Aptamer-Based Label-free Fluorescent Assay

Aptamers are selected in vitro by systematic evolution of ligands by exponential enrichment (SELEX), and are single-stranded DNA or RNA molecules that can bind various target ligands with high affinity and specificity. A variety of analytical techniques based on aptamers have been developed, including colorimetric assay, fluorescence assay, electrochemical aptasensor and so on. Among these detection methods, the analysis based upon fluorescence has the advantages of simplicity, rapidity, less expensive and more suitable for automation; however, these fluorescence assays require fluorophore-labeled aptamers. Such step would not only make experiments relatively more expensive and complex, but may also affect the binding affinity between the targets and aptamers and influence the sensitivity for detection. Therefore, rapid, simple and sensitive label-free fluorescent detection methods were developed herein based on aptamers and fluorescent dyes. The method can be used for the detection of toxins and has great significance for food safety. The main contents are summarized as follows:(1) A simple and universal aptamer-based label-free approach for highly sensitive and selective fluorescence detection of a broad range of targets including inorganic ions, proteins and small molecules was developed by using PicoGreen to transduce the fluorescent signal of the double strand DNA duplex formed between free aptamer and its complementary strand. Before each target analyte assay, the concentration of PicoGreen used, the intercalation time between Picogreen and the respective dsDNA duplexes and also each aptamer and complementary strand hybridization time were optimized. For potassium ions (K+) detection, the limit of detection (LOD) is 1μmol/L and the linear range is 1 μmol/L-1 mol/L.K+ sensitivity test experiment was repeated except adding 10 mmol/L,100 mmol/L and 200 mmol/L Na+ into each assay, respectively, and it is still capable of monitoring K+ variations from 1 μmol/L to 1 mol/L. The concentrations of K+in real urine samples measured by our method and Atomic Absorption Spectrometryhave high similarities. For thrombin detection, the LOD is 10 U and the linear range is 10-1000 U. In the presence of 1 mg/mL bovine serum albumin in thrombin sensitivity test experiment, the linearity of the fluorescence response to concentration of thrombin is from 10 U to 1000 U. For adenosine detection, the LOD is 0.5 μmol/L and the linear range is 0.5 μmol/L-5 mmol/L. Adenosine in 20-fold diluted fetal bovine serum was successfully detected with a wide linear range from 0.5 μmol/L to 5 mmol/L and a detection limit of 0.5 μmol/L.(2) The simple and rapid aptamer-based label-free approach by using ultra-sensitive double-strand DNA specific dyes PicoGreen was used for the detection of ochratoxin A (OTA). The results showed that as low as 1 ng/mL of OTA could be detected with a dynamic range of more than 5 orders of magnitude which from 1 ng/mL to 100 μg/mL. With the specificity of aptamer, the assay exhibited high selectivity for OTA against two non-specific targets (N-acetyl-l-phenylalanine and zearalenone). More control experiments using chloramphenicol’s aptamer and its complementary strand were performed under the condition similar to OTA’s aptamer. These results clearly indicated that the aptamer-based fluorescence aptasensor is highly specific for OTA determination. In 1% beer, concentration of OTA down to 1 ng/mL is detected and the linear range is wide from 1 ng/mL to 100 μg/mL.(3) A simple and sensitive method for the label-free fluorescent detection of thrombin with high sensitivity and specificity by taking advantage of the fluorescent probe perylene tetracarboxylic acid diimide (PTCDI) derivatives was developed. Before target analyte assay, the concentration of PTCDI (5 nmol/L,50 nmol/L,500 nmol/L,1μmol/L,2μmol/L, 5μmol/L, 10μmol/L, and the volume of aptamer (5 μL,10μL,15μL,20 μL) used were optimized. The concentration of PTCDI is 500 nmol/L, the volume of aptamer (5 μmol/L) is 20 μL. This detection method exhibits high sensitivity with a detection limit of 40 pmol/L and the linear range is 40-1600 pmol/L. To determine the specificity of this method, the presence of other proteins (Bovine Serum Albumin, Lysozyme, Mouse IgG)) with the same concentration exhibited negligible effect on fluorescence intensity. The results clearly indicated that the aptamer-based fluorescence aptasensor is highly specific for thrombin determination.