this technique because of their vulnerability, high cost and suboptimal sensitivity. Here, a docking-aided rational tailoring (DART) strategy was established at three levels and in two dimensions for the refinement of malachite green (MG) DNA aptamers. Guided by in silico molecular docking, coarse and fine tailoring were conducted at three levels each, to significantly enhance fluorescence activation intensity and binding affinity in two dimensions. Empowered by the results of the rational tailoring, a mechanistic view of the MG DNA aptamer-target interaction was thoroughly analyzed via four types of interactions. To meet the demand for point-of-care testing (POCT), a label-free and ratiometric fluorescent aptasensor was developed leveraging the tailored MG aptamer, based on the binding site competition-equilibrium effect via the introduction of a reference dye. This sensitive, specific, low-cost and rapid aptasensor subsequently demonstrated outstanding detection performance, achieving an ideal signal response range of 5 nmol·L−1 - 6 μmol·L−1 and a low limit of detection (LOD) of 1.49 nmol·L−1. The DART strategy and systematic exploration of the MG DNA luminescent aptamers herein will provide a valuable reference in the field of aptamer tailoring, biosensing and bioimaging. The proposed label-free ratiometric aptasensor also provides a highly generalizable strategy for hazardous biomolecular detection.

A Novel and Label-Free Chemiluminescence Detection of Zearalenone Based on a Truncated Aptamer Conjugated with a G-Quadruplex DNAzyme

Zearalenone (ZEN), one of the most frequently occurring mycotoxin contaminants in foods and feeds, poses considerable threat to human and animal health, owing to its acute and chronic toxicities. Thus, rapid and accurate detection of ZEN has attracted broad research interest. In this work, a novel and label-free chemiluminescence aptasensor based on a ZEN aptamer and a G-quadruplex DNAzyme was constructed. It was established on a competitive assay between ZEN and an auxiliary DNA for the aptamer, leading to activation of the G-quadruplex/hemin DNAzyme and subsequent signal amplification by chemiluminescence generation after substrate addition. To maximize the detection sensitivity, numerous key parameters including truncated aptamers were optimized with molecular docking analysis. Upon optimization, our aptasensor exhibited a perfect linear relationship (R2 = 0.9996) for ZEN detection in a concentration range of 1-100 ng/mL (3.14-314.10 nM) within 40 min, achieving a detection limit of 2.85 ng/mL (8.95 nM), which was a 6.7-fold improvement over that before optimization. Most importantly, the aptasensor obtained a satisfactory recovery rate of 92.84-137.27% and 84.90-124.24% for ZEN-spiked wheat and maize samples, respectively. Overall, our label-free chemiluminescence aptasensor displayed simplicity, sensitivity, specificity and practicality in real samples, indicating high application prospects in the food supply chain for rapid detection of ZEN.

A label-free fluorescent aptasensor based on a novel exponential rolling circle amplification for highly sensitive ochratoxin A detection

Rapid and sensitive analysis of ochratoxin A (OTA) plays an important role in food safety. Here, an aptasensor based on novel exponential rolling circle amplification (ERCA) was proposed for ultrasensitive and label-free fluorescence detection of OTA. The attachment of OTA to its aptamer could release H and rapidly hybridize with CT to initiate rolling circle amplification (RCA). The amplicons could further displace H from APH to initiate recycled RCA, achieving exponential growth of amplification products that contained G4 dimers for lighting up ThT. Benefiting from the exponential amplification efficiency of the ERCA strategy and the high fluorescence quantum yield of G4 dimer/ThT, this strategy exhibited a wide linear range from 10 fg/mL to 10 ng/mL with a detection limit of 4.3 fg/mL. In addition, the aptasensor displayed satisfactory recoveries in real sample analysis. We believe that this novel aptasensor possesses promising application prospects in food safety and medicine detection.

Simultaneous screening of multiple diarrhetic shellfish poisons with group-specific split aptamers and silver nanocluster beacon

Poisoning events concerning diarrhetic shellfish poisons (DSPs) are increasing continually. It is extremely necessary to develop simple analysis methods for screening simultaneously different types of DSPs from food-related samples. Okadaic acid (OA) and its analogues, i.e., dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2), are the prevalent DSPs. Herein, a facile and label-free fluorescent aptasensor targeting the three DSPs was constructed with a pair of group-specific split aptamers and silver nanocluster beacon. In presence of the targets, the DNA templates attached at the ends of the split aptamers would be dragged close to trigger enhanced fluorescence signals from silver nanoclusters. The aptasensor offered high sensitivity and good selectivity, with limit of detection of 2.282 nmolL-1, 19.38 nmolL-1, and 13.61 nmolL-1 for OA, DTX-1, and DTX-2, respectively. Moreover, the applicability of aptasensor was well verified with shellfish and seawater samples. This study provides good reference for further exploration on analysis methods for food-related molecules.