Noncompetitive Determination of Small Analytes by Sandwich-Type Lateral Flow Assay Based on an Aptamer Kissing Complex.

Here, we present the proof-of-concept of a lateral flow assay (LFA) that is capable of detecting small-molecule targets in a noncompetitive manner by deploying a sandwich-type format based on the aptamer kissing complex (AKC) strategy. A fluorescently labeled hairpin aptamer served as the signaling agent, while a specific RNA hairpin grafted onto the strip served as the capture element. The hairpin aptamer switched from an unfolded to a folded form in the presence of the target, resulting in kissing interactions between the loops of the reporter and the capture agents. This design triggered a target-dependent fluorescent signal at the test line. The AKC-based LFA was developed for the detection of adenosine, achieving a detection limit in the micromolar range. The assay revealed the presence of the same analyte in urine. The method also proved effective with another small molecule (theophylline). We believe that the AKC-based LFA approach could overcome many of the shortcomings associated with conventional signal-off methods and competitive processes.

Aptamer-based colorimetric and lateral flow assay approaches for the detection of toxic metal ions, thallium(i) and lead(ii).

Thallium(i) and lead(ii) ions are heavy metals and extremely toxic. These metals are environmental pollutants, posing a severe risk to the environment and human health. In this study, two approaches were examined using aptamer and nanomaterial-based conjugates for thallium and lead detection. The first approach utilized an in-solution adsorption-desorption approach to develop colorimetric aptasensors for the detection of thallium(i) and lead(ii) using gold or silver nanoparticles. The second approach was the development of lateral flow assays, and their performance was tested with thallium (limit of detection is 7.4 µM) and lead ion (limit of detection is 6.6 nM) spiked into real samples. The approaches assessed are rapid, inexpensive, and time efficient with the potential to become the basis for future biosensor devices.

Adsorption-desorption nano-aptasensors: fluorescent screening assays for ochratoxin A.

In this study, a FRET-based fluorescent aptasensor for the detection of ochratoxin A (OTA) was optimized based on the quenching efficiency of single-walled carbon nanotubes (SWCNTs) and the binding affinity of aptamers. OTA aptamers were conjugated with quantum dots and adsorbed to the surface of both acid-modified and unmodified SWCNTs. The maximum fluorescence quenching efficiency of the SWCNTs were compared. Acid-modified SWCNTs (amSWCNTs) have moderate quenching efficiency, providing an optimal sensitivity for qualitative fluorescence-enhancement biosensor assays. The binding parameters of the QD-modified OTA aptamers (1.12.2 and A08min) on the surface of amSWCNTs were compared. Based on our results, the A08min aptamer is a better candidate for OTA detection. Using the A08min aptamer, the SWCNT method had a limit of detection (LOD) of 40 nM. The amSWCNT method had a significantly lower LOD of 14 nM. Turn-on fluorescent nano-aptasensors are emerging as an effective diagnostic tool for simple detection of mycotoxins. Nanocomplexes designed for the detection of mycotoxins in solution and paper-based tests have proven to be useful.

An aptamer-based colorimetric lateral flow assay for the detection of human epidermal growth factor receptor 2 (HER2).

An aptamer-based colorimetric lateral flow assay was developed for the detection of human epidermal growth factor receptor 2 (HER2). In this study, two approaches were examined using HER2 binding aptamers and gold nanoparticles. The first method used was a solution-based adsorption-desorption colorimetric approach wherein aptamers were adsorbed onto the gold nanoparticle surface. Upon the addition of HER2, HER2 binds specifically with its aptamer, releasing the gold nanoparticles. Addition of NaCl then induces the formation of gold nanoparticle aggregates. This leads to a color change from red to blue and a detection limit of 10 nM was achieved. The second method used an adsorption-desorption colorimetric lateral flow assay approach wherein biotin-modified aptamers were adsorbed onto the gold nanoparticle surface in the absence of HER2. In the presence of HER2, HER2 specifically binds with its aptamer leading to release of the gold nanoparticles. These solutions were applied to the lateral flow assay format and a detection limit of 20 nM was achieved. Both colorimetric and lateral flow assays are inexpensive, simple, rapid to perform and produce results visible to the naked-eye.

Correction to: Comparison of turn-on and ratiometric fluorescent G-quadruplex aptasensor approaches for the detection of ATP.

Regrettably, before online publication the figure of Scheme 2 has been pasted twice as Scheme 1.

Comparison of turn-on and ratiometric fluorescent G-quadruplex aptasensor approaches for the detection of ATP.

Two fluorescent aptasensor methods were developed for the detection of ATP in biochemical systems. The first method consisted of a label-free fluorescent "turn-on" approach using a guanine-rich ATP aptamer sequence and the DNA-binding agent berberine complex. In the presence of ATP, the ATP preferentially binds with its aptamer and conformationally changes into a G-quadruplex structure. The association of berberine with the G-quadruplex results in the enhancement of the fluorescence signal of the former. The detection limit of ATP was found to be 3.5 µM. Fluorescence, circular dichroism and melting temperature (Tm) experiments were carried out to confirm the binding specificity and structural changes. The second method employs the ratiometric fluorescent approach based on the Forster resonance energy transfer (FRET) for the detection of ATP using berberine along with a quencher (AuNRs, AgNPs) and a fluorophore (red quantum dots (RQDs), carbon dots (CDs)) labeled at 5' and 3' termini of the ATP-binding aptamer sequence. Upon addition of ATP and berberine, ATP specifically binds with its aptamer leading to the formation of G-quadruplex, and similarly, berberine also binds to the G-quadruplex. This leads to an enhancement of fluorescence of berberine while that of RQD and CDs were significantly quenched via FRET. The respective detection limits calculated were 3.6 µM and 3.8 µM, indicating these fluorescent aptasensor methods may be used for a wide variety of small molecules. Graphical abstract.

Label-free aptasensors based on fluorescent screening assays for the detection of Salmonella typhimurium.

We report two label-free fluorescent aptasensor methods for the detection of S. typhimurium. In the first method, we have used a ''turn off'' approach in which the aptamer is first intercalated with SYBR Green I (SG), leading to a greatly enhanced fluorescence signal. The addition of S. typhimurium (approximately 1530-96938 CFU/mL), which specifically binds with its aptamer and releases SG, leads to a linear decrease in fluorescence intensity. The lowest detection limit achieved with this approach was in the range of 733 CFU/mL. In the second method, a ''turn on'' approach was designed for S. typhimurium through the Förster resonance energy transfer (FRET) between Rhodamine B (RB) and gold nanoparticles (AuNPs). When the aptamer and AuNPs were mixed with RB, the fluorescence of RB was significantly quenched via FRET. The aptamer adsorbs to the AuNP surface to protect them from salt-induced aggregation, which leads to the fluorescence quenching of RB in presence of AuNPs. Upon the addition of S. typhimurium, S. typhimurium specifically binds with its aptamer and loses the capability to stabilize AuNPs. Thus, the salt easily induces the aggregation of AuNPs, resulting in the fluorescence recovery of the quenched RB. S. typhimurium concentrations ranging from 1530 to 96938 CFU/mL with the detection limit of 464 CFU/mL was achieved with this methodology. Given these data, some insights into the molecular interactions between the aptamer and the bacterial target are provided. These aptasensor methods also may be adapted for the detection of a wide variety of targets.