CRISPR-Cas12a-based colorimetric aptasensor for aflatoxin M1 detection based on oxidase-mimicking activity of flower-like MnO2 nanozymes.

Given the highly mutagenic and carcinogenic nature of Aflatoxin M1 (AFM1), the quantity assessment of AFM1 residues in milk and dairy products is necessary to maintain consumer health and food safety. Herein, CRISPR-Cas12a-based colorimetric aptasensor was developed using the catalytic activity of flower-like nanozymes of MnO2 and trans-cleavage property of CRISPR-Cas12a system to quantitatively detect AFM1. The basis of the developed colorimetric aptasensor relies on whether or not the CRISPR-Cas12a system is activated, as well as the contrast in oxidase-mimicking capability exhibited by flower-like MnO2 nanozymes when AFM1 is absent or present. When AFM1 is not present in the sample, single-stranded DNA (ssDNA) is degraded by the activated CRISPR-Cas12a, and the solution turns into yellow due to the catalytic activity of the nanozymes. While, in the attendance of AFM1, ssDNA degradation does not occur due to the inactivation of the CRISPR-Cas12a. Therefore, with the adsorption of the ssDNA on the MnO2 nanozymes, their catalytic activity decreases, and the solution color becomes pale yellow due to less oxidation of the chromogenic substrate. In this aptasensor, the relative absorbance changes increased linearly from 6 to 160 ng L-1, and the detection limit was 2.1 ng L-1. The developed aptasensor displays a selective detection performance and a practical application for quantitative analysis of AFM1 in milk samples. The results of the introduced aptasensor open up the way to design other selective and sensitive aptasensors for the detection of other mycotoxins by substitution of the used sequences.

Signal-off nanozyme-based colorimetric aptasensor for sensitive detection of ampicillin using MnO2 nanoflowers and gold nanoparticles.

The combination of nanomaterials possessing distinct characteristics and the precision of aptamers facilitates the creation of biosensors that exhibit exceptional selectivity and sensitivity. In this manuscript, we present a highly sensitive aptasensor that utilizes the distinctive characteristics of MnO2 nanoflowers and gold nanoparticles to selectively detect ampicillin (AMP). In this aptasensor, the mechanism of signal change is attributed to the difference in the oxidase-mimicking activity of MnO2 nanoflowers in the presence of a free sequence. The inclusion of AMP hindered the creation of a double-stranded DNA configuration through its binding to the aptamer, resulting in an observable alteration in absorbance. The relative absorbance varied linearly with the concentration of AMP in the range of 70 pM to 10 nM with a detection limit of 21.7 pM. In general, the colorimetric aptasensor that has been developed exhibits exceptional selectivity and remarkable stability. It also demonstrates favorable performance in human serum, making it a highly reliable diagnostic tool. Additionally, its versatility is noteworthy as it holds great potential for detecting various antibiotics present in complex samples by merely replacing the utilized sequences with new ones.

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.

Time-resolved Fluorescence DNA-based Sensors for Reducing Background Fluorescence of Environment.

The fluorescence assay is one of the popular methods that is applied for detection of different targets. However, this method may show low sensitivity and high background in biological samples due to the natural fluorescence of different compounds in complicated samples. In addition, it inevitably affects the detection results accuracy. A fundamental solution to this problem is the use of the time-resolved fluorescence technique (TRF). The main component of this technique is the use of long fluorescence lifetime reagents. In this review, various time-resolved fluorescent reagents such as complexes of lanthanide ions, lanthanide-doped inorganic nanoparticles; Mn-doped ZnS quantum dots (QDs) and pyrene excimer are introduced. Moreover, TRF sensors, especially TRF aptasensors (DNA-based sensors) are discussed. This review will give new ideas for researchers to develop novel high-sensitive TRF sensors that can remove or decrease background fluorescence and use them for the detection of various targets in complicated samples without treatment.

A novel turn-off fluorescent aptasensor for ampicillin detection based on perylenetetracarboxylic acid diimide and gold nanoparticles.

Herein, a novel turn-off fluorescent aptasensor was developed for selective detection of ampicillin (AMP) at picomolar level based on 3,4,9,10-perylenetetracarboxylic acid diimide (PTCDI) as an affordable and low-cost fluorophore. This aptasensor was designed using aptamer, its complementary strand (CS) and gold nanoparticles (AuNPs). The principle of the sensing method is a decrease in the fluorescence intensity of PTCDI in the presence of free CS. Following the addition of AMP, Aptamer/CS-modified AuNPs releases CS and so, the fluorescence intensity of PTCDI is reduced. The designed analytical method indicated a good linear range from 100 pM to 1000 pM and a limit of detection (LOD) of 29.2 pM was obtained. Furthermore, the sensing strategy indicated satisfactory results for the detection of AMP in the spiked human serum samples. By changing the sequences of aptamer and its CS, the presented analytical approach can be easily applied for detection of other antibiotics.

A fluorometric assay for oxytetracycline based on the use of its europium(III) complex and aptamer-modified silver nanoparticles.

An ultrasensitive assay is described for the determination of oxytetracycline (OTC) at nanomolar levels. The method is using silver nanoparticles (AgNPs) that were first modified with OTC-binding aptamer and then exposed to the OTC-Eu(III) complex. The pink fluorescence of the OTC-Eu(III) complex on the AgNPs is almost completely quenched. On addition of OTC, it will compete with the OTC-Eu(III) complex for binding to the aptamer on the AgNPs. The OTC-Eu(III) complex is released and becomes strongly fluorescent, with excitation/emission peaks at 385/620 nm. The resulting assay was validated in terms of linearity and linear range, sensitivity, selectivity, detection limit and accuracy. Under optimum conditions, response is linear in the 10 to 500 nM OTC concentration range, and the limit of detection is 1.9 nM. The method was applied to the determination of OTC in spiked milk and tablets samples, and it gave satisfactory results. Graphical abstract Schematic presentation of the assay. In the presence of oxytetracycline (OTC), it will compete with the OTC-europium(III) complex for binding to the silver nanoparticles (AgNPs)-aptamer conjugate. The OTC-Eu(III) complex is released and strong pink fluorescence is observed.