Label-free Aptasensor Research Articles

Label-free and low-background fluorescent structure-switching aptasensor for sensitive detection of staphylococcal enterotoxin A based on graphene oxide-assisted separation of ssDNA

Label-free and low-background fluorescent structure-switching aptasensor for sensitive detection of staphylococcal enterotoxin A based on graphene oxide-assisted separation of ssDNA

Conformational change-based fluorometric aptasensor for sensitive cadmium(II) detection in fruits and vegetables.

Cadmium (Cd2+) is a highly toxic heavy metal that can accumulate in the human body through contaminated food and water, posing great health risks. In this study, a label-free fluorescent aptasensor based on SYBR Green I (SGI) for the rapid and sensitive detection of Cd2+ in food samples was designed. The aptasensor utilizes a Cd2+-specific aptamer (Cd-(21)) and its complementary strand (CSCd-(21)) to form a double-stranded DNA (dsDNA) structure in the absence of Cd2+. SGI intercalates into the dsDNA, resulting in a strong fluorescence signal. In the presence of Cd2+, the aptamer undergoes a conformational change, preventing the formation of dsDNA and leading to a decrease in fluorescence intensity. Under optimized conditions, the aptasensor exhibited a linear response to Cd2+ concentrations ranging from 0.11 to 157.37 ng mL-1, with a limit of detection (LOD) of 0.07 ng mL-1. The aptasensor demonstrated high specificity and was successfully applied to detect Cd2+ in fruits and vegetables, with satisfactory recovery rates (95-111%). The proposed aptasensor provides a promising tool for the rapid and sensitive detection of Cd2+ in food.

A label-free aptasensor based on electrodeposition of gold nanoparticles on silver-based metal-organic frameworks for measuring ochratoxin A in black and red pepper.

Since ochratoxin A (OTA) is immunotoxic, teratogenic and carcinogenic, it is very important to monitor this compound in food samples. In the present work, the development and fabrication of a label-free electrochemical aptasensor based on the gold nanoparticles/silver-based metal-organic framework (AuNPs/Ag-MOF) for the determination of ochratoxin A (OTA) is introduced. The aptasensor was fabricated by electrodeposition of AuNPs on a glassy carbon electrode modified with Ag-MOF. The characteristics of the synthesized Ag-MOF were determined by field emission scanning electron microscopy (FESEM), energy-dispersive X-ray spectroscopy (EDS), Fourier transform infrared spectroscopy (FTIR) and UV-Visible spectroscopy. The aptamer was immobilized on the modified electrode and then OTA was incubated on it. The process of different stages of the aptasensor construction has been confirmed by two methods of electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) and using [Fe(CN)6]3-/4- as a redox probe. The EIS method has also been used for the OTA quantitative determination. The difference in charge transfer resistance (Rct) before and after the interaction of OTA with the immobilized aptamer was considered as the analytical response of the aptasensor. Using the developed aptasensor, it is possible to measure OTA in the concentration range of 1.0 × 10-3 to 200.0 ng mL-1 with a detection limit of 2.2 × 10-4 ng mL-1. Finally, the ability of the aptasensor to measure OTA in red and black pepper was investigated and completely satisfactory results were obtained.

MXene quantum dot functionalized bimetallic MOFs as a label-free aptasensor for highly sensitive detection of exosomes

A schematic illustration of the construction of a CuNi-MOFs@MQDs-based aptasensor for exosome sensing.

A label-free ratiometric fluorescent aptasensor based on a peroxidase-mimetic multifunctional ZrFe-MOF for the determination of tetrodotoxin.

A Fe/Zr bimetal-organic framework (ZrFe-MOF) is utilized to establish a ratiometric fluorescent aptasensor for the determination of tetrodotoxin (TTX). The multifunctional ZrFe-MOF possesses inherent fluorescence at 445nm wavelength, peroxidase-mimetic activity, and specific recognition and adsorption capabilities for aptamers, owing to its organic ligand, and Fe and Zr nodes. The peroxidation of o-phenylenediamine (OPD) substrate generates fluorescent 2,3-diaminophenazine (OPDox) at 555nm wavelength, thus quenching the inherent fluorescence of ZrFe-MOF because of the fluorescence resonance energy transfer (FRET) effect. TTX aptamers, which are absorbed on the material surface without immobilization or fluorescent labeling, inhibit the peroxidase-mimetic activity of ZrFe-MOF. It causes the decreased OPDox fluorescence at 555nm wavelength and the inverse restoration of ZrFe-MOF fluorescence at 445nm wavelength. With TTX, the aptamers specifically bind to TTX, triggering rigid complex release from ZrFe-MOF surface and reactivating its peroxidase-mimetic activity. Consequently, the two fluorescence signals exhibit opposite changes. Employing this ratiometric strategy, the determination of TTX is achieved with a detection limit of 0.027ng/mL and a linear range of 0.05-500ng/mL. This aptasensor also successfully determines TTX concentrations in puffer fish and clam samples, demonstrating its promising application for monitoring trace TTX in food safety.

A Label-Free Aptasensor for Turn-On Fluorescent Detection of Aflatoxin B1 Based on an Aggregation-Induced-Emission-Active Probe and Single-Walled Carbon Nanohorns.

The determination of the aflatoxin B1 (AFB1) content has received widespread attention in the context of food safety, which is a global public health issue. Accordingly, a label-free and turn-on fluorescent AFB1 determination method is developed herein with an ss-DNA aptamer as the recognition element, 4, 4-(1E,1E)-2, 2-(anthracene-9, 10-diyl) bis(ethene-2, 1-diyl) bis(N, N, N-trimethylbenzenaminium iodide) (DSAI) as the aggregation-induced emission (AIE) fluorescent probe, and single-walled carbon nanohorns (SWCNHs) as the selective part with a fluorescence quenching effect. In the presence of AFB1, the AFB1-specific aptamer undergoes a structural transformation and switches from being a random helix to a folded structure. DSAI's fluorescence is protected as a result of the resistance of the transformed aptamer adsorbed on the SWCNHs' surface. Because DSAI's fluorescence is not quenchable, the fluorescence intensity is calculated as a function of the AFB1 concentration. By simply mixing DSAI, aptamer, AFB1 samples, and SWCNHs, the method can be carried out in 2 h, with a limit of detection (LOD) of 1.83 ng/mL. It has a high selectivity in the presence of other mycotoxins, and its performance is confirmed in soybean sauce with a known concentration of AFB1. The LOD was 1.92 ng/mL in the soy sauce samples and the recovery ranged from 95 to 106%, implying that the presented aptasensor has great potential for food analysis.

A label-free 5CB-based aptasensor for highly selective optical detection of amoxicillin residue in freshwater samples

ABSTRACT A novel application of thermotropic liquid crystal (LC) 5CB in an optical aptasensor for the sensitive detection of amoxycillin (AMOX) antibiotic has been reported. In the studied system, the glass substrates were treated with dimethyloctadecyl [3–(trimethoxysilyl) propyl] ammonium chloride (DMOAP) and an aqueous solution of cationic surfactant cetyltrimethylammonium bromide (CTAB) was used to obtain a homeotropically aligned 5CB monolayer at the LC-aqueous interface resulting in a uniformly dark appearance. A highly specific amoxycillin aptamer (AMXA) was immobilised on the glass substrate as a target recognition probe for AMOX molecules. The specific binding of AMXA and AMOX molecules induces orientational changes in LC ordering resulting in a change in LC optical textures observed under a Polarized Optical Microscope (POM). A low limit of detection (LOD) of 10 nM for AMOX was obtained using this sensing system. Additionally, a high specificity for AMOX was also observed during comparative analysis. Finally, the developed biosensor was used to successfully detect AMOX in environments of potential application like tap water and lake water samples. Thus, the developed LC-based aptasensor can be a simple, sensitive and label‐free platform for monitoring real‐time levels of AMOX in freshwater environments.

Evolution of high-affinity aptamer probe for label-free and probe conformation switch-based fluorescent biosensing of β-carotene in seaweeds

β-Carotene is an important bioactive component showing great activities and receiving great attention in recent years. The traditional methods for the accurate detection of β-carotene in food are time-consuming. There is an urgent need to establish rapid quantification methods for β-Carotene. Herein, a label-free fluorescent aptasensor which was based on the conformation-switch of the aptamer probe was developed to detect β-carotene in seaweeds. Firstly, a post-SELEX strategy based on extraction and terminal-fixation was established to evolve the anti-β-carotene aptamer to achieve high affinity. An aptamer probe (β-2-TF) with a 177.5-fold affinity increment was obtained and then applied to develop a probe conformation-switch-based fluorescent aptasensor with the assistance of SYBR Green I (SGI). Under the optimal conditions, the aptasensor exhibited good accuracy and selectivity, with a linear detection range of 62.5–4000 nM and a limit of detection (LOD) as low as 39.64 nM. The developed strategies can act as robust references for the analytical method development in the food field.

Femtomolar detection of the heart failure biomarker NT-proBNP in artificial saliva using an immersible liquid-gated aptasensor with reduced graphene oxide

Measuring NT-proBNP biomarker is recommended for preliminary diagnostics of the heart failure. Recent studies suggest a possibility of early screening of biomarkers in saliva for non-invasive identification of cardiac diseases at the point-of-care. However, NT-proBNP concentrations in saliva can be thousand time lower than in blood plasma, going down to pg/mL level. To reach this level, we developed a label-free aptasensor based on a liquid-gated field effect transistor using a film of reduced graphene oxide monolayer (rGO-FET) with immobilized NT-proBNP specific aptamer. We found that, depending on ionic strength of tested solutions, there were different levels of correlation in responses of electrical parameters of the rGO-FET aptasensor, namely, the Dirac point shift and transconductance change. The correlation in response to NT-proBNP was high for 1.6 mM phosphate-buffered saline (PBS) and zero for 16 mM PBS in a wide range of analyte concentrations, varied from 1 fg/mL to 10 ng/mL. The effects of transconductance and Dirac point shift in PBS solutions of different concentrations are discussed. The biosensor exhibited a high sensitivity for both transconductance (2 µS/decade) and Dirac point shift (2.3 mV/decade) in diluted PBS with the linear range from 10 fg/mL to 1 pg/mL. The aptasensor performance has been also demonstrated in undiluted artificial saliva with the achieved limit of detection down to 41 fg/mL (∼4.6 fM).

Label-free and sensitive determination of toxic Cd(II) in environmental waters using a Fe3O4-PEI-Au based electrochemical aptasensor

A novel electrochemical strategy was presented using gold nanoparticles decorated on polyethyleneimine-modified iron oxide nanoparticles (Fe3O4-PEI-Au) as a nanocomposite modifier for sensitive and selective detection of Cd(II). The aptasensor was fabricated by immobilizing a thiol-terminated Cd(II) aptamer onto a screen-printed carbon electrode modified with the Fe3O4-PEI-Au nanocomposite. This nanocomposite provided high surface area for enhanced aptamer loading, excellent biocompatibility, and good conductivity to promote the signal amplification at the sensing interface. Differential pulse voltammetry was used for quantitative measurement of Cd(II), demonstrating a dynamic linear detection range from 0.04 nM to 25 nM. The limit of detection was found to be 0.01 nM, which is lower than the safe limit in drinking water defined by the U.S. Environmental Protection Agency. The label-free aptasensor showed high selectivity, reproducibility, stability and reliability for real-time Cd(II) monitoring in environmental water samples. This work successfully demonstrates the development of a novel electrochemical aptasensor utilizing a Fe3O4-PEI-Au nanocomposite modifier for sensitive and selective detection of trace levels of Cd(II) in water resources.

Development of a label-free impedimetric aptasensor based on Zr-MOF and titaniom carbide nanosheets for detection of L-tryptophan

This study primarily focuses on the L-Tryptophan (Trp) biomarker assay, with particular attention given to its objectives. The investigation centers on the potential implications of imbalanced Trp levels and its associated metabolites, which have been attributed to the spectrum of both psychological and physiological disorders, encompassing conditions such as cancer. Therefore, the swift and accurate detection of this amino acid is of paramount importance in cancer monitoring, as it plays a crucial role in preventing the metastasis and spread of cancer cells.Thus, an electrochemical aptasensor was designed based on nanocomposite of AgNPs@UiO-66/Ti3C2 (MXene) as immobilization strategy for the ultrasensitive detection of Trp. Zirconium metal–organic frameworks (Zr-MOFs) were employed as carriers for silver nanoparticles to facilitate subsequent binding with aptamers. Markedly, the obtained results show that the constructed aptasensor can specifically detect Trp in the two concentration range from 1fM to 1 nM and 1 nM to 200 nM and with a low detection limit of 0.35 fM. When applied to real samples, the experimental results demonstrated positive recovery rates. Consequently, a discerning and sensitive electrochemical aptasensor devoid of labeling agents was successfully fabricated for the explicit purpose of Trp detection.

A label-free electrochemical aptasensor based on Ti3C2Tx-Ag/Au nanoparticles as a signal amplification strategy for CRP detection

C-reactive protein (CRP) is an essential biomarker that is associated to acute inflammation and infectious illnesses. This study developed a label-free electrochemical aptasensor utilizing Ti3C2Tx-silver/gold nanoparticles (Ti3C2Tx-Ag/Au NPs) for the first time for CRP detection. Based on the unique capacity of Ti3C2Tx of self-adsorbing and reducing silver ions (Ag+), Ag signal probes with strong redox activity and good electrical conductivity were synthesized. Electrodeposition of Au NPs on the electrode surface modified with Ti3C2Tx-Ag, not merely improved the adsorption capacity of the immobilized aptamer and prevented Ag loss; it also collaborated with Ti3C2Tx-Ag to create a dual-amplified signal strategy. Aptamer molecules bind to Au NPs through the Au-S bond for precise capture of CRP. The detection was performed by differential pulse voltammetry (DPV), the current was proportional to the logarithm of CRP antigen, and the linear range of CRP detection was 0.1–200 ng mL−1 with a limit of detection (LOD) 41 pg mL−1. Simultaneously, the label-free aptasensor presented superior detection selectivity, reproducibility, and stability. This study provided an ideal electrochemical biosensor for determining CRP in serum, with an excellent prospect for clinical diagnostic applications at the point of care testing (POCT).

Label-free competitive time-resolved fluorescent aptasensor for the detection of Pb2+ using ssDNA-sensitized fluorescence of Tb3+ ions

Lead (Pb2+) is one of the most toxic heavy metals, and its environmental pollution and serious damages is a global concern. Therefore, it is necessary to develop effective sensing methods. This study describes a new strategy for the design of label-free competitive time-resolved fluorescent (TRF) aptasensor for detecting Pb2+ ions. The sensing principle of this aptasensor is the competition between Pb2+ and Tb3+ ions to bind to the guanine/thymine-rich sequence (lead aptamer) and the sensitized luminescence of Tb3+ by this sequence. The developed TRF aptasensor demonstrated a good linear detection range from 2.5 nM to 150 nM and a limit of detection (LOD) of 645 pM. In addition, the proposed TRF aptasensor has a high selectivity towards Pb2+, and it has also been successfully utilized to detect this ion in milk and human serum samples. This TRF aptasensor offers advantages such as short analysis time, simple operation, low cost, being label-free, and surpassing the interference of background fluorescence of biological samples due to its TRF characteristic. In this paper, we propose a Tb3+/guanine/thymine-rich sequence system for designing different aptasensors against diverse targets by applying a proper guanine-rich sequence.

A label-free aptasensor for colorimetric detection of food toxin: Mediation of catalytically active gold nanozymes and smartphone imaging strategy

A portable colorimetric aptasensor was developed on the surface of a plastic sheet for on-site detection of acrylamide. The mechanism of aptasensor is based on the disruption of the aptamer complex with its complementary strand on the sensing zone, and subsequently, the catalytic activity of gold nanoparticles (AuNPs) for the reduction process of 4-nitrophenol (4-NP) in the presence of sodium borohydride (NaBH4). A yellow-to-colorless change of the sample solution revealed the target presence, easily discernible by the naked eye. The acrylamide quantification was accomplished using the smartphone imaging readout technique. The aptasensor detected the acrylamide concentration in the range of 0.01–500 nmol L−1 with a detection limit of 0.0024 nmol L−1. Coffee, potato chips, bread, and lake water samples were successfully analyzed by the aptasensor for their acrylamide content. The introduced aptasensor can pave a facile, cost-effective, portable, and user-friendly sensing tool for food safety control and environmental monitoring.

AuPt Nanodonuts as a Novel Coreaction Accelerator and Luminophore for a Label-free Electrochemiluminescence Aptasensor.

Novel and effective coreaction accelerators are of great importance in electrochemiluminescence (ECL) systems. In this work, novel AuPt nanodonuts, i.e., SnS2 quantum dots (QDs)/Cys-AuPt heterogeneous nanorings (NRs), serve as both a highly effective coreaction accelerator and the luminophore in a label-free ECL aptasensor. The novel AuPt nanodonuts were formed by decorating SnS2 QDs onto AuPt NR surfaces, which would promote the production of more coreactant intermediate in the SnS2 QDs/K2S2O8 system. As a result, the ECL performance was greatly improved. Meanwhile, l-cysteine (l-Cys) played an important role in the combination between AuPt NRs and SnS2 QDs, and the nanodonuts served as the matrix to load numerous lincomycin (Lin) aptamers. Under optimal conditions, the ECL aptasensor exhibited ultrasensitive detection of Lin from 1 to 0.1 pg/mL with a limit of detection (LOD) of 0.7 fg/mL (1.72 fM)..

Fluorescent polythymidine-templated copper nanoclusters aptasensor for sensitive detection of tropomyosin in processed shrimp products

Tropomyosin (TM) is the main allergen in shellfish. Developing a novel, simple and accurate method to track and detect TM in food products is necessary. In this work, a label-free fluorescent aptasensor based on polythymidine (poly(T))-templated copper nanoclusters (CuNCs) was designed for sensitive detection of TM in processed shrimp products. Magnetic beads (MBs), aptamer and cDNA were used to construct an MBs-aptamer@cDNA complex as a detection probe, and with the presence of TM, the poly(T)-templated CuNCs attached at the end of the cDNA as the fluorescent signal was released from the complex to turn on the fluorescence. Under optimal conditions, the poly(T)-templated CuNCs aptasensor achieved a linear range from 0.1 to 50 μg/mL (R2 = 0.9980), a low limit of detection of 0.0489 μg/mL and an excellent recovery percentage of 105.29%-108.91% in the complex food matrix, providing a new approach for food safety assurance.

Label-free colorimetric sensor for Pb2+ determination using catalytic activity of MnO2 nanoflowers and elongated aptamer

In this study, a label-free aptasensor utilizing colorimetric properties was developed to detect Pb2+ with high sensitivity. The approach involved applying modified aptamer which enhanced the oxidase-mimicking activity of MnO2 nanoflowers. This innovative method provides an efficient means for monitoring Pb2+ ions without requiring any labeling techniques. The fundamental principle of this aptasensor is based on the adsorption of a modified aptamer onto MnO2 nanoflowers' surface, which in turn increases their affinity for chromogenic substrates and enhances their catalytic activity. The proposed aptasensor exploits the high sensitivity due to the extension of the aptamer sequence length by terminal deoxynucleotidyl transferase (TdT). Under optimum experimental conditions, the developed colorimetric aptasensor indicated a linear detection range from 4 to 80 nM with a limit of detection (LOD) of 1.4 nM. Moreover, the aptasensor successfully monitored Pb2+ in the drinking water, milk and human serum samples. Henceforth, the colorimetric aptasensor exhibited in this study possesses several benefits such as uncomplicated operation, cost-effectiveness, label-free detection and remarkable sensitivity. Thus rendering it a suitable option for analyzing intricate samples.

A label-free aptasensor for fluorescent detection of carbendazim based on a G-quadruplex-specific probe

A label-free aptasensor for fluorescent detection of carbendazim based on a G-quadruplex-specific probe

A colorimetric aptasensor for detecting ochratoxin A based on label-free aptamer and gold nanozyme.

In this study, the aptamer of ochratoxin A (OTA) increased the negative charge density on the surface of gold nanoparticles (AuNPs) and promoted the release of hydroxyl radicals and Au3+ to enhance the peroxidase-like activity of the AuNPs. The OTA bound only to the aptamer and did not adsorb non-specifically to the AuNPs. Based on these two conclusions, a label-free colorimetric aptasensor was successfully developed, enabling the precise detection of OTA within the concentration range of 10-600nM, with a remarkably low detection limit of 6.20nM. The colorimetric aptasensor was applied to detect OTA in oats, corn, soybeans, rice, and glutinous rice.

Fluorometric detection of alpha-fetoprotein based on the use of a novel organic compound with AIE activity and aptamer-modified magnetic microparticles

BackgroundLiver cancer is one of the most common cancers in the world, and it seriously threatens human life and health. Alpha-fetoprotein (AFP), as a carcinogenic glycoprotein, is an important serum marker for detecting liver cancer. Therefore, the accurate and sensitive determination of AFP is crucial for the early diagnosis and treatment of liver cancer. To this end, a label-free fluorescence aptasensor for detecting AFP based on the use of a novel organic Compound D with an aggregation-induced emission activity and aptamer-modified magnetic microparticles was constructed. ResultsCompound D could combine with the complementary short chain of the aptamer (CSC-Apt) of AFP to form the D/CSC-Apt complex and realize the fluorescence enhancement of Compound D. Then, magnetic particles modified by the Apt of AFP (Apt-Fe3O4) were prepared. When AFP (or nontarget substance) and D/CSC-Apt were successively added to the Apt-Fe3O4 solution, Apt-Fe3O4 selectively bound to AFP or the D/CSC-Apt complex. Magnetic separation technology showed the changes in the fluorescence intensity of the supernatant. The research results revealed a good linear relationship between the changes in the fluorescence intensity of the supernatant and concentration of AFP within the concentration range of 10–10000 pg mL−1. The proposed aptasensor could achieve high-sensitivity and high-specificity detection of AFP, and its limit of detection was 3 pg mL−1 (S/N = 3). Significance and noveltyThe sensor combines the advantages of high selectivity of Apt, high sensitivity of fluorescence analysis, AIE effect and good water solubility of Compound D, and rapid separation using magnetic separation technology. And it can be directly used for the detection of AFP in actual serum samples with high accuracy, whereas most of the methods reported in the literature can only detect AFP in spiked serum samples.