A FRET based ultrasensitive fluorescent aptasensor for 6'-sialyllactose detection.

As a kind of human milk oligosaccharide, 6'-sialyllactose (6'-SL) plays an important role in promoting infant brain development and improving infant immunity. The content of 6'-SL in infant formula milk powder is thus one of the important nutritional indexes. Since the lacking of efficient and rapid detection methods for 6'-SL, it is of great significance to develop specific recognition elements and establish fast and sensitive detection methods for 6'-SL. Herein, using 6'-SL specific aptamer as the recognition element, catalytic hairpin assembly as the signal amplification technology and quantum dots as the signal label, a fluorescence biosensor based on fluorescence resonance energy transfer (FRET) was constructed for ultra-sensitive detection of 6'-SL. The detection limit of this FRET-based fluorescent biosensor is 0.3 nM, and it has some outstanding characteristics such as high signal-to-noise ratio, low time-consuming, simplicity and high efficiency in the actual sample detection. Therefore, it has broad application prospect in 6'-SL detection.

Screening of a Sialyllactose-Specific Aptamer and Engineering a Pair of Recognition Elements with Unique Fluorescent Characteristics for Sensitive Detection of Sialyllactose.

A single-stranded DNA (ssDNA) aptamer specific for 6'-sialyllactose (6'-SL) was screened through magnetic separation-based SELEX and post-SELEX truncation and used to construct unique aptamer bio-dots for sensitive detection of 6'-SL. Eighteen rounds of screening were conducted during the SELEX process. The ssDNA aptamer Apt9 (Kd = 152.3 nM) with a length of 79 nucleotides (nt) was demonstrated as the optimal aptamer candidate after affinity and specificity evaluation. Then, Apt9 was truncated and optimized according to secondary structure and molecular docking. A 35 nt truncated aptamer Apt9-1 (Kd = 91.75 nM) with higher affinity than Apt9 was finally obtained. Furthermore, Apt9-1 was used to synthesize bio-dots as a new recognition element of 6'-SL, and the aminobenzene boric acid functionalized carbon dots were employed as the other recognition element. With the respective fluorescent characteristics, the two quantum dots (QDs) were made a pair to construct a 6'-SL fluorescent biosensor. The linear detection range of the biosensor is 10 µM to 5 mM, and the detection limit is 0.9 µM. With the advantages of time-saving, high efficiency, and simplicity in the actual sample detection, the screened aptamer and dual-QD-based biosensor have broad application prospects in 6'-SL detection.

Fluorescent biosensor based on FRET and catalytic hairpin assembly for sensitive detection of polysialic acid by using a new screened DNA aptamer.

A fast and efficient dsDNA library-immobilized magnetic bead-based SELEX technique was employed for selection of the aptamers against polysialic acid (PSA). Overall twelve rounds of screening, the pooled library was subjected to high-throughput sequencing. Five aptamer candidates with low Gibbs binding free energy and high abundance were selected for affinity evaluation. Apt3 was demonstrated to be the optimal aptamer for PSA with Kd of 114.0 nM. Furthermore, an ultrasensitive fluorescence resonance energy transfer (FRET)-based biosensor for PSA was constructed by employing the newly selected aptamer and catalytic hairpin assembly (CHA) amplification strategy. The linear detection range for PSA is from 10 pM to 1 µM and the limit of detection is 0.63 pM. The fluorescent biosensor is able to detect the target in the complex biological samples, which indicates that Apt3 has good application prospect for the biological detection and clinical diagnostics.

A FRET-based detection of N-acetylneuraminic acid using CdSe/ZnS quantum dot and exonuclease III-assisted recycling amplification strategy.

N-acetylneuraminic acid (Neu5Ac) is widely spread in many biologically significant glycans of mammals, commonly as a terminal α-glycoside. It is of great significance to develop analytical techniques for detection of Neu5Ac. Herein, a high-sensitive fluorescent biosensor for Neu5Ac has been developed based on FRET between CdSe/ZnS quantum dots (QDs) and BHQ2, as well as exonuclease III (Exo III)-assisted recycling amplification strategy. Employing the specially designed three-level FRET systems and fluorescent signal recovery mechanism, together with five-step recycling signal amplification chain reactions, an ultralow detection limit of 24 fM was achieved. Meanwhile, good linear response ranges within 0.2-12.5 pM and 12.5-1000 pM were founded. The assay has excellent performance in real sample detection, and thus offers great potential for detection of sialic acids modified glycans/lipids in the fields of medical diagnosis and food testing.